WO2011133432A9 - Methods to detect and isolate cancer stem cells - Google Patents

Abstract

Methods are provided herein for identifying and/or isolating viable stem cells, such as cancer stem cells. These methods include the use of a nucleotide or analogue thereof labeled with a detectable marker, wherein the detectable marker can be detected in a viable cell. More than one nucleotide or analogue thereof labeled with a detectable marker can be utilized.

Description

METHODS TO DETECT AND ISOLATE CANCER STEM CELLS

PRIORITY CLAIM

This claims the benefit of U.S. Provisional Application No. 61/342,642, filed April 16, 2010, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

This relates to the field of cancer biology, specifically to methods for detecting and/or isolating stem cells, such as cancer stem cells, and assessing the drug sensitivity of cancer stem cells.

BACKGROUND

The cancer stem cell hypothesis suggests that tumors contain a small subpopulation of cells which are exclusively responsible for cancer initiation, maintenance and therapeutic failure, called cancer stem cells (CSC). Although, CSC were first identified in hematologic malignancies, recent studies have suggested the presence of CSC in solid organs (breast, brain, pancreas, colon, and liver). CSC have been identified using non-specific markers such asCD133, CD24, CD44, CD90, although these markers are present on other cells. The ability to efflux the Hoechst dye, defined as the "side-population," has also been used to identify CSC. Collectively, these methods resulted in heterogenic populations enriched with putative CSC. However, cells identified using these methods generally were not able to initiate tumors with less than 100 cells.

The gold standard for testing CSC is xenogeneic transplantation into immunosuppressed mice. It tests CSC capacity to initiate tumors in a non-human- environment. Quintana et al. (Nature 456: 593-598, 2008) demonstrated that xenogeneic transplantation of melanoma cells into a more immunosuppressed animal (NOD/SCID-IL2reg^"/ ) results in a higher frequency of cancer- initiating-cells. These data raised questions regarding the ability of xenotransplantation to differentiate between CSC, progenitors and non- stem-cancer-cells. Growing human cells in a mouse might select for a sub-population of cancer cells capable of growing as xenografts and not necessarily reflect CSC capacity to grow and metastasize in humans. Thus, methods are needed to isolate CSC directly from variety of cancers using specific functional properties of stem cells and avoiding xenotransplantation. SUMMARY

Stem cells, including cancer stem cells (CSC) can self -renew and produce more differentiated cells. Stem cells can undergo asymmetric cell division (ACD). ACD with non-random chromosomal cosegregation (NRCC) segregates the older template DNA strands into daughter stem cells, and newly synthesized DNA into daughter cells destined for differentiation. The asymmetric cell division can be used to identify and/or isolate stem cells, including cancer stem cells.

Methods that utilize single and dual-color chromosomal DNA labeling in viable cells are described herein. This methodology can be used for the isolation of viable stem cells, or other cells (such as cancer stem cells) undergoing ACD-NRCC. In some embodiments, these methods utilize fluorescence activated cell sorting

(FACS). These methods include the use of a nucleotide or analogue thereof labeled with a detectable marker, wherein the detectable marker can be detected in a viable cell. More than one nucleotide or analogue thereof labeled with a detectable marker can be utilized.

In some embodiments, the method for identifying a stem cell, such as cancer stem cells uses two detectable markers. These methods include synchronizing the cell cycle of cells in a population of cells, such as cancer cells, and labeling the population of cells undergoing cell cycle division by exposing the cells to a first nucleotide or an analogue thereof labeled with a first detectable marker for one cell cycle, wherein the first detectable marker can be detected in a viable cell, such as a cancer cell. The first nucleotide or analogue thereof is incorporated into DNA of the cells during a first DNA replication, and cells are isolated that include the first nucleotide or analogue in their DNA. The first nucleotide or analogue thereof labeled with the first detectable marker is then removed from the population of cells, and the population of cells is allowed to undergo cytokinesis. The population of cells undergoing cell cycle division is then labeled again by exposing the cells to a second nucleotide or analogue thereof labeled with a second detectable marker, wherein the second detectable marker can be detected in a viable cell, for the duration of one cell cycle. The first detectable marker and the second detectable marker are different. The second nucleotide or analogue thereof is incorporated into the DNA of the cells during a second DNA replication. The second nucleotide or analogue thereof is then removed from the cells, and the cells are allowed to undergo cytokinesis. At least one viable cell is identified that comprises only the second nucleotide or analogue thereof incorporated into the DNA of the cell, in the absence of the first nucleotide or analogue thereof incorporated into the DNA of the cell, by detecting the presence of the second detectable marker and the absence of the first detectable marker in the viable cell. In this manner the stem cell, such as the cancer stem cell, is identified and/or isolated.

In additional embodiments, methods are provided for identifying a stem cell, such as a cancer stem cell, that include labeling a population of synchronized cancer cells undergoing cell cycle division by exposing the cells to a nucleotide or analogue thereof labeled with a detectable marker prior to a first round of DNA replication, and allowing the cells to undergo a single cell division. The nucleotide or analogue thereof is incorporated into chromosomes of the cells, and the detectable marker can be detected in viable cells. The nucleotide or analogue thereof labeled with the detectable marker that is not incorporated into the chromosomes of the cells is removed after one round of DNA replication, and the cells are allowed to divide for at least five additional cell divisions, such as but not limited to five, six or seven cell divisions, in the absence of the nucleotide or analogue thereof labeled with a detectable marker. Viable cells comprising the nucleotide or analogue thereof incorporated into the chromosomes of the cancer cells are identified by detecting the detectable marker, which detects the stem cell, such as the cancer stem cell.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1C are schematic diagrams. Asymmetric cell division with non- random chromosomal cosegregation (ACD-NRCC). Figure 1A. ACD-NRCC proposes that each chromosome in a stem-cell (SC) has one template-DNA strand that is conserved after numerous asymmetric divisions (AS YD). It undergoes the fewest rounds of replication. SCs maintain "immortal template-DNA strands" in order to avoid propagation and accumulation of DNA replication errors in these high fidelity chromosomes. As a result, mutations can be deliberately isolated into the daughter-cells destined to differentiate and ultimately be eliminated. Figure IB. To identify ASYD with chromosomal cosegregation, an in vitro chromosomal double- labeling procedure was developed in which cells are followed for two cell cycles. Prior to each round of DNA replication, the cells are labeled sequentially with two different nucleotides: Cy5-dUTP (vertical lines) then ALEXA FLUOR® 555-dUTP (horizontal lines). When random segregation of chromosomes, or symmetric division (SYD), occurs both nuclei in the couplet will incorporate both Cy5-dUTP and ALEXA FLUOR® 555-dUTP (represented on Figure IB by a cell including an inner filled circle). However, non-random cosegregation, or ASYD, occurs then one nucleus will incorporate both Cy5-dUTP and ALEXA FLUOR® 555-dUTP (bright circle around three cells at the end outcome; represented on Figure IB by a cell including an inner filled circle) while the other nuclei, containing the immortal template-DNA strands will incorporate only ALEXA FLUOR® 555-dUTP after the second division (dark circle around one cell at the end outcome; represented on Figure IB by a cell including an inner circle with horizontal lines). Figure 1C. To further confirm that the preserved immortal template-DNA strands after multiple divisions, an in vitro long-term chromosomal label-retaining procedure (SOM- methods) was developed in which cells were followed for six cell cycles. Prior to the first round of DNA replication, the cells were labeled with a single nucleotide, Cy5-dUTP, light grey cells. FACS sorting produced greater than 99% pure sample that was proprogated in culture for five more cell cycles and then the long-term Cy5- dUTP-labeled cells with the immortal template DNA strands were isolated via FACS sorting.

Figure 2 is a schematic diagram of asymmetric division (ASYD ~ "self- renewal") with non-random chromosomal cosegregation in human cancer via a chromosomal double-labeling technique. Twelve hours after plating, human liver cancer cells (PLC/PRF/5) were stained with 0.5μΜ CFSE (100% CFSE, dark grey cells cell, top section). Ten hour later, cells were harvested and prepared for initial nucleotide labeling. Cells were microporated with Cy5-dUTP (light grey DNA strand) (ΙΟΟμΜ). Thirteen hours following microporation (35hrs total ~ one complete cell cycle), the initial FACS sort was performed. A subpopulation of cells were isolated that contained 50% CFSE (one cell division) and 100% Cy5. This subpopulation of cells was sorted to a purity of 99.9%. The 50%CFSE+/Cy5+ sorted cells were immediately placed in culture using AFS media at 37°C. Twenty- two hours following the 50%CFSE+/Cy5+ sort, the cells were harvested for the second round of dUTP-labeling with ALEXA FLUOR® 555-dUTP (bottom section). Once again, the cells were harvested and prepared for final nucleotide labeling. Cells were microporated with Alex-fluor 555-dUTP (ΙΟΟμΜ). Eighteen hours following microporation (75 hrs total ~ two complete cell cycles), the final FACS sort was performed. Cells are initially sorted to ensure the second division occurred, which is confirmed by 25% CFSE staining. The heterogenic population of cells are then sorted into two groups: 25% CFSE+/Alexa555+ cells and 25% CFSE+/Alexa555+/Cy5+ cells. The 25% CFSE+/Alexa555+ subpopulation represents the cells in which non-random chromosomal cosegregation occurred and the immortal strand-DNA was preserved. The 25% CFSE+/Alexa555+/Cy5+ subpopulation represents the cells in which random chromosomal segregation has occurred. The cells were then prepared for microscopy.

Figures 3A-3I is a set of plots of data from FACS sort analysis to distinguish symmetric and asymmetric division. Figures 3A-3B. Following staining with CFSE (0.5μΜ), cells were microporated to incorporate the control, unlabeled dUTP (ΙΟΟμΜ) or the initial labeled nucleotide, Cy5-dUTP (ΙΟΟμΜ). Following the 1^st complete cell cycle (~35hrs), the initial FACS sort was performed. One hour prior to the FACS sort, control cells were incubated in 0.5μΜ CFSE to provide the 100% CFSE controls for the FACS sorting. Cell viability was determined based on light scatter using FSC-A vs. SSC-A (93%) and then SSC-A vs. FSC-W (95%). Cells were then gated based on 50% CFSE (amount remaining after one cell division) and 55% Cy5+ cells. Figure 3C. This population of cells was sorted to 99% purity. The 50% CFSE+/Cy5+ sorted cells were immediately placed in culture using AFS media at 37°C. Twenty-two hours following the 50% CFSE+/Cy5+ sort, the cells were harvested for the second round of dUTP-labeling with ALEXA FLUOR® 555- dUTP. Once again, the cells were microporated to incorporate either unlabeled dUTP (ΙΟΟμΜ) or Alex-fluor 555-dUTP (ΙΟΟμΜ). Following the 2^nd complete cell cycle (~75hrs), the final FACS sort was performed. The 100% CFSE control cells that were prepared for the initial sort were now used as the 50% CFSE control group for the final sort. Once again, cell viability was determined based on light scatter using FSC-A vs. SSC-A (95%) and then SSC-A vs. FSC-W (94%). Figures 3D-3F. Cells were then gated based on 25% CFSE (amount remaining after two cell divisions) and 65% Cy5+ cells and 85% Alexa555+ cells. There were two groups isolated: 25% CFSE+/Alexa555+ cells and 25% CFSE+/Alexa555+ /Cy5+ cells. Figure 3G. The 25% CFSE+/Alexa555+ cells sorted subpopulation had an

Alexa555+ purity of 98%. Figures 3H-3I. The double-labeled sorted subpopulation, 25% CFSE+/Alexa555+ /Cy5+, had an Alexa555+ purity of 97% and a Cy5+ purity of 95%. Following the final FACS sort, the cells were placed in placed in culture using AFS media at 37°C.

Figure 4 is a series of digital images showing three dimensional fluorescent confocal microscopy with surface rendering to capture symmetric division

(differentiation) and asymmetric division (self -renewal) in human liver cancer (PLC/PRF/5) using double-labeling technique. Following CFSE staining and Cy5- dUTP labeling, CFSE+( white)/Cy5-dUTP+ (grey) were isolated after the first round of replication via FACS sorting. This population of Cy5+ cells was greater than 99.9% pure. Following a second round of labeling with ALEXA FLUOR® 555-dUTP (red) the cells were observed for a second round of replication. After the 2^nd cell cycle, the cells were sorted to distinguish symmetrically divided cells, CFSE+(white)/Cy5+(light grey)/Alexa555+(dark grey) cells and asymmetrically divided cells, CFSE+(white)/Alexa555+(dark grey) cells. Symmetric division is illustrated by cells fixed in cytokinesis containing two nuclei labeled with both nucleotides (light and dark grey). Symmetric division illustrated by cells fixed in cytokinesis containing two nuclei labeled with only one nucleotide (dark grey). Asymmetric division is illustrated by cells fixed in cytokinesis containing one nucleus with the "immortal template-DNA strand" (dark grey) and the other nucleus containing both nucleotides (light and dark grey). The DAPI (black) staining reveals the same nuclear space without an intervening membrane. Three-dimensional reconstruction movies were created demonstrating no intervening cell membrane between the two nuclei indicating localization of both nuclei within the same cytoplasmic space during cytokinesis.

Figure 5 is a schematic diagram of symmetric (self-renewal) division with non-random chromosomal cosegregation in human cancer cells (PLC/PFR/5) via a long-term labeling retaining technique. Cells were harvested and prepared for microporation using Cy5-dUTP (ΙΟΟμΜ). Following 1^st complete cell cycle, Cy5+ cells were sorted via FACS analysis. A subpopulation of Cy5+ cells was placed in culture at 37°C. Cells were proprogated in culture for six cycles. Following completion of the 6^th cell cycle, the cells were then sorted again for Cy5+ (long-term label retaining cells which contain the immortal strand-DNA) and Cy5- cells.

Figures 6A-6C are a set of plots from a FACS sort analysis to isolate long- term label retaining cells. Cells were microporated to label with the control, unlabeled dUTP (ΙΟΟμΜ) or the labeled nucleotide, Cy5-dUTP (ΙΟΟμΜ).

Following the 1^st complete cell cycle (~35hrs), the initial FACS sort was performed. Cell viability was determined based on light scatter using FSC-A vs. SSC-A (95%) and then SSC-A vs. FSC-W (97%, no shown). Cells were then gated based on 50% Cy5+ cells as shown in Figure 6A. As shown in Figure 6B, this population of cells was sorted to 99% Cy5+ purity. The cells were then propagated in culture for six cell cycles. The final FACS sort was performed. Once again, cell viability was determined based on light scatter using FSC-A vs. SSC-A (96%) and then SSC-A vs. FSC-W (95%). In Figure 6C, cells were then gated based on 0.5% Cy5+ cells.

Figures 7A-7B are a graph and a table showing that tumor initiation capacity varies with different anatomical sites of transplantation raising questions whether xenogeneic transplantation is the optimal gold standard for testing putative cancer stem cells. The sine qua none of stem cells (SC) is their ability to differentiate into more mature progeny and to self -renew. The current gold standard for testing putative CSC is xenogeneic transplantation into immunosuppressed mice. It tests putative CSC's capacity to initiate tumors in a non-human environment. To test this concept, three different pancreatic cell lines were transplanted in various anatomical sites of NUDE/SCID mice (front and hind limbs, intrahepatic and intravenous via the tail- vein). Figure 7 A presents data showing that transplantation in various anatomical sites results in statistically different rates of tumor initiation (p<0.001), as shown above and in Figure 7B. lx 10⁶ cells were injected into either front limbs, hind limbs, liver, or tail veins. Limb injections were done subcutaneously. Liver injection involved laparotomy and injection into the liver parenchyma under direct visualization. Xenotransplantation via the tail vein resulted in no tumor initiation in all three cell lines tested. The study above used panc-1, a pancreatic cancer cell line. Similar results were obtained for two other pancreatic cancer cell lines, SW1990 and Su86.86. Front limb injection produces the fastest tumor growth among all three cell lines.

Figure 8 is a table and graphs classification of human cancer cell line (PLC/PFR/5) doubling time with various condition: control (PLC only), FACS sorting (PLC Sort Only), microporation (PLC Micro only) and microporation with unlabeled-dUTP. In 15 to 18 wells of 6-well plates, 5 x 10e4 cells were plated per well and allowed to attach for 24 hours. On each time point, 8 hours apart, the numbers of live cells were determined in three wells. Acquired numbers were averaged and converted into percentage relative to the average acquired at the first time point. These percentages were plotted using an Excel spreadsheet. A best fit exponential trend line with y-axis interception at 100% was generated using regression analysis of this trend line the doubling time was computed. Correlation value R2>0.9 was considered adequate for computations of doubling times.

Doubling times were calculated for all cell conditions tested.

Figure 9 is a graph and a digital image of CFSE (carboxyfluorescein diacetate, succinimidyl ester)-staining to assess cell proliferation. CFSE passively diffuses into cells and the label is inherited by daughter cells after each cell division. It is colorless and non-fluorescent until the acetate groups are cleaved by

intracellular esterases to yield highly fluorescent carboxyfluorescein succinimdyl ester groups. These ester groups react with intracellular amines and form fixed fluorescent conjugates that are retained. Human cancer cell (PLC/PRF/5) are evaluated at baseline (right), 35hrs after plating (center) and 70hrs after plating (left). A digital image is also shown of three-dimensional confocal microscopy surface rendering of a cell stained with CFSE. The nucleus appears dark (DAPI) while CFSE-stained cytoplasm appears light (pseudo-colored in order to distinguish from Cy5 and Alexa555 coloring-schematic).

Figure 10 is a graph of cell cycle synchronization and analysis of human cancer cell line (PLC/PFR/5) to determine the ideal technique to ensure optimal nucleotide incorporation in which the cells are predominantly in the Gl/S phases.

The cells were plated lxlO⁵ cells in 24- well plates using AFS media. Cells were plated simultaneously. Control cells were evaluated at 20, 22, 24, 26, 28, 32, 34 and 38 hours after initial plating. Experimental groups consisted of serum- starvation for 24-48 hours and/or aphidicolin (2μg/ml) treatment to induce an Sl- phase arrest. Twelve-hours after plating, cells were serum-starved (SS) alone (12, 24, 36, 48, 60 and 72 hours post-initial plating), SS then returned to AFS media, SS (24 and 48hrs) then treated with aphidicolin and finally treated with aphidicolin 20 hrs after initial plating. At the indicated timepoints, cells were harvested and with 1 % PFA prior to staining with a 1 : 1 ratio of Vindelov' s PI and plain PI (200μL· per sample). FACS analysis was performed in order to assess the G1/S/G2 phases within each condition. All conditions were performed in triplicate. The x-axis shows the various conditions. The y-axis the percentage of each cell-cycle phase: Average % Gl (blue), Average % S (red) and Average % G2 (green). Given that similarity in the 20 hours-post initial plating control sample and the 'gold standard' serum-starvation with aphidicolin arrest yielded compatible results, the cells were exposed to chemical alteration when performed the double-labeling technique.

Figure 11. In vivo study using the isolated long-term, label-retaining (LTLR) Cy5+ was conducted given on the current accepted "gold standard" for confirmation of putative CSC. Non-label retaining cells (Cy5- control) and LTLR- Cy5+ cells were injected into nude/SCID mice. The cells were prepared in 25% Matrigel (10 cells/ΙΟΟμΙ total volume) and injected subcutaneously. One week prior to the injections, each mouse was injected with a subcutaneous transponder in order for the injection and subsequent tumor measurements to be performed in a blinded fashion over 16 weeks. Tumor growth was monitored biweekly at two dimensions with a ruler. Tumors were harvested if the product of the largest diameters was greater than 100mm or at the 16- week endpoint. Tumors were generated with only 10 long term label retaining cancer stem cells.

Figures 12A and 12B are graphs showing cancer derived LRC express higher levels of stem cell associated genes than non-LRC, In Figure 12A, the real time qRT- PCR array analysis of human stem cell genes for HCC cells show that the pluripotent transcription factor Sox2 gene is increased surprisingly by 38.9 + 13.1 (p=0.035) fold in LRC verse non-LRC. Also increased are the stem cell genes of BMPl, Neurog2 and DTX1 as well as the lineage gene CD8A. Figure 12B shows the same analysis for WNT pathway genes show that the stem cell genes of FGF4, WNTl, WNT6 and FOXN1 as well as the lineage gene FSHB are increased in LRC verse Non-LRC. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Methods are provided herein that can be used to identify viable stem cells, such as cancer stem cells. Stem cells undergo asymmetric cell division (AS YD). Each chromosome in a stem cell (SC) contains one DNA strand that is conserved throughout multiple asymmetric divisions (see, for example Figure 1A). It is this conserved template, the "immortal strand," which undergoes the fewest divisions. Without being bound by theory, using this mechanism a SC is able to avoid the accumulation of mutations from DNA replication errors by preferentially segregating the replication errors into the daughter-cell fated to further differentiate. The methods that are disclosed herein use ASYD to identify stem cells, such as cancer stem cells (CSC).

It is demonstrated herein that asymmetric cell division can be detected in viable cells. Described are double-labeling techniques, which use two markers, and long-term label retaining techniques, that use at least one marker. The isolation of stem cells, such as cancer stem cells, allows the use of these cells in screening methods, in order to identify agents that specifically target these cells. The present methods have sufficient sensitivity and specificity to identify a homogenous population of stem cells, such as cancer stem cells.

Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in

Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology : a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

ALEXA FLUOR®: Dyes that are synthesized through sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. Sulfonation makes ALEXA FLUOR® dyes negatively charged and hydrophilic. ALEXA FLUOR® dyes are generally more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission. In one example, ALEXA FLUOR® 555 is a yellow-green dye that absorbs light at 555 nm and emits at 565. It is approximately 1250 g/mol and has an ε (cm^_1M^_1) of 150,000.

Breast cancer: A neoplastic condition of breast tissue that can be benign or malignant. The most common type of breast cancer is ductal carcinoma. Ductal carcinoma in situ is a non-invasive neoplastic condition of the ducts. Lobular carcinoma is not an invasive disease but is an indicator that a carcinoma may develop. Infiltrating (malignant) carcinoma of the breast can be divided into stages (I, ΠΑ, ΠΒ, ΠΙΑ, ΙΠΒ, and IV).

Breast carcinomas lose the typical histology and architecture of normal breast glands. Generally, carcinoma cells overgrow the normal cells and lose their ability to differentiate into glandular like structures. The degree of loss of differentiation in general is related to the aggressiveness of the tumor. For example, "in situ" carcinoma by definition retains the basement membrane intact, whereas as it progresses to "invasive", the tumor shows breakout of basement membranes. Thus one would not expect to see, within breast carcinomas, staining of a discrete layer of basal cells as seen in normal breast tissue. For a discussion of the physiology and histology of normal breast and breast carcinoma, see Ronnov-Jessen et al., Physiol Rev 76, 69-125, 1996).

Breast cancers can be divided into groups based on their expression profiles. Basal-type carcinomas usually are negative for expression of estrogen receptor (ER) and negative for expression of HER2 (erbB2) and progesterone receptor (PR), and thus are referred to as "triple-negative breast cancers" or "TNBC." This type of breast cancer is also denoted ER7HER27PR^" and represents about 15-20 % of all breast cancer, and generally cannot be treated using Her2 targeted or estrogen targeted therapies. It is believed that the aggressive nature of this cancer is correlated with an enrichment for cancer stem cells (CSC) with a CD44⁺CD24^"/l0 phenotype. In some embodiments, basal carcinomas are negative for expression of progesterone receptor (PR), positive for expression of epidermal growth factor receptor (EGFR), and positive for expression of cytokeratin 5 (CK5). This phenotype is denoted as follows: ER7PR7HER27CK5⁺/EGFR⁺.

Cancer: A malignant neoplasm. Generally, malignant neoplasms have undergone characteristic anaplasia with loss of differentiation, increase rate of growth, invasion of surrounding tissue, and are capable of metastasis. For example, thyroid cancer is a malignant neoplasm that arises in or from thyroid tissue, and breast cancer is a malignant neoplasm that arises in or from breast tissue (such as a ductal carcinoma). Residual cancer is cancer that remains in a subject after any form of treatment given to the subject to reduce or eradicate thyroid cancer. Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. Cancer includes, but is not limited to, solid tumors.

Cancer Stem Cells (CSCs): Cancer cells found within solid tumors or hematological cancers that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer, and also have the ability to self-renew. Cancer stem cells are tumorigenic (tumor- forming). CSCs can generate tumors through the stem cell processes of self -renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. CSCs have recently been identified in several solid tumors, including cancers of the brain, breast, colon, ovary, pancreas and prostate, as well as in hematologic cancers.

Carboxy fluorescein succinimidyl ester (CFSE): A membrane permeable fluorescent dye that can measure cell proliferation using flow cytometry. Upon entry into a living cell, esterases remove the acetate groups resulting in membrane- impermeable CFSE that crosslinks to intracellular proteins. Through the use of flow cytometry, proliferation of the labeled cell sample can be quantitated. The relative fluorescent intensity decreases by half with each round of cell division.

Cell cycle: The series of events that takes place in a cell leading to its division and duplication (replication). The cell cycle can be divided in two brief periods: interphase-during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA and t mitosis (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells." The cell cycle includes four distinct phases: Gi phase (interphase or growth phase), S phase (DNA synthesis, chromosome replication), G₂ phase (growth phase) and M phase (mitosis, including karyokinesis and cytokines). In karyokinesis the cell's chromosomes are divided between the two daughter cells, and during cytokinesis the cell's cytoplasm divides in half forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called Go phase. Cells in a population that are "synchronized" are at the same phase of the cell cycle, such as Gi, S, G₂ or M.

Chemotherapeutic agent: As used herein, any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one embodiment, a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors. In one embodiment, a chemotherapeutic agent is radioactive molecule, a DNA intercalating agent, an antimetabolite, a natural product that can kill dividing cells, or an alkylating agent. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

Colon cancer: Colorectal cancer, also called large bowel cancer, includes cancerous growths in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Many colorectal cancers are thought to arise from adenomatous polyps in the colon. These mushroom-like growths are usually benign, but some may develop into cancer over time. The majority of the time, the diagnosis of localized colon cancer is through colonoscopy. Therapy is usually through surgery, which in many cases is followed by

chemotherapy. The first symptoms of colon cancer are usually vague, such as bleeding, weight loss, and fatigue (tiredness). Local (bowel) symptoms are rare until the tumor has grown to a large size. Generally, the nearer the tumor is to the anus, the more bowel symptoms there will be.

Cy5 and Cy3: Reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are green (-550 nm excitation, -570 nm emission), while Cy5 is fluorescent in the red region (-650/670 nm). They are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically linked to either nucleic acids or protein molecules. Labeling is done for visualization and uantification purposes. The chemical structures of these dyes are:

The R groups do not have to be identical. In the dyes as used they are short aliphatic chains one or both of which ends in a highly reactive moieties such as N- hydroxysuccinimide or maleimide. ALEXA FLUOR® dyes or DYLIGHT

FLUOR™, can be used interchangeably with Cy dyes in most biochemical applications.

Cytokinesis: The process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells. It usually initiates during the late stages of mitosis, and sometimes meiosis, splitting a binucleate cell in two, to ensure that chromosome number is maintained from one generation to the next.

Detectable Marker: A marker having a characteristic, such as color, intensity, excitation wavelength, emission spectra, luminescence or other

characteristic that permits it to be detected.

DNA Replication: A fundamental process occurring in all living organisms to copy their DNA. Each strand of the original double- stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two DNA molecules have been produced from a single double-stranded DNA molecule.

Fluorescent Marker: A marker that can be detected by its fluorescent emission spectrum. Exemplary markers can be detected by a blue argon laser (488 nm), a red diode laser (635 nm), and a violet laser (405 nm). Exemplary fluorescent markers are as follows.

Blue argon laser (488 nm):

1. Green (usually labeled FL1): fluorescein isothiocyanate (FITC), ALEXA FLUOR® 488, CFSE, CFDA-SE, DyLight™ 488

2. Orange (usually FL2): phycoerythrin (PE), propidium iodide (PI)

3. Red channel (usually FL3): Cy5.5, PE-ALEXA FLUOR® 700, Cy5

4. Infra-red PE-ALEXA FLUOR® 750, PE-Cy7

Red diode laser (635 nm): APC, APC-Cy7, APC-EFLUOR™ 780, ALEXA FLUOR® 700, Cy5, Draq-5

Violet laser (405 nm): Pacific Orange, Amine Aqua, Pacific Blue, DAPI (4',6-diamidino-2-phenylindole), ALEXA FLUOR® 405, EFLUOR™ 450 , EFLUOR™ 605, EFLUOR™ 625, EFLUOR™ 650

Fluorescence Activated Cell Sorting (FACS): A technique for counting, examining and separating microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of cancer and the isolation of populations of lymphocytes, but has many other applications in both research and clinical practice.

A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle from 0.2 to 150

micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle.

A flow cytometer generally has several main components: (1) a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing; (2) a measuring system, such as those that allow measurement of impedance (or conductivity) and optical systems - lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low- power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals; (3) a detector and Analogue-to-Digital Conversion (ADC) system which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer; (4) an amplification system (either linear or logarithmic); and (5) a computer for analysis of the signals. FACS instruments usually have multiple lasers and fluorescence detectors (such as up to 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers.

Using fluorescent activated cell sorting (FACS), a heterogeneous mixture of biological cells can be separated into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately-prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains a charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. FACS can be used to identify and isolate cancer stem cells, as described herein.

Gating: The data generated by FACS can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology and the identification of stem cells.

The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software, e.g., FLOJO™, or CELLQUEST PRO™.

Generation (of a cell): The duration of time necessary for a cell to undergo one complete cell cycle, including both nuclear division and cytokinesis. The length of a generation is cell-type specific, and can be determined empirically using techniques well known in the art. For example, many cycling mammalian cells typically divide every 16-24 hours.

Isolated (biological component): An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and

extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An "isolated" cell has been substantially separated (purified) from other cells in a sample. In some embodiments, isolated cells are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% pure.

Lymphoma: A cancer that begins in the lymphocytes and presents as a solid tumor of lymphoid cells. Lymphomas are generally treatable with chemotherapy, and in some cases radiotherapy and/or bone marrow transplantation, and can be curable, depending on the histology, type, and stage of the disease. The WHO classification is a generally accepted system for the classification of lymphoma and is based upon the foundations laid within the "Revised European- American

Lymphoma classification" (REAL). This system attempts to group lymphomas by cell type (i.e. the normal cell type that most resembles the tumor) and defining phenotypic, molecular or cytogenetic characteristics. There are three large groups: the B cell, T cell, and natural killer cell tumors. Hodgkin's lymphoma, although considered separately within the WHO classification, is now recognized as being a tumor of lymphocytes of the mature B cell lineage.

Lymphomas include mature B cell lymphomas such as chronic lymphocytic leukemia/Small lymphocytic lymphoma, B-cell prolymphocyte leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macro globulinemia), splenic marginal zone lymphoma, plasma cell neoplasms: plasma cell myeloma,

plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, and Burkitt lymphoma/leukemia.

Lymphomas also include mature T cell and natural killer cell neoplasms, such as T cell prolymphocyte leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma (nasal type), enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, and anaplastic large cell lymphoma.

Mammal: This term includes both human and non-human mammals.

Similarly, the term "subject" includes both human and veterinary subjects. Membrane Permeable: A molecule that can pass through a cell membrane in response to a concentration gradient. Active transport is not required to move a membrane permeable substance into (or out of) a living cell.

Non-viable cells: Cells that are in the process of dying or are dead. These cells do not divide. Non-viable cells include necrotic and apoptotic cells.

Nucleotide: A nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar (either ribose or 2'-deoxyribose), and one to three phosphate groups. Together, the nucleobase and sugar comprise a nucleoside. The phosphate groups form bonds with either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site most common. Cyclic nucleotides form when the phosphate group is bound to two of the sugar's hydroxyl groups. Ribonucleotides are nucleotides where the sugar is ribose and deoxyribonucleotides contain the sugar deoxyribose. Nucleotides can contain either a purine or pyrimidine base. In vivo, the purine bases in DNA are adenine and guanine, while the pyrimidines are thymine and cytosine. RNA uses uracil in place of thymine. Nucleotides include ATP, CTP, GTP, TTP and UTP.

Nucleotide analogue: Any analogue of a nucleotide base which can be incorporated into replicating cellular DNA. Thus, a nucleotide analogue can be an adenosine, guanosine, uridine, cytosine or thymidine analog. In several

embodiments, detectable nucleotide analogs can be detected in viable cells; the cells do not need to be preserved (fixed), or killed to detect the nucleotide analogue incorporated into the DNA of the cell. Thus, the nucleotide analogue is not 5- bromo-deoxyuridine (BrdU) or any other halogenated nucleotide base analogues such as iodo-deoxyuridine or chloro-deoxyuridine, or other nucleotide analogues which can be incorporated into replicating cellular DNA following permeabilization of the plasma membrane but cannot be visualized until the cell is killed. Exemplary nucleotide analogs of use in the present method are those labeled with a fluorescent marker, such as but not limited to Cy5, Cy3 and Alexa555.

Pharmaceutical agent or drug: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. Pharmaceutical agents include, but are not limited to, chemo therapeutic agents and anti-infective agents. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in the methods and compositions disclosed herein are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions {e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Separating: In reference to the separation of a stem cell, such as a cancer stem cell, from other cells, such as a non-cancerous stem cell. More generally, the term refers to the purification of one cell type from another cell type, as used herein, refers to spatially segregating cells of the different cell types from each other so as to yield a fraction that is relatively enriched in a first cell type, with respect to a second cell type, and another fraction that is relatively enriched in a second cell type, with respect to a first cell type. In certain embodiments, cell types (e.g., cancer stem cells and/or progenitor cells and non-cancerous stem cells and/or progenitor cells) can be separated from each other such that the segregated fractions of the respective cell types are enriched in the desired cells by at least a factor of about 5, in some embodiments by at least a factor of about 10, in some embodiments by at least a factor of about 100, in some embodiments by at least a factor of about 1000, in some embodiments by at least a factor of about 10⁴, in some embodiments by at least a factor of about 10⁵, in some embodiments by at least a factor of about 10⁶, and in yet other embodiments the desired cells in the segregated fraction are free of cells of the undesired type.

Stem cell: A cell that can generate a fully differentiated functional cell of a more than one given cell type and can self-renew. The role of stem cells in vivo is to replace cells that are destroyed during the normal life of an animal. Generally, stem cells can divide without limit and are totipotent or pluripotent. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation. A nervous system (NS) stem cell is, for example, a cell of the central nervous system that can self-renew and can generate astrocytes, neurons and oligodendrocytes. Cancer stem cells are defined above.

Suspension of Cells or Cellular suspension: A mixture of cells suspended in a carrier liquid. The carrier liquid may be naturally part of the biological sample from which the cells derive, for example blood is a suspension of blood cells suspended in plasma, or, for cells which are not normally present in a suspension, the carrier liquid can be any suitable diluent or medium. A cellular suspension can include a plurality of stem cells of one or more specific and desired types, for example cancer stem cells, lympho-hematopoietic stem cells. For example, for such an embodiment in the context of cancer treatment, diagnostics, or research, the methods described herein can be used to generate cellular suspension including a plurality of cancer stem cells.

Synchronizing (a population of cells in the cell cycle): A process by which cells at different stages of the cell cycle in a culture are brought to the same phase. Cell synchronization can be induced by either chemical or physical methods. Centrifugation and FACS are two physical methods can used to synchronize cells. Chemical methods include nutritional methods such as serum starvation and treatment with thymidine, aminopterin, hydroxyurea and cytosine arabinoside. Thymidine or aphidicolin halt the cell in the Gi phase. Treatment with colchicines and treatment with nocodazole halt the cell in M phase and treatment with 5- fluorodeoxyuridine halts the cell in S phase.

Totipotent or totipotency: A cell's ability to divide and ultimately produce an entire organism including all extraembryonic tissues in vivo. In one aspect, the term "totipotent" refers to the ability of the cell to progress through a series of divisions into a blastocyst in vitro. The blastocyst comprises an inner cell mass (ICM) and a trophectoderm. The cells found in the ICM give rise to pluripotent stem cells (PSCs) that possess the ability to proliferate indefinitely, or if properly induced, differentiate in all cell types contributing to an organism. Trophectoderm cells generate extra-embryonic tissues, including placenta and amnion.

As used herein, the term "pluripotent" refers to a cell's potential to differentiate into cells of the three germ layers: endoderm (e.g., interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g., muscle, bone, blood, urogenital), and ectoderm (e.g., epidermal tissues and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type including germ cells. However, PSCs alone cannot develop into a fetal or adult animal when transplanted in utero because they lack the potential to contribute to all extraembryonic tissue (e.g., placenta in vivo or trophoblast in vitro).

PSCs are the source of multipotent stem cells (MPSCs) through spontaneous differentiation or as a result of exposure to differentiation induction conditions in vitro. The term "multipotent" refers to a cell's potential to differentiate and give rise to a limited number of related, different cell types. These cells are characterized by their multi-lineage potential and the ability for self-renewal. In vivo, the pool of MPSCs replenishes the population of mature functionally active cells in the body. Among the exemplary MPSC types are hematopoietic, mesenchymal, or neuronal stem cells.

Transplantable cells include MPSCs and more specialized cell types such as committed progenitors as well as cells further along the differentiation and/or maturation pathway that are partly or fully matured or differentiated. "Committed progenitors" give rise to a fully differentiated cell of a specific cell lineage.

Tumor: An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease" refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.

The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant."

Examples of hematological tumors include leukemias, including acute leukemias (such as 1 lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic

myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma,

pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In several examples, a tumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

Viable: Cells that are alive. Generally a viable cell will survive and can divide when induced to undergo cell division under appropriate culture conditions in vitro.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Methods for Identifying and/or Isolating Viable Stem Cells Methods are provided herein for identifying and/or isolating viable stem cells. The stem cell can be any stem cell of interest, including totipotent, pluripotent and multipotent stem cells. Generally, the stem cell can self-renew and give rise to daughter cells that produce all the cells of the originating tissue. The stem cells can be precursor cells that give rise to the all cells of a specific tissue type. Thus, the stem cells can be hemapoietic stem cells or the stem cells of any tissue type, such as neuronal stem cell, endocrine stem cells, liver stem cells, colon stem cells, mesenchymal stem cells, amongst others. In some embodiments, the stem cells are cancer stem cells.

Thus, the methods can identify and/or isolate viable stem cells, including totipotent, pluripatent and mulipotent stem cells from a sample. The sample can be primary tissue sample, such as a sample of white blood cells or a tissue biopsy from an individual, or the sample can be cells that are propagated in vitro. In one embodiment, the sample includes cancer cells.

These methods disclosed herein can also be used to identify cancer stem cells, including both stem cells from hematologic tumors and solid tumors. In specific examples, the cancer is a breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer. In other examples, the cancer stem cells are isolated from leukemias, including acute leukemias (such as l lq23-positive acute leukemia, acute

lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodysplasia. In additional examples, the cancer stem cells are isolated from solid tumors, such as sarcomas and carcinomas. These cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,

mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma,

meduUoblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In several examples, a tumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

The methods disclosed herein utilize synchronized populations of cells. Cell synchonization can be induced by either chemical or physical methods.

Centrifugation and FACS are two physical methods can used to synchronize cells. Chemical methods include nutritional methods such as serum starvation and treatment with thymidine, aminopterin, hydroxyurea and cytosine arabinoside.

Thymidine or aphidicolin halt the cell in the Gi phase. Treatment with colchicine and treatment with nocodazole halt the cell in M phase and treatment with 5- fluorodeoxyuridine halts the cell in S phase. In one embodiment, serum starvation is utilized. Thus, in one example, cancer cells are isolated from a subject, and the cells are synchronized. In another example, cancer cells that have been propagated in vitro are utilized, such as cell lines, and the cells are synchronized.

One of skill in the art can readily determine the length of a cell cycle for a specific cell type. The cell cycle can be divided in two brief periods: interphase- during which the cell grows and mitosis (M) phase, during which the cell divides. The cell cycle includes four distinct phases: Gi S, G₂ phase, and M phase (mitosis, including karyokinesis and cytokines). One of skill in the art can readily determine the length of the cell cycle, such as by determining the amount of time it takes for the number of cells to double in a population. Techniques for determining cell number are routine, and include any counting technique, including the use of a hemocytometer or FACS.

The methods disclosed herein utilize nucleotides, or analogs thereof labeled with a detectable marker that can be detected in a living cell. The nucleotide or analogue thereof can be any nucleotide that can be detected in a viable stem cell, such as a cancer stem cell. Nucleotides include, but are not limited to fluorescent labeled ATP, CTP, TTP, UTP and GTP or an analogue thereof that can be detected in a viable cell, such as a cancer cell. In some specific examples, the nucleotide is not bromodeoxyuridine, iododeoxyuridine, chlorodeoxyuridine or H thymidine. In one specific, non-limiting example, the nucleotide is UTP.

The detectable marker can be any marker that can be detected in a viable cell. In some embodiments, the marker is a fluorescent marker. Suitable fluorescent markers include:

Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, Texas red; Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine

Oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole boron-dipyrromethene

ALEXA FLUOR® (Invitrogen) - a trade grouping and do not share structures DYLIGHT FLUOR® (Thermo Scientific, Pierce)

FLUOPROBES® (Interchim) - Tetrapyrrole derivatives: porphin, phtalocyanine, bilirubin

Cascade yellow, azure B, acridine orange, DAPI, Hoechst 33258, lucifer yellow, piroxicam, quinine and anthraqinone, squarylium, oligophenylenes, etc.

Examples of fluorescent markers also include:

Probe Ex (nm) Em (nm) MW

Hydroxycoumarin 325 386 331

Aminocoumarin 350 445 330

Methoxycoumarin 360 410 317

Cascade Blue (375);401 423 596

Pacific Blue 403 455 406

Pacific Orange 403 551

Lucifer yellow 425 528

NBD 466 539 294

R-Phycoerythrin (PE) 480;565 578 240 k

PE-Cy5 conjugates 480;565;650 670

PE-Cy7 conjugates 480;565;743 767

Red 613 480;565 613

PerCP 490 675

TruRed 490,675 695

FluorX 494 520 587

Fluorescein 495 519 389

BODIPY-FL 503 512

TRITC 547 572 444

X-Rhodamine 570 576 548

Lissamine Rhodamine B 570 590

Texas Red 589 615 625

AUophycocyanin (APC) 650 660 104 k

APC-Cy7 conjugates 650;755 767 ALEXA FLUOR® DYES

Probe Ex (nm) Em (nm) ! MW Quencher

ALEXA FLUOR® 350 343 442 1410

ALEXA FLUOR®405 401 421 1028

ALEXA FLUOR®430 434 540 1702

ALEXA FLUOR®488 499 519 1643 QY 0.92

ALEXA FLUOR®500 503 525 1700

ALEXA FLUOR™ 514 517 542 1714

ALEXA FLUOR®532 530 555 1724 QY 0.61

ALEXA FLUOR®546 561 572 1079 QY 0.79

ALEXA FLUOR®555 553 568 1 1250 QY 0.1

ALEXA FLUOR®568 579 603 1792 QY 0.69

ALEXA FLUOR®594 591 618 1820 QY 0.66

ALEXA FLUOR®610 610 629 1 1285

ALEXA FLUOR®633 632 648 1200

ALEXA FLUOR®647 652 668 1 1300 QY 0.33

ALEXA FLUOR®660 663 691 1 1100

ALEXA FLUOR®680 680 702 1 1150

ALEXA FLUOR®700 696 719 1 1400

ALEXA FLUOR®750 752 776 1300

ALEXA FLUOR®790 782 804 1 1750

Cy Dyes

These markers can be used to label nucleotides and nucleotide analogs, including ATP, UTP, CPT, GTP, TTP and analogs thereof. In one embodiment, the nucleotide or analogue thereof is labeled with a detectable maker and is incorporated into DNA of a viable cell during S phase of the cell cycle. Cells that incorporate the nucleotide or analogue thereof with the fluorescent marker can then be identified and/or isolated using fluorescent activated cell sorting (FACS).

FACS employs a plurality of color channels, low angle and obtuse light- scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells. Any FACS technique may be employed as long as it is not detrimental to the viability of the desired cells (for exemplary methods of FACS see U.S. Patent No. 5, 061,620, herein incorporated by reference). Flow cytometers of use can be a flow cytometer that detects the cells by scattering light and/or fluorescence. Exemplary flow cytometers are BD

FACSARRAY® (Becton Dickinson), BD FACSCANTO® with HTS (Becton Dickinson), BD FACSCALIBUR® with HTS (Becton Dickinson), and Beckman COULTER CYTOMICS® FC500 MPL. However, one of skill in the art can readily identify other flow cytometers of use from other suppliers.

In some embodiments, cell viability is also assayed. There are a variety of suitable cell viability assays which can be used, including, but not limited to, light scattering, viability dye staining, and exclusion dye staining.

In one embodiment, a membrane permeable cell proliferation dye is utilized. In some examples, the dye is fluorescent. These dyes include, but are not limited to carboxyfluorescein diacetate, succinimidyl ester. In some examples, a cell proliferation dye is used that is fluorescent and can be identified using fluorescence activated cell sorting.

A light scattering assay can also be used as a viability assay; these assays are well known in the art. When viewed in the FACS, cells have particular

characteristics as measured by their forward and 90 degree (side) light scatter properties. These scatter properties represent the size, shape and granule content of the cells. These properties account for two parameters to be measured as a readout for the viability. Briefly, the DNA of dying or dead cells generally condenses, which alters the 90° scatter; similarly, membrane blebbing can alter the forward scatter. Alterations in the intensity of light scattering, or the cell-refractive index indicate alterations in viability. Thus, these properties can be used to identify viable cells.

In some embodiments, the method is a dual label method. The method can be used to identify stem cells, including cancer stem cells. Thus, the method for identifying a stem cell includes obtaining a population of synchronized cells, such as cancer cells, and labeling the population of cancer cells undergoing cell cycle division by exposing the cancer cells for only one cell cycle, to a first nucleotide or an analogue thereof labeled with a first detectable marker, wherein the first detectable marker can be detected in a viable cell. The first nucleotide or analogue thereof is incorporated into DNA of the cancer cells during only a first round of DNA replication.

Following DNA replication, the first nucleotide or analogue thereof is removed from the population of cells, and the population of cells, such as cancer cells is allowed to undergo cytokinesis. Cells, such as cancer cells that include the first nucleotide analogue incorporated into the DNA are isolated, and these cells are exposed to a second nucleotide or analogue thereof labeled with a second detectable marker for the duration of only one cell cycle. In these embodiments, the first detectable marker and the second detectable marker are different, but can both be detected in viable cells. However, the first nucleotide or analogue thereof can be different or the same as the second nucleotide or analogue thereof.

The second nucleotide or analogue thereof is incorporated into the DNA of the cells, such as the cancer cells during only a second DNA replications. The second nucleotide or analogue thereof is then removed following only one round of DNA replication, and the cells are allowed to undergo cytokinesis.

At least one viable cell, such as a cancer cell is then identified that includes only the second nucleotide or analogue thereof incorporated into the DNA of the cell, in the absence of the first nucleotide or analogue thereof incorporated into the DNA of the cell. This can be achieved by detecting the presence of the second detectable marker and the absence of the first detectable marker in the viable cell, such as a cancer cell. In this manner, stem cells, such as cancer stem cells are identified and/or isolated.

In some embodiments, the method is a long term label method. These methods include labeling a population of synchronized cells, such as cancer cells, undergoing cell cycle division by exposing the cells to at least one nucleotide or analogue thereof labeled with a detectable marker prior to a first round of DNA replication. The cells are allowed to undergo a single cell division, wherein the at least one nucleotide or analogue thereof is incorporated into chromosomes of the cells, and wherein the detectable marker can be detected in a viable cell. Following only one cell division, any free nucleotide or analogue thereof labeled with the detectable marker (any nucleotide or analogue thereof that is not incorporated into the chromosomes of the cells) is removed. The cells are allowed to divide for at least five additional cell divisions in the absence of the nucleotide or analogue thereof labeled with a detectable marker. In some examples, the cells are allowed to divide for 5, 6, 7, 8, 9 or 10 cell divisions. In other examples, the cells are allowed to divide for 5-8 cell divisions, 5-7 cell divisions or 5-6 cell divisions. Viable cells that include the nucleotide or analogue thereof incorporated into the chromosomes of the cells are identified and/or isolated by detecting the detectable marker, thereby detecting the stem cell, such as a cancer stem cell. Additional Methods for Identifying Cancer Stem Cells

The methods for isolating and/or identifying viable stem cells, such as cancer stem cells, can also include detecting one or more markers of interest. In some embodiments, the methods include detecting the expression of one or more the markers listed below:

Gene

Gene name

symbol

BMP1 Bonemorphogeneticprotein 1

BMP3 Bonemorphogeneticprotein 3

CD8A CD8a molecule

CD8B CD8b molecule

CXCL12 Chemokine (C-X-C motif) ligand 12

(stromal cell-derived factor 1)

CYP2C8 Cytochrome P450, family 2, subfamily C,

polypeptide 8

DTX1 Deltex homolog 1

DTX2 Deltex homolog 2

FGF1 Fibroblast growth factor 1

FGF4 Fibroblast growth factor 4

FGFR1 Fibroblast growth factor receptor 1

FOXN 1 Forkhead box N 1

FSHB Follicle stimulating hormone, beta

polypeptide

KRT15 Keratin 15

LEF1 Lymphoid enhancer-binding factor 1

MME Membrane metallo-endopeptidase

NANOG Nanog homeobox

NEUROG2 Neurogenin 2

SOX17 SRY (sex determining region Y)-box 17

SOX2 SRY (sex determining region Y)-box 2

WNT1 Wingless-type MMTV integration site

family, member 1

WNT6 Wingless-type MMTV integration site family, member 6

WNT8A Wingless-type MMTV integration site

family, member 8A

In some embodiments, the methods include measuring the expression of Sox2 and/or Neurog2. Exemplary amino acid sequences for Sox2 are provided in GENBANK® Accession No. NP_003097.1 (March 27, 2011) and EAW78354.1 (February 4, 201), which are incorporated by reference herein. Exemplary amino acid sequences for Neurog2 are provided in GENBANK® Accession No. NP_076924.1 (March 13, 2011) and AAH36847.1 (July 15, 2006), which are incorporated by reference herein. An increase in Sox2 and/or Neurog2 indicates that the cell is a stem cell, such as a cancer stem cell.

In additional embodiments, the methods include detecting the expression of

Myc, Nanog, CD44 and/or BMP-1, or any combination of these markers. An exemplary amino acid sequence for Myc is provided in GENBANK® Accession No. CAA25015.2 (November 14, 2006), incorporated by reference herein. An exemplary amino acid sequence for Nanog is provided in GENBANK® Accession No.

AAP49529.1 (May 27, 2004, incorporated by reference herein). An exemplary amino acid sequence for CD44 is provided in GENBANK® Accession No.

ACI46596.1 (October 19, 2008, incorporated by reference herein) and in

GENBANK® Accession No. CAA44602.1 (September 9, 2004), incorporated by reference herein. An exemplary amino acid sequence for bone morphogenic protein 1 (BMP-1) is provided in GENBANK® Accession No. AAC41710.1

(January 10, 1995), incorporated by reference herein. An alteration in Myc, Nanog, CD44 and/or BMP-1 indicates that the cell is a stem cell, such as a cancer stem cell.

Generally, any method can be utilized provided it can detect the expression of target gene mRNA or protein as compared to a control. One of skill in the art can readily identify an appropriate control, such as a sample a cell known not to be a stem cells, a sample from a somatic cells line, or a known amount of nucleic acid. Statistically normal levels can be determined for example, from differentiated cells or cells that are not stem cells. An increase or a decrease in a marker of interest can be, for example, about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, change (increase or decrease) in the expression of a particular nucleic acid or protein. Alterations, including increases or decreases in the expression of nucleic acid molecules can be detected using, for instance, in vitro nucleic acid amplification and/or nucleic acid hybridization. Alterations, including increases or decreases in the expression of proteins, can be detected using a variety of assays, including but not limited to immunoassays. The results of such detection methods can be quantified. In one embodiment, nucleic acid based methods are utilized. These methods include serial analysis of gene expression (SAGE techniques), RT-PCR, quantitative PCR, real time PCR, Northern blot, dot blots, micro arrays, amongst others. The methods described herein may be performed, for example, by utilizing at least one specific nucleic acid probe, which may be conveniently used. In one embodiment, this assay is performed in a medical laboratory on a sample of cells. Nucleic acid reagents that are specific to the nucleic acid of interest, can be readily generated given the sequences of these genes for use as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR in situ hybridization:

protocols and applications, Raven Press, NY).

A differential display procedure can be utilized based on Northern analysis and/or RT-PCR. In one embodiment, the methods disclosed herein include the use of an ordered array of nucleic acids representing thousands of genes on a solid support. mRNA from the cells of interest are used to create a labeled, first strand cDNA probe that is then hybridized to the microarray. In one embodiment, two mRNA samples are directly compared to the same microarray by incorporating different labels into the cDNA probes derived from the samples. The extent of hybridization of the probes to each nucleic acid sequence on the microarray is then quantitated and the ratio of the pixel intensities for each label is used as a measure of the relative mRNA expression in the two samples.

The array can be a high density array, such that the array includes greater than about 100, greater than about 1000, greater than about 16,000 and most greater than about 65,000 or 250,000 or even greater than about 1,000,000 different oligonucleotide probes. The oligonucleotide probes generally range from about 5 to about 50 nucleotides, such as about 10 to about 40 nucleotides in length or from about 15 to about 40 nucleotides in length.

The location and sequence of each different oligonucleotide probe sequence in the array is known. Moreover, in a high density array, the large number of different probes occupies a relatively small area so that there is a probe density of greater than about 60 different oligonucleotide probes per cm , such as greater than about 100, greater than about 600, greater than about 1000, greater than about 5,000, greater than about 10,000, greater than about 40,000, greater than about 100,000, or greater than about 400,000 different oligonucleotide probes per cm . The small surface area of the array (such as less than about 10 cm , less than about 5 cm , less than about 2 cm ) permits extremely uniform hybridization conditions (temperature regulation, salt content, etc.) while the extremely large number of probes allows parallel processing of hybridizations.

Generally, the methods of monitoring gene expression using array technology involve (1) providing a pool of target nucleic acids comprising RNA transcript(s) of one or more target gene(s), or nucleic acids derived from the RNA transcript(s); (2) hybridizing the nucleic acid sample to an array of probes (including control probes), that can be a high density array; and (3) detecting the hybridized nucleic acids and calculating a relative expression (transcription) level.

In order to measure the transcription level of a gene or genes, it is desirable to provide a nucleic acid sample comprising mRNA transcript(s) of the gene or genes, or nucleic acids derived from the mRNA transcript(s). As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template, such as a cDNA ("first strand" transcribed from the mRNA). Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript. Detection of such products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, suitable samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, and the like.

Generally, the transcription level (and thereby expression) of one or more genes in a sample is quantified, so that the nucleic acid sample is one in which the concentration of the mRNA transcript(s) of the gene or genes, or the concentration of the nucleic acids derived from the mRNA transcript(s), is proportional to the transcription level (and therefore expression level) of that gene. The hybridization signal intensity should also be proportional to the amount of hybridized nucleic acid. Generally, the proportionality is relatively strict (for example, a doubling in transcription rate results in approximately a doubling in mRNA transcript in the sample nucleic acid pool and a doubling in hybridization signal), one of skill will appreciate that the proportionality can be more relaxed and even non-linear. Thus, for example, an assay where a 5 fold difference in concentration of the target mRNA results in a 3 to 6 fold difference in hybridization intensity can be sufficient. Where more precise quantification is required, controls can be run to correct for variations introduced in sample preparation and hybridization as described herein. In addition, serial dilutions of "standard" target mRNAs can be used to prepare calibration curves according to methods well known to those of skill in the art. Of course, where simple detection of the presence or absence of a transcript is desired, controls or calibrations may not be required.

In one embodiment, a nucleic acid sample is utilized, such as the total mRNA isolated from a biological sample. The biological sample can be from any cells sample of interest, such as a cells isolated from a subject with cancer.

Nucleic acids (such as mRNA) can be isolated from the sample according to any of a number of methods well known to those of skill in the art. Methods of isolating total mRNA are well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology:

Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid

Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993) and Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993). In one example, the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method, and polyA+ mPvNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley- Interscience, N.Y. (1987)). In another example, oligo-dT magnetic beads may be used to purify mRNA (Dynal Biotech Inc., Brown Deer, WI).

The nucleic acid sample can be amplified prior to hybridization. If a quantitative result is desired, a method is utilized that maintains or controls for the relative frequencies of the amplified nucleic acids. Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that can be used to calibrate the PCR reaction. The array can then include probes specific to the internal standard for quantification of the amplified nucleic acid.

Suitable amplification methods include, but are not limited to, polymerase chain reaction (PCR) (see Innis et al., PCR Protocols, A guide to Methods and Application, Academic Press, Inc. San Diego, 1990), ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560, 1989; Landegren et al., Science 2 \:\Q11 , 1988; and Barringer, et al., Gene 89: 117, 1990), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. U.S.A. 86: 1173, 1989), and self- sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. U.S.A. 87: 1874, 1990). In one embodiment, the sample mRNA is reverse transcribed with a reverse transcriptase and a primer consisting of oligo dT and a sequence encoding the phage T7 promoter to provide single stranded DNA template (termed "first strand"). The second DNA strand is polymerized using a DNA polymerase. After synthesis of double-stranded cDNA, T7 RNA polymerase is added and RNA is transcribed from the cDNA template. Successive rounds of transcription from each single cDNA template results in amplified RNA.

Methods of in vitro polymerization are well known to those of skill in the art (see, for example, Sambrook, supra; Van Gelder et al., Proc. Natl. Acad. Sci. U.S.A. 87: 1663-1667, 1990). The direct transcription method provides an antisense

(aRNA) pool. Where antisense RNA is used as the target nucleic acid, the oligonucleotide probes provided in the array are chosen to be complementary to subsequences of the antisense nucleic acids. Conversely, where the target nucleic acid pool is a pool of sense nucleic acids, the oligonucleotide probes are selected to be complementary to subsequences of the sense nucleic acids. Finally, where the nucleic acid pool is double stranded, the probes may be of either sense as the target nucleic acids include both sense and antisense strands.

The protocols include methods of generating pools of either sense or antisense nucleic acids. Indeed, one approach can be used to generate either sense or antisense nucleic acids as desired. For example, the cDNA can be directionally cloned into a vector (for example Stratagene's pBluscript II KS (+) phagemid) such that it is flanked by the T3 and T7 promoters. In vitro transcription with the T3 polymerase will produce RNA of one sense (the sense depending on the orientation of the insert), while in vitro transcription with the T7 polymerase will produce RNA having the opposite sense. Other suitable cloning systems include phage lambda vectors designed for Cre-loxP plasmid subcloning (see, for example, Palazzolo et al., Gene 88:25-36, 1990).

In one embodiment, the nucleic acid from the sample can be immobilized, for example, to a solid support such as a membrane, including nylon membranes or nitrocellulose, or a plastic surface such as that on a microtitre plate or polystyrene beads. Labeled nucleic acid probes that specifically bind the gene(s) of interest are bound to the immobilized sample. The labels include radiolabels, enzymatic labels, and binding reagents (such as avidin or biotin). Detection of the annealed, labeled nucleic acid reagents is accomplished using standard techniques well known to those in the art.

In one embodiment, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels can be incorporated by any of a number of methods. In one example, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In one embodiment, transcription amplification, as described above, using a labeled nucleotide (such as fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to the original nucleic acid sample (such as mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example, nick translation or end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).

Detectable labels suitable for use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (for example DYNABEADS™), fluorescent dyes (for example, fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example, 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (for example, horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent No. 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,939,350; U.S. Patent No. 3,996,345; U.S. Patent No. 4,277,437; U.S. Patent No. 4,275,149; and U.S. Patent No. 4,366,241.

Means of detecting such labels are also well known. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

The label may be added to the target (sample) nucleic acid(s) prior to, or after, the hybridization. So-called "direct labels" are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, so-called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected (see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., 1993).

Nucleic acid hybridization simply involves providing a denatured probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus, specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer

mismatches.

One of skill in the art will appreciate that hybridization conditions can be designed to provide different degrees of stringency. In a one embodiment, hybridization is performed at low stringency in this case in 6xSSPE-T at 37°C (0.005% Triton X-100) to ensure hybridization and then subsequent washes are performed at higher stringency (e.g., lxSSPE-T at 37°C) to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25xSSPE-T at 37°C to 50° C) until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level control, normalization control, mismatch controls, etc.).

In general, there is a tradeoff between hybridization specificity (stringency) and signal intensity. Thus, in one embodiment, the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity. Thus, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest. These steps have been standardized for commercially available array systems.

Methods for evaluating the hybridization results vary with the nature of the specific probe nucleic acids used as well as the controls provided. In one

embodiment, simple quantification of the fluorescence intensity for each probe is determined. This is accomplished simply by measuring probe signal strength at each location (representing a different probe) on the array (for example, where the label is a fluorescent label, detection of the amount of florescence (intensity) produced by a fixed excitation illumination at each location on the array). Comparison of the absolute intensities of an array hybridized to nucleic acids from a "test" sample (such as from cells isolated using the methods disclosed herein) with intensities produced by a "control" sample (such as from cells known not to be cancer stem cells) provides a measure of the relative expression of the nucleic acids that hybridize to each of the probes.

In several embodiments, an amount of one or more polypeptides are measured. Both monoclonal and polyclonal antibodies, and fragments thereof, can also be utilized to detect and quantify the proteins. This can be accomplished, for example, by immunohistochemistry, immunoassay (such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), Western blotting, flow cytometric or fluorimetric detection. The antibodies (or fragments thereof) can be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection. In situ detection includes contacting a sample comprising cells with labeled antibody, and detecting binding of the antibody to cells in the sample. A wide variety of methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Generally, immunoassays typically include incubating a biological sample including the cells of interest, in the presence of antibody, and detecting the bound antibody by any of a number of techniques well known in the art. The biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of

immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the antibody that binds the protein of interest. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. If the antibody is directly labeled, the amount of bound label on solid support can then be detected by conventional means. If the antibody is unlabeled, a labeled second antibody, which detects that antibody that specifically binds the protein of interest (see above) and/or the antibody can be used.

By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present disclosure. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet or test strip. In one embodiment, proteins are isolated from a biological sample. In one embodiment, an enzyme linked immunosorbent assay (ELISA) is utilized to detect the protein (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA),"

Diagnostic Horizons 2: 1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, Md.; Voller et al., J. Clin. Pathol. 31:507-520, 1978; Butler, Meth. Enzymol. 73:482-523, 1981; Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). In this method, an enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by

spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

However, detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fingerprint gene wild-type or mutant peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). In another example, a sensitive and specific tandem

immunoradiometric assay may be used (see Shen and Tai, J. Biol. Chem., 261:25, 11585-11591, 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fhiorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Any method known to those of skill in the art can be used to detect and quantify proteins. Thus, in additional embodiments, a spectrometric method is utilized. Spectrometric methods include mass spectrometry, nuclear magnetic resonance spectrometry, and combinations thereof. In one example, mass spectrometry is used to detect the presence of a protein of interest in a biological sample, (see for example, Stemmann, et al., Cell Dec 14;107(6):715-26, 2001;

Zhukov et al., "From Isolation to Identification: Using Surface Plasmon Resonance- Mass Spectrometry in Proteomics, PharmaGenomics, March/ April 2002, available on the PharmaGenomics website on the internet). Methods for Identifying Agents of Use in Treating Cancer

The methods disclosed herein can be used to identify an agent that can be used to treat a subject with cancer. Specifically, cancer stem cells from the subject are isolated using the methods disclosed herein. The cancer stem cells can then be contacted with an agent of interest, such as a chemotherapeutic agent, in order to determine if it will affect the cancer stem cells. For example, the death of cancer cells upon treatment with an agent of interest can be assessed.

Examples of chemotherapeutic agents of interest are alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine,

cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum Π also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-fluorouracil(FU), Fludarabine, Hydrea, Idarubicin, Ifosfamide,

Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. The agent can also be a polypeptide or a nucleic acid molecule, such as an siRNA.

Appropriate agents of interest can be contained in libraries, for example, synthetic or natural compounds in a combinatorial library. Numerous libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, such as antisense oligonucleotides and oligopeptides, also are known. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different compounds.

Libraries (such as combinatorial chemical libraries) useful in the disclosed methods include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37:487-493, 1991; Houghton et al., Nature, 354:84-88, 1991; PCT Publication No. WO 91/19735), encoded peptides

(e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Natl. Acad. Sci. USA, 90:6909-6913, 1993), vinylogous polypeptides (Hagihara et al, J. Am. Chem. Soc, 114:6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et ah, J. Am. Chem. Soc, 114:9217-9218, 1992), analogous organic syntheses of small compound libraries (Chen et ah, J. Am. Chem. Soc, 116:2661, 1994), oligocarbamates (Cho et ah, Science, 261: 1303, 1003), and/or peptidyl phosphonates (Campbell et ah, J. Org. Chem., 59:658, 1994), nucleic acid libraries (see Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Springs

Harbor Press, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nat. Biotechnol, 14:309-314, 1996; PCT App.

No. PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et ah, Science, 274: 1520-1522, 1996; U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33, 1993; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones and methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514) and the like. Libraries may include a varying number of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.

In one convenient embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds and testing them to determine their effect on cancer stem cells. Such combinatorial libraries are then screened in one or more assays as described herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity (such as a toxic effect on cancer stem cells, or decreasing the ability of the cancer stem cells to divide). In one example an agent of use is identified that causes the death of stem cells, such as cancer stem cells. In another example an agent of use is identified that decreases the proliferation rate of stem cells, such as cancer stem cells.

The compounds identified using the methods disclosed herein can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. In some instances, pools of candidate agents may be identify and further screened to determine which individual or subpools of agents in the collective have a desired activity. Methods for Isolating Bone Marrow without Cancer Stem Cell

Methods are also provided herein for treating a subject with a lymphoma or a leukemia. These methods include, isolating bone marrow from the subject, and isolating cancer stem cells from the bone marrow using the methods disclosed herein. However, the cancer stem cells are not retained. Bone marrow cells that are not labeled using the methods disclosed herein are collected. Thus, the use of the methods results in the production of bone marrow depleted of cancer stem cells. The bone marrow depleted of cancer stem cells then can be reintroduced into the subject, who is treated to eliminate any remaining bone morrow. In this manner, the subject is treated. The subject can be any subject, such as a human or veterinary subject.

In some examples, the subject has a leukemia, such as an acute leukemia (such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia). In other examples, the subject has a chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodysplasia.

In some embodiments, the dual label method is utilized. Thus, the method includes obtaining a population of synchronized bone marrow cells and labeling the population of cancer cells undergoing cell cycle division by exposing the cells to a first nucleotide or an analogue thereof labeled with a first detectable marker for one cell cycle, wherein the first detectable marker can be detected in a viable cell. The first nucleotide or analogue thereof is incorporated into DNA of the cells during a first round of DNA replication.

Following DNA replication, the first nucleotide or analogue thereof is removed from the population of cells the first nucleotide or analogue thereof labeled with the first detectable marker and the population of cells is allowed to undergo cytokinesis. Cells that include the first nucleotide analogue incorporated into the DNA are isolated, and the cells are exposed to a second nucleotide or analogue thereof labeled with a second detectable marker for the duration of one cell cycle. In these embodiments, the first detectable marker and the second detectable marker are different, but can both be detected in viable cells. However, the first nucleotide or analogue thereof can be different or the same as the second nucleotide or analogue thereof. The second nucleotide or analogue thereof is incorporated into the DNA of the cells, such as the cells during a second round of DNA replication. The second nucleotide or analogue thereof is removed following this round of DNA replication, and the cells are allowed to undergo cytokinesis. Cancer cells are identified that includes only the second nucleotide or analogue thereof incorporated into the DNA of the cell, in the absence of the first nucleotide or analogue thereof incorporated into the DNA of the cell. These cells are removed from the population of cells. Any remaining cells, such as cells that include the first nucleotide or analogue thereof and the second nucleotide or analogue thereof are reintroduced into the subject. This can be achieved by detecting the presence of the second detectable marker and the absence of the first detectable marker in the viable cell, such as a cancer cell. In this manner, bone marrow cells that do not include cancer stem cells are identified and/or isolated. These cells can be transferred back into the subject, such as following depletion of the endogenous bone marrow, such as by total body irradiation.

In some embodiments, the long term label method is utilized. These methods include labeling a population of synchronized cells, such as cancer cells, undergoing cell cycle division by exposing the cells to at least one a nucleotide or analogue thereof labeled with a detectable marker prior to a first round of DNA replication. The cells are allowed to undergo a single cell division, wherein the at least one nucleotide or analogue thereof is incorporated into chromosomes of the cells, and wherein the detectable marker can be detected in a viable cell. Following one cell division, the at least one nucleotide or analogue thereof labeled with the detectable marker that is not incorporated into the chromosomes of the cells is removed. The cells are allowed to divide for at least five additional cell divisions in the absence of any nucleotide or analogue thereof labeled with a detectable marker. Viable cells that include the nucleotide or analogue thereof incorporated into the chromosomes of the cells by detecting the detectable marker, are discarded, and any bone marrow cells that do not include the nucleotide or analogue thereof are isolated. In this manner, bone marrow cells that do not include cancer stem cells are identified and/or isolated. These cells can be transferred back into the subject, such as following bone marrow depletion, such as is achieved by total body irradiation.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Stem cells can undergo symmetric division or asymmetric cell division (ACD). There are several forms of ACD. ACD with non-random chromosomal cosegregation (NRCC) is proposed to segregate the older template DNA strands into daughter stem cells, and newly synthesized DNA into daughter cells destined for differentiation. Long term label retaining cells (LRC) can be the result of either slow cycling and/or ACD-NRCC. Using single and dual-color chromosomal DNA labeling in live cells, methodology is described for the isolation of live LRC and cells undergoing ACD-NRCC, such as by FACS. The validation of the methods was done by confocal microscopy. These methods can be used to identify any stem cell, including, but not limited to, a cancer stem cell.

The hallmark characteristic of CSC is both the ability to make more progeny (self-renewal), as well as produce differentiated cells (Clevers, H., Nature Genet, 37, 1027-1028, 2005). Asymmetric division (ASYD) via chromosomal cosegregation provides a strategy in which a CSC can achieve both with a single division. More than thirty years ago, the immortal- strand-hypothesis (ISH) was introduced to provide a method for self-renewal (Cairns, J., Nature, 255(5505): 197-200, 1975). The ISH proposes that each chromosome in a stem cell (SC) contains one DNA strand that is conserved throughout multiple asymmetric divisions (Figure la). It is this conserved template, the "immortal strand," which undergoes the fewest divisions. As a result, the SC is able to avoid the accumulation of mutations from DNA replication errors by preferentially segregating the replication errors into the daughter-cell fated to differentiate and ultimately be eliminated (Cairns, supra, 1975). This principle has been demonstrated in various long-term label retaining studies which demonstrated the cosegregation of H -thymidine-labled DNA strands following multiple divisions (Lark, K.G., et al., Science, 154(753): 1202-5, 1966); Potten, C.S., et al. Cell, 1978. 15(3): 899-906, 2002). Additional double-label studies in murine models both in vivo and in vitro have also been successful (see Potten et al, supra, 2002; Smith, G.H., Development, 2005. 132(4): 681-687; 2005); Karpowicz, P., et al., J. Cell Biol, 170(5): 721-732, 2005); Shinin, V., et al., Nat Cell Biol, 8(7): 677-87, 2006); Merok, J.R., et al., Cancer Res, 62(23): 6791-6795 (2002).; Rambhatla, L., et al., Cancer Res 65(8): 3155-3161 , 2005). However, identification of putative stem cells, such as CSC based on their capacity to asymmetrically divide via chromosomal cosegregation has yet to be demonstrated in humans or animals. The methods disclosed below provide allow the detection of stem cells, such as cancer stem cell.

It is demonstrated herein that ASYD occurs in human cancer cells using two novel in vitro methodologies. These methods include the use of a double-labeling technique (see, for example, Figure lb) and a long-term, label-retaining technique (see, for example, Figure lc).

The CSC model suggests that current non-surgical treatments of solid organ cancers (SOCs) are destined to fail given that they target rapidly dividing cells and, consequently, have little impact on the CSC within the tumor (Hart and El-Deiry, / Clin Oncol, 26: 2901-2910, 2008). The ability to isolate solid-organ-cancer-stem cells (SOCSCs), allow the identification of appropriate a therapeutic treatment options could be developed for SOCs. The present methods have sufficient sensitivity and specificity to identify a homogenous population of stem cells, such CSC.

Example 1

Materials and Methods

Cells and Media: Human Hepatocellular carcinoma (HCC) PLC/PRF/5 ATCC, CRL-8024; 45% DMEM, 45% Ham's F-12 supplemented with 10% FCS (Invitrogen Corp, Grand Isle, NY) without antibiotics.

Growth curves and doubling times: In order to maximize the quantitative and qualitative detection rate of asymmetrically dividing cells, it was required that the cell cycle be synchronized according to the cell lines' doubling time with and without microporation and dUTP-labeling. In 15 to 18 wells of 6-well plates, 5 x 10e4 cells were plated per well and allowed to attach for 24 hours. On each time point, 8 hours apart, the numbers of live cells were determined in three wells.

Acquired numbers were averaged and converted into percentage relative to the average acquired at the first time point. These percentages were plotted using Excel spreadsheet.

A best fit exponential trend line with y-axis interception at 100% was generated using regression analysis of this trend line the doubling time was computed (Figure 8). Correlation value R2>0.9 was considered adequate for computations of doubling times. Doubling times were calculated for all cell conditions tested.

Cell proliferation: Extensive experiments for optimization of cell

proliferation were performed using the Cell Trace CFSE (carboxyfluorescein diacetate, succinimidyl ester) Cell Proliferation Kit (Invitrogen C34554) according to manufacturing recommendations (Figure 9). The concentration was optimized to account for cell death due to toxicity as well as adequate emission-distinction via FACS analysis that did not have significant overlap with the Alexa555-dUTP emission spectrum. Cells were plated in antibiotic-free serum (AFS) media for 12 hours prior to staining with CFSE. Cells were washed with DPBS and incubated at 37°C for 15 minutes in 0.5μΜ CFSE. Cells were washed with DBPS and then incubated in AFS media. Cells were then analyzed via FACS analysis after various timepoints to establish one and two cell-divisions, 50% and 25% CFSE staining respectively. See below for further details.

Cell cycle synchronization and analysis: Optimization experiments were performed to determine the ideal technique to ensure optimal nucleotide

incorporation in which the cells are predominantly in the Gl/S phases. Human cancer cells (PLC/PFR/5) were plated Ixl0e5 cells in 24-well plates using AFS media. Cells were plated simultaneously. Control cells were evaluated at 20, 22, 24, 26, 28, 32, 34 and 38 hours after initial plating. Experimental groups consisted of serum-starvation for 24-48 hrs and/or aphidicolin (2μg/ml, Sigma A0781) treatment to induce an Sl-phase arrest. Twelve-hours after plating, cells were serum-starved (SS) alone (12, 24, 36, 48, 60 and 72hrs post-initial plating), SS then returned to AFS media, SS (24 and 48hrs) then treated with aphidicolin and finally treated with aphidicolin 20 hrs after initial plating. At the indicated timepoints, cells were harvested and with 1% PFA prior to staining with a 1: 1 ratio of Vindelov's PI and plain PI (200μL· per sample). FACS analysis was performed in order to assess the G1/S/G2 phases within each condition. All conditions were performed in triplicate (Figure 10)

Microporation: Extensive experiments for optimization of fluorophore labeled-dUTP incorporation via microporation using the MicroPorator MP- 100 (BTX-Harvard Apparatus). Manufacturing guidelines were followed using both the l0μL· and ΙΟΟμί tip kits. Various instrument settings, cell concentrations and labeled-dUTP concentrations were optimized for the HCC cell line. The selected dUTPs used were unlabeled dUTP (Amersham 28406542), Cy5-dUTP (Amersham PA55032) and Alexa Fluor 555-dUTP (Invitrogen A32762). Cells were plated in AFS media for 22 hours prior to harvesting for microporation. Prior to

microporation, cells were trypsinized, washed in DPBS and resuspended in R buffer at a concentration of 1.5e5 cells per l0μL· for the l0μL· tips and 5e6 cells per ΙΟΟμί for the ΙΟΟμί tips. All dUTPs were used at a final concentration of lOOmM. Cells were loaded into 10 μΐ or 100 μΐ tips and placed into the microporation chamber containing 3 ml of microporation buffer. The cells were microporated at 1400 V for 20 millisecond and 2 pulsations, then immediately plated in AFS media for culture at 37°C.

Chromosomal double-labeling technique: Extensive experiments for the optimization of nucleotide incorporation efficiency via microporation and CFSE staining for proliferation were performed.

Cells were plated in AFS media for 12 hours. Cells were washed with DPBS and incubated at 37°C for 15 minutes in 0.5μΜ CFSE. Cells were washed with DBPS and then incubated in AFS media for 10 hours. The cells were harvested with trypsin/EDTA and prepared for microporation using l0μL· tips. Per each l0μL· tip, 1.5e5 cells were microporated to label with either unlabeled dUTP or Cy5-dUTP at concentrations of ΙΟΟμΜ and ΙΟΟμΜ, respectively. Four hours post-microporation, cells were washed with DPBS and fresh AFS media was added. Thirteen hours following microporation, one complete cell cycle, the initial FACS (BDFacsAriall, BD Biosciences) sort was performed. One hour prior to FACS sort, control cells were incubated in 0.5μΜ CFSE to provide the 100% CFSE controls for the FACS sorting. Cell viability was determined based on light scatter using FSC-A vs. SSC-A and then SSC-A vs. FSC-W. Cells were then gated based on 50% CFSE (amount remaining after one cell division) and 55% Cy5+ cells. This population of cells was sorted to 98% purity. The ½ CFSE+/Cy5+ sorted cells were immediately placed in culture using AFS media at 37°C. Approximately 4 hours following plating, the cells were washed with DPBS and fresh AFS media was added. Twenty-two hours following the ½ CFSE+/Cy5+ sort, the cells were harvested for the second round of dUTP- labeling with ALEXA FLUOR® 555-dUTP. Once again, the cells were trypsinized and prepared for microporation using l0μL· tips. Per each l0μL· tip, 1.5e5 cells were microporated to label with either unlabeled dUTP or ALEXA FLUOR® 555-dUTP at concentrations of ΙΟΟμΜ and ΙΟΟμΜ, respectively. Four hours post-microporation, cells were washed with DPBS and fresh AFS media was added. Eighteen hours following microporation, the final FACS (BDFacsAriall, BD Biosciences) sort was performed after completion of the second round of replication. The 100% CFSE control cells prepared for the initial sort were used as the 50% CFSE control group for the final sort. Once again, cell viability was determined based on light scatter using FSC-A vs. SSC-A and then SSC-A vs. FSC-W. Cells were then gated based on 25% CFSE (amount remaining after two cell divisions) and 65% Cy5+ cells and 85% Alexa555+ cells. There were two groups isolated: ¼ CFSE+/Alexa555+ cells and ¼ CFSE+/Alexa555+/Cy5+ cells. The ¼

CFSE+/Alexa555+ cells sorted subpopulation had an Alexa555+ purity of 98%. The double-labeled sorted subpopulation, ¼ CFSE+/Alexa555+/Cy5+, had an

Alexa555+ purity of 97% and a Cy5+ purity of 95%. Following the final FACS sort, the cells were placed in placed in culture using AFS media at 37°C. The cells were then prepared for microscopy. See below for details. Chromosomal long-term label-retaining technique: Human liver cancer cells (PLC/PRF/5) were labeled with Cy5-dUTP by microporation. Microporation was completed according to the manufacturer's instruction. Twenty-two hours after plating, cells cultured in AFS media were trypsinized, harvested and washed with DPBS once. Prior to microporation, 5e6 cells were resuspended in 108 μΐ of R

Buffer mixed with 12 μΐ of Cy5-dUTP (100 μΜ). Cells were loaded into a 100 μΐ tip and placed into the microporation chamber containing 3 ml of microporation buffer, and microporated at 1400 V for 20 millisecond twice, then transferred immediately to AFS media for culture at 37°C. After one complete cell cycle, Cy5+ cells were sorted by FACS (BDFacsAriall, BD Biosciences). Cell viability was determined based on light scatter using FSC-A vs. SSC-A and then SSC-A vs. FSC-W. A population of Cy5+ cells comprising approximately 50-55% of the beginning population was sorted to greater than 99% purity. Cells were propagated in culture for six cell cycles. Following completion of the 6^th cell cycle, the cells were then sorted again for Cy5+ (long-term label retaining cells) and Cy5- control cells. The sorted cells were fixed for confocal microscopy. Sorted cells were either fixed for confocal microscopy (see below for details) or injected into nude/SCID mice (SHO, Jackson Lab) subcutaneously in 25% of Matrigel (10 cells/injection in 100 μΐ total volume). There were ten mice in each group. Each mouse was injected with a transponder (Bio Medic Data Systems, Inc) to track the mouse ID. Tumor growth was monitored weekly at two dimensions with a ruler and in a blinded manner for 16 weeks. Mice with tumors were examined and photographed.

Fluorescence confocal microscopy: Following microporation and FACS sorting, cells were plated at various stages in collagen IV-coated 8-well chamber slides (Ibidi 80822). Following a four-hour incubation period, the cells were washed with DPBS and fixed with 4% PFA for 15 minutes at room temperature. The cells were washed with DPBS and incubated at 37°C for one hour. Several drops of Vectashield/DAPI stain (Vector Laboratories H-1200) were placed in each chamber and then stored at 4°C prior to confocal images acquired. Confocal images were sequentially acquired with Zeiss AIM software on a Zeiss LSM 510 Confocal system (Carl Zeiss Inc.) with a Zeiss Axiovert 100M inverted microscope and 50 mW argon UV laser tuned to 364 nm, a 25 mW Argon visible laser tuned to 488 nm, a 1 mW HeNe laser tuned to 543 nm, and a 5 mW HeNe laser tuned to 633 nm. A 63x Plan- Apochromat 1.4 NA oil immersion objective was used at digital zoom settings of 1 or 2. Emission signals after sequential excitation of DAPI (blue), FITC (pseudo- colored white), ALEXA FLUOR® 555 (red) and Alexa Fluor 568 (pseudo-colored green) by 364 nm, 488 nm, 543 nm or 633 nm laser lines were collected with a BP 385-470 filter, BP 505-550 filter, LP 560 filter or LP 650 filter, respectively, using individual photomultipliers. Z-stacks consisted of 30 to 50 slices at 0.38μιη intervals and these stacks were used with Bitplane's (Zurich, Switzerland) Imaris software (v6.0) for surface rendering. In some cases, a cutting plane was used to expose internal surfaces or outer surfaces were made transparent. Three- dimensional video imaging can be seen for a symmetrically dividing

Alexa555+/Cy5+ cell, a symmetrically Alexa555+ cell, and an asymmetrically dividing cell.

Example 2

Chromosomal Double-Labeling Technique:

In order to guarantee that two cell divisions have occurred, classification of the doubling-time of the cell and a marker for proliferation is paramount. Following various conditions, the doubling-time of the liver cancer cell line (PLC/PRF/5) was approximately 35 hours (Figure 9). Cell proliferation was monitored using a cytoplasmic stain, CFSE (carboxyfluorescein diacetate, succinimidyl ester, 0.5μΜ) prior to the first round of DNA replication. Following two cell divisions, cytoplasmic dye-protein complexes within the CFSE-labeled cells are retained by the cells throughout mitosis and then passed onto daughter cells after each division at a fixed percentage (Figure 10).

To identify asymmetric division with chromosomal co segregation, cells were labeled sequentially with two different fluorophore-labeled nucleotides prior to each round of replication: Cy5-dUTP (pseudo-colored green) then ALEXA FLUOR® 555-dUTP (red) (Figure 2). The optimal time for incorporation of nucleotides is when cells are predominately in the Gl-S phase which was found to be approximately 22 hours following initial plating (Figure 11). Twelve hours after plating, human liver cancer cells (PLC/PRF/5) were stained with 0.5μΜ CFSE (100% CFSE). The cells were given ample time to recover prior to transfection with the initial fluorophore-labeled dUTP, Cy5-dUTP (ΙΟΟμΜ), via microporation. Following one complete cell cycle (~35hrs), the initial FACS sort was performed.

The aim of the initial sort was to isolate cells that have completed one cell division (containing one half of the CFSE, 50% CFSE) and were labeled with Cy5- dUTP (Cy5+). Control cells were stained with 0.5μΜ CFSE to provide the 100% CFSE controls for the FACS sorting. A cell viability marker was not feasible due to the spectrum overlap of CFSE and ALEXA FLUOR® 555-dUTP with various makers such as PI or 7-AAD. Therefore, cell viability was selected via light scatter properties yielding greater than 90% viability following the initial CFSE/Cy5+ FACS sort. Cells were then gated based on 50% CFSE-staining which accounted for approximately 94% of the viable cells (Figures 3a). Following Cy5-labeling, 95% of the cells were Cy5+ when compared to the unlabeled-dUTP controls; however, to ensure the best post-sort purity, the gating was set using only those cells which were 55% Cy5+ (Figures 3b). As a result the post-50% CFSE+/Cy5+ cells had a 98% Cy5+ purity (Figure 3c).

The aim of the final sort was to isolate cells that completed two cell divisions (containing one quarter of the CFSE, 25% CFSE) and have divided asymmetrically (labeled with ALEXA FLUOR® 555-dUTP only (Alexa555+)) or symmetrically (labeled with both Cy5-dUTP (Cy5+) and ALEXA FLUOR® 555-dUTP only (Alexa555+)). Proliferation controls from the initial sort (100% CFSE-staining) were now used at the new controls for the final sort into order to set the gating for 50% CFSE (one cell division) and 25% CFSE (two cell divisions). Once again, viability was assessed using light scatter and yielded a presort population greater than 90% viable following the second round of fluorophore-labeling with ALEXA FLUOR® 555-dUTP. Cells were then gated based on 25% CFSE-staining which accounted for 95% of the viable cells (Figure 3d). Following the Alexa555- labeling, 85% of the cells were Alexa555+ (Figure 3e) and 65% were Cy5+ (Figure 3f) when compared to the unlabeled-dUTP controls. Two select populations were then sorted to yield asymmetrically divided cells (25% CFSE/Alexa555+) and symmetrically divided cells (25% CFSE/Alexa555+/Cy5+). The post-sort analysis demonstrated 98% Alexa555+ purity in the 25% CFSE/Alexa555+ population and 95% Cy5+ purity and 97% Alexa555+ purity in the 25% CFSE/Alexa555+/Cy5+ population.

Further investigation of the double-labeling technique was achieved with confocal microscopy using three-dimensional surface rendering to capture SYD and ASYD. The post-sort 50% CFSE/Cy5+ population was seen with cytoplasmic CFSE-staining (pseudo-colored white), nuclear DAPI-staining (blue) and Cy5- labeled nucleotides (pseudo-colored green) (Figure 4a). Following the second round of fluorophore-labeling with Alexa555-dUTP and a second cell division, two subpopulations were sorted to distinguish symmetrically divided cells,

CFSE+(white)/Cy5+(green)/Alexa555+(red) cells (Figure 4b) and asymmetrically divided cells, CFSE+(white)/Alexa555+(red) cells (Figure 4c). Although the assay was designed to detect single cells, there were couplets that were captured following cell fixation. As a result, cytokinesis was observed in three different couplets: SYD in which two nuclei labeled with both fluorophores (green and red, Figure 4e); SYD in which two nuclei labeled with only a single labeled-nucleotide (red, Figure 4f); and ASYD in which one nucleus containing the "immortal template-DNA strand" (red) and the other nucleus containing both labeled-nucleotides (green and red) (Figure 4g). Three-dimensional reconstruction movies from panels 4e-4g demonstrate no intervening cell membrane between the two nuclei indicating localization of both nuclei within the same cytoplasmic space during cytokinesis (SOM-Movies 1-3, d-f respectively).

Example 3

Chromosomal Long-Term, Label-Retaining Technique

A second methodology was developed that utilized the incorporation of a single fluorophore-labeled nucleotide in order to assess long-term label retention and confirm existence of the "immortal-template DNA strand." If cells are initially labeled with Cy5-dUTP prior to the first cell cycle and then isolated, a subpopulation of 100% Cy5+ cells is created that can be isolated, such as by FACS.

Thus, only one label was used to label one strand of replicating chromosomal DNA during one cell cycle. If labeling is uniform and detection is very sensitive, and assuming that after each cell division the Cy5+ labeled DNA is diluted by approximately 50% (although, potentially, it might be more than 50% but unlikely to follow a complete Gaussian distribution), it was calculated that potentially after 6 and 8 cell-cycles approximately 1.56% and 0.39% of the cells will be Cy5+ (Figure 5).

Human liver cancer cells (PLC/PRF/5) were microporated to incorporate the control, unlabeled dUTP (ΙΟΟμΜ) or the labeled- nucleotide, Cy5-dUTP (ΙΟΟμΜ). Following one complete cell cycle (35 hours), the initial FACS sort was performed. Cell viability was determined by light scatter and yielded greater than 95% viability, similar to the initial sort of the double-labeling technique. In order to ensure the purest subpopulation of Cy5+ cells, a higher threshold, 50% positivity, was used for gating the Cy5+ cells (Figure 5a). This subpopulation of cells was sorted to 99% Cy5+ purity (Figure 5b). This Cy5+ subpopulation was then propagated in culture for six cell cycles and FACS analysis was used to sort the persistent Cy5+ cells. Cell viability was determined by light scatter and yielded greater than 95% viability. The long-term, label-retaining (LTLR) Cy5+ subpopulation was gated based on Cy5+ positivity of 0.5% (Figure 5c). The LRC-(Cy5+) that were detected after 6 and 8 cell generations was 5.0% and 1.54% to 5.0%, respectively. These represent approximately 3-fold greater than the theoretical calculated values, respectively. Furthermore, 95.35+0.01% and 88.40+0.08% of the LRC and the non-LRC expressed the Ki-67 antigen indicating that LRC are as proliferative as non-LRC cells.

An in vivo study was then conducted using LTLR-Cy5+ in order to assess the currently accepted "gold standard" for confirmation of putative CSC. Non-label retaining cells and LTLR-Cy5+ cells were fixed for confocal microscopy or injected into nude/SCID mice (SHO, Jackson Laboratory).

Cells sorted using LRC retained labels and cells sorted as non-LRC do not retain labels. F urthermore, after using FACS to sort LRC and non-LRC, the sorting results were validated by testing 15,000 post FACS cells individually, each of LRC and non-LRC. Multi- spectral flow cytometry (ImageStreamX Cytometer, Amnis Corp) was used to examine LRC and non-LRC in real time as they go through the nozzle of the FACS machine. Seven experiments were performed in various cancers. It was found that 1.54% to 5.0% of the recovered cells were label retaining cells (LRC). A two tailed Wilcoxon signed rank test was used, with a p=0.016. Therefore, the findings were statistically significant, indicating the LRC could be the result of ACD-NRCC.

The cells were prepared in 25% Matrigel (10 cells/ ΙΟΟμΙ total volume) and injected subcutaneously. Each mouse was injected with a subcutaneous transponder in order for the injection and subsequent tumor measurements to be performed in a blinded fashion over 16 weeks. Tumor growth was monitored biweekly at two dimensions with a ruler (Figure 11). After 16 weeks, the Cy5- Control group had no evidence of tumor growth in any of the ten mice; however, 5/10 mice (50%) in the LTLR-Cy5+ experimental group developed tumors.

Stem cells can differentiate into more mature daughter cells and, self -renew. SCs guarantee self -renewal by undergoing two types of division: asymmetric (ASYD) and symmetric (SYD) divisions. Solid organ cancer- stem-cells (SOCSCs) predominately divide asymmetrically, they also divide symmetrically in order to increase their progeny. This study demonstrates ASYD via non-random

chromosomal cosegregation in human cancer cells.

The long-term labeling (LTLR) technique confirms the hypothesis that the immortal DNA strand is labeled with the fluorophore-labeled nucleotides while the non-immortal DNA strand should continue to lose the fluorophore-labeling after multiple generations. This is further substantiated by the in vivo pilot study which showed evidence of tumor growth with LTLR cells while the unlabeled cells lack tumor- initiating capacity of the LRTR cells.

Identification and isolation of CSCs has both therapeutic and prognostic value. The techniques disclosed herein allow the isolation of a highly homogenous population of putative CSCs. Example 4

A subpopulation of Label-Retaining- Cancer- Cells (LRCC) is not quiescent and undergoes active cell-division The presently disclosed methods for isolation of live LRCC were

used to demonstrate that a subpopulation of LRCC is not quiescent, actively dividing, and exhibit stem-cells and pluripotency gene expression profile.

Label retaining cancer cells (LRCC) have greater tumor initiating capacity than non-LRCC in-vivo.

Label retaining cells (LRC) could theoretically result from either relative quiescence/slow-cycling or asymmetric-cell-division with non-random- chromosomal-cosegregation (ACD-NRCC). To test if some cancer-derived LRC (LRCC) are undergoing active cell division, LRCC and non-LRCC were isolated from three HCC cell lines (PLC/PRF/5, HuH-7 and SK-Hep-1) and three primary cell lines generated freshly from surgical specimens i (two colon cancers and one pancreatic cancer). The relative percentages of the LRCC ranged from 0.2% to 2.5% (n=6).

First, LRCC and non-LRCC were tested for evidence of cell cycling. Ki67 is a marker of cells undergoing through the cell cycle. It is a non-specific cell cycle marker indicating that cells are in Gl, S, G2 or M phase. Phospho-histone-3 (pHH3) is a specific mitotic marker. FACS analysis revealed that 95% vs. 88% of the LRCC and non-LRCC are Ki67 positive (p=0.009), respectively. Additionally, 16% vs. 8.7% of the LRCC vs. non-LRCC are positive for pHH3 (p=0.059), respectively. These results indicate that there is no statistical difference in the proportion of cells undergoing through the cell-cycle or cells in active mitosis between the LRCC and non-LRCC.

To confirm these results, cell cycle analysis was performed by Vindelov's propidium iodide method. It was found that LRCC undergo active cell division: 55%, 20% and 16% of the LRCC are in G1/G0, S and G2/M phases, respectively. In comparison to the non-LRCC, there is no statistical difference in the proportion of LRCC that are in G1/G0, S and G2/M phases, p=0.2, p=0.6 and p=0.3, respectively. These results further support the previous findings indicating that at least a subpopulation of LRCC is not quiescent and undergoes active cell division.

To further confirm these results, the LRCC were compared to the non-LRCC for expression of key cell-cycle check point genes. Using qRT-PCR cell-cycle super array, it was shown that there is no statistical difference in the expression of

CCNA2, CCND1, CCND2, CCND3, CCNE1, CDC2, CDK2, CDK4 and CDK6 between LRCC and non-LRCC. These genes represent key transition point genes through the various cell-cycle phases. Interestingly, CCND2, a gene expressed during the mid Gl -transition phase and exit from G0-G1 phase was expressed 4-fold higher in the LRCC than in the non-LRCC but achieved only a statistical trend

(p=0.06). Finally, to further validate these findings, the LRCC and the non-LRCC were sorted and cultured separately. The real time the cell-cycle duration (doubling times) of the LRCC was tested and compared to the cell-cycle duration of the non- LRCC. The cell-cycle duration of LRCC was 34 +8.8 hours (n=18, 3 experiments per each of 6 different cancers), and the cell-cycle duration of the non-LRCC was 36 +9.2 (n=18). These results were not statistically different, validating the previous results.

Thus, using several layers of evidence (cell proliferation markers, mitotic markers, cell-cycle phase- specific markers, cell-cycle check point genes and real time measurements of cell-cycle duration), it was demonstrated that a subpopulation of LRCC is actively dividing. Therefore, if some LRCC are not quiescent and are actively dividing how do they retain DNA labels? These findings demanded further investigation of the alternative hypothesis: Label-retaining-cells are generated by asymmetric-cell division with non-random chromosomal cosegregation (ACD- NRCC). Example 5

LRCC undergo asymmetric cell division with non-random chromosomal cosegregation

A subpopulation of LRCC undergoes active cell division mitigating the slow- cycling/quiescence hypothesis. Therefore, the alternative hypothesis was tested: LRCC are generated, at least in part by asymmetric cell division with non-random chromosomal cosegregation (ACD-NRCC). LRCC were isolated from early passage cells generated from three fresh surgical specimens and were cultured for several generations in a controlled chamber capable of continuous confocal-microscopic- cinematography. LRCC were isolated as described above.

Cancer cells were grown for one cell cycle in serum free media and underwent a double-thymidine arrest to increase the probability of cells being synchronously in G1-G0 phase at the inception of the experiment. Subsequently, to release the cells from the cell cycle arrest into active cycling, complete media was added DNA synthesis was allowed to occur with the DNA nucleotide analogue Cy5- dUTP. After incorporation of Cy5-dUTP into the DNA, cells were grown for one more cell cycle in culture, and subsequently, using FACS, Cy5-dUTP positive cancer cells were sorted with >99 purity. Cy5-dUTP positive cancer cells were then placed in collagen Γ coated chamber slides and their nuclei were labeled with the vital stain Cyto9. Cells were followed for multiple generations. Subsequently, after 8 cell doubling times, continuous confocal-microscopic-cinematography of live cells undergoing cell divisions. At time t=0 minutes, a single cell containing a single nucleus containing DNA labeled with Cy5-dUTP could be seen.

Three hundred cell divisions in 3 different experiments were observed. Of these 3 (3/300, 1%) were ACD-NRCC. In subsequent experiments the relative proportion of cells undergoing ACD-NRCC was XX%- YY%. LRCC undergoing ACD-NRCC is a rare phenomena but statistically significant. Example 6

LRCC exhibit greater tumor initiating capacity than non-LRCC

LRCC undergoing ACD-NRCC is statistically significant but biologically a rare phenomenon. Thus, to further understand the biological implications and the potential stem-cell nature of LRCC, the tumor initiating capacity was determined in immunodeficient mice. LRCC and non-LRCC were isolated as described above from one HCC cell line (PLC/PRF/5 ) and one cell-line generated from fresh surgical specimens (CSCL-04-Ke, pancreatic cancer). All in-vivo experiments were done in a double blinded fashion where the scientist who measured the developing tumors, the scientist who isolated the cells and injected the mice, and the scientist who interpreted the blinded data were three different persons. Moreover, mice were scrambled blindly within cages and we used coded electronic transponders to track the mice. All experiments were terminated at a predetermined date that was set at 16 weeks. Ten cells were transplanted from each of the three cancer cell types into 10 Nude/SCID mice per each group. Each of the LRCC and non-LRCC had a total of 20 mice per group. It was found that LRCC exhibited superior tumor initiating capacity when compared to non-LRCC. Specifically, 14/20 vs. 2/20 of the mice generated tumors (p=0.0005, Fisher's exact test). The LRCC generated faster and larger tumors than the non-LRCC, 8 weeks vs. 14 weeks. In summary, in various cancers there is a unique subpopulation of cells that actively divide and retain labels over prolonged period of time after asymmetric cell division with non-random chromosomal cosegregation; these cells are LRCC (Label Retaining Cancer Cells). LRCC have superior tumor initiating capacity in-vivo and possess stem like properties. Example 7

Gene expression analysis suggests that LRCC have stem-like and pluripotency profile

To further understand the potential stem cell nature of LRCC at the molecular level, live LRCC and non-LRCC were isolated their gene expression profiles were compared. qRT-PCR SUPERARRAY™ (SABiosciences) analysis using three platforms: Wnt gene array (86 genes), Stem-Cells gene array (82 gens) and Pluripotency gene array (12 genes). Cell lines that have been maintained for long time in culture potentially have different gene expression profile from freshly generated cells isolated from surgical specimens. Therefore, the analysis of the HCC cell lines from the freshly isolated cancer cells was performed separately (3 HCC cell lines and 3 freshly generated cancer cells).

Analysis of the Stem-Cells SUPERARRAY™ gene expression in the three HCC cell lines (PLC/PRF/5, HuH-7 and SK-Hep-1) revealed that there is

statistically significant upregulation of Sox2 (39 +13 folds, p=0.035), BMPl (2.7 +0.4 folds, p=0.002). The results show that the pluriotent transcription factor SRY (sex determining region Y)-box 2 (Sox2) gene is increased surprisingly by 38.9 + 13.1 (p=0.035) fold in LRC verse non-LRC (Figure 5A). Sox2 is essential to maintain human embryonic stem cell pluripotency and make Induced Pluripotent Stem (iPS) cells from somatic cells (Park et al., Nature 451, 141-146, 2008) and required for neural stem cell self-renewal. FGF is required for human embryonic stem cell pluripotency (Pera and Tarn, Proceedings of the National Academy of Sciences of the United States of America 107, 2195-2200, 2010). It was found that the Sox2 target gene FGF4 is increased by 20.3 + 9.2 (p=0.0011) fold (Figure 5B). It was also found that bone morphogenetic protein 1 (BMPl) is increased by 2.7 ± 0.4 (p=0.0021) fold (Figure 5A). BMPl is a zinc metalloproteinase with no homology with other BMPs. However it is an important regulator of BMP2, BMP4 and BMP7, which are involved in stem cell pluripotency and dorsal- ventral patterning during early embryonic development (Lee et al., 2009; Pera and Tarn, 2010). The wnt signaling pathway was suggested to play a role in pluripotency (Pera and Tarn, supra, 2010) and functions in stem cells of all three germ layers (Balciunaite et al., Nat Immunol 3, 1102-1108, 2002). It was found that WNT1 and WNT6 are increased by 8.6 + 2.1 (p=0.013) and 14.4 + 5.7 (p=0.0033) fold, respectively (Figure 5B). The wnt pathway target gene FOXN1 (Balciunaite et al., supra, 2002) is increased by 32.1 + 17.7 (p=0.0040) fold (Figure 6B). Knockout of FOXN1 is responsible for the congenital athymia and hairlessness phenotypes in nude mouse and rat due to stem cell defects of thymus epithelia and hair follicle (Balciunaite et al., supra, 2002). In agreement with FOXN1 increase, the cytotoxic T lymphocyte surface marker CD8A is increased by 5.1 + 0.2 (p=0.0015) fold (Figure 12A).

Another wnt pathway target gene pituitary follicle stimulating hormone beta polypeptide (FSHB) is also increased by 18.9 + 9.7 (p=0.0037) fold (Figure 12B). Notch signaling pathway is important in maintaining neural, intestinal and lymphoid stem cell fates (Izon et al., Immunity 16, 231-243, 2002). It was found that a Notchl antagonist, human Deltex 1 (DTX1, a Drasophila DTX2 hololog, (Izon et al., supra, 2002), is increased by 2.6 + 0.2 (p=0.00043) fold (Figure 12A). Neurogenin 2 (Neurog2), a neural stem cell marker, was increased by 7.0 + 0.6 (p=0.020) fold. Taken together this data show that LRC express higher levels of stem cell associated genes than non-LRC.

To further verify the findings in LRC, another method was used for the isolation of live cells undergoing ACD-NRCC and symmetric cell division, specifically by DNA double labeling with florescent nucleotide analogs Cy5-dUTP and Alexa555-dUTP and FACS sorting. Live cells undergoing ACD-NRCC and symmetric cell division were isolated from HCC cell lines PLC/PRF/5 and HuH-7, the same human stem cell and wnt pathway gene expression analysis was performed by real time qRT-PCR using the SUPERARRAY™. As described above, Sox2 and Neurog2 genes were increased by 4.7 + 0.8 (p=0.05) and 3.8 + 0.3 (p=0.02) fold, respectively, in cells undergoing ACD-NRCC verse cells undergoing symmetric cell division. Similar to BMP1 in LRC, the growth and differentiation factor 3 (GDF3), which maintains human stem cell pluripotency by inhibiting BMP signaling and promoting Nodal signaling (Pera and Tarn, supra, 2010), wass increased remarkably by 49.2 + 11.1 (p=0.049) fold. Similarly, the wnt signaling pathway activator Casein kinase 2 alpha 1 polypeptide (CSNK2A1) and target gene TCF7 are increased by 61.7 + 59.1 (p=0.024) and 7.4 + 1.3 (p=0.0082) fold, respectively. The neural stem cell gene Achaete-Scute complex homolog 2 (ASCL2) and endoderm stem cell marker Soxl7 were increased by 3.8 + 0.3 (p=0.023) and 13.1 + 6.5 (p=0.038) fold, respectively. In addition, lineage genes encoding structural proteins cardiac muscle actin 1 alpha (ACTCl) and collagen type I alpha 1 (COLlAl) were increased by 3.8 + 0.3 (p=0.023) and 2.9 + 0.2 (p=0.001) fold, respectively, and a negative stem cell marker gap junction protein alpha 1 (GJA1) was decreased by 2.7 + 0.2 (p=0.0001) fold. Interestingly cyclin D2 (CCND2) was increased and retinoblastoma 1 (Rbl) was decreased by 5.4 + 0.67 (p=0.0046) and -3.8 + 0.28 (0.011), respectively. The results with cells undergoing ACD-NRCC further support that LRC express higher levels of stem cell associated genes than non-LRCC do. The following Table summarizes the up-regulated genes:

Table 1. Cancer derived LRC express up-regulated stem cell-associated genes

Fold

Gene regulation _{R va|ue Gene function}

Gene name

symbol (Value ±

s.e.m .)

BMP1 Bonemorphogeneticprotein 1 2.69 ± 0.40 0.0021 BMP signaling antagonist BMP3 Bonemorphogeneticprotein 3 20.95 ± 0.64 0.0029 BMP signaling antagonist CD8A CD8a molecule 7.14 ± 1 .1 1 0.028 CTL antigen

recognition CD8B CD8b molecule -1 1 .00 ± 1 .00 0.0025 CTL antigen

recognition CXCL12 Chemokine (C-X-C motif) 3.05 ± 0.10 Activate

ligand 12 (stromal cell-derived

factor 1 ) lymphocytes

CYP2C8 Cytochrome P450, family 2, -3.89 ± 0.26 0.021 Promote apoptosis subfamily C, polypeptide 8

DTX1 Deltex homolog 1 2.54 ± 0.13 0.025 Notch signaling antagonist

DTX2 Deltex homolog 2 2.57 ± 0.16 0.00043 Notch signaling antagonist

FGF1 Fibroblast growth factor 1 13.00 ± 0.13 0.048 Promote FGF

signaling

FGF4 Fibroblast growth factor 4 20.30 ± 9.15 0.001 1 Promote FGF

signaling

FGFR1 Fibroblast growth factor 4.34 ± 1 .17 0.0039 Promote FGF receptor 1 signaling

FOXN1 Forkhead box N1 32.05 ± 17.68 0.0040 Thymus epithelia and hair follicle developement

FSHB Follicle stimulating hormone, 18.90 ± 9.73 0.0037 Follicle stimulating beta polypeptide

KRT15 Keratin 15 3.00 ± 0.12 0.037 Cyto skeleton

constituent of epithelial cells

LEF1 Lymphoid enhancer-binding 4.1 1 ± 0.40 0.047 Wnt effector

factor 1 transcription factor MME Membrane metallo- 25.27 ± 3.59 0.012 Endopeptidase endopeptidase

inactivating hormones

NANOG Nanog homeobox 1.77 ± 0.86 0.0040 Pluripotency

transcription factor

NEUROG2 Neurogenin 2 6.99 ± 0.57 0.020 Neural stem cell marker

SRY (sex determining region -16.33 ± 3.33 0.013 Endoderm stem cell Y)-box 17 marker

SRY (sex determining region 38.89 ± 13.06 0.035 Pluripotency Y)-box 2 transcription factor

Wingless-type MMTV 8.63 ±2.09 0.013 Promote Wnt integration site family, signaling member 1

Wingless-type MMTV 14.41 ± 5.68 0.0033 Promote Wnt integration site family, signaling member 6

Wingless-type MMTV 10.50 ± 2.95 0.032 Promote Wnt integration site family, signaling member 8A

Example 8

Materials and Methods for Examples 4-7

Fresh primary human cancer cells and cancer cell lines: After obtaining consent patients were enrolled on the tissue procurement protocol. Short term fresh primary human cancer cells were generated through serial transplantation of spheroids into Nude mice from patient tumors.

DNA double labeling procedure: To detect ACD-NRCC, DNA double labeling with Iodo-deoxyuridine (IdU, Sigma) and chloro-deoxyuridine (CldU,

Sigma) was performed with modifications as described (Conboy et al., PLoS Biol 5, el02, 2007). Extensive experiments for optimization of nucleotide incorporation efficiency were performed. According to the cell doubling times determined (see Figure 8), cells were first synchronized in serum-free media (SFM), and then grown in growth media containing the first thymidine analog either IdU or CldU at 5 uM, and in SFM. At the completion of the first cell cycle, cells were plated singly in collagen IV-coated 8-well chamber slides in growth media containing the second thymidine analog either CldU or IdU. Before the completion of the second cell cycle, growth media was replaced with SFM containing Cytochalasin-D (2 μΜ, Sigma) to arrest cells at cytokinesis. Arresting cells at cytokinesis allowed couplets (two nuclei within same cytoplasmic space) to be observed at division.

Immunofluorescence staining: Immunofluorescence staining was performed as previously described with modifications (Conboy et al., supra, 2007). The specificity of the anti-CldU and anti-IdU primary antibodies, and the secondary antibody was confirmed.

Fluorescence confocal microscopy: In order to detect couplets of cells arrested in cytokinesis, couplets were only scored that were well isolated from other cells. Confocal images were acquired using a Zeiss LSM 510 NLO Confocal system. Z-stacks were used with Bitplane's (Zurich, Switzerland) IMARIS® software (v6.0) for surface rendering. T o clearly define the positions of two nuclei in the same cytoplasmic space, a cutting plane was used to expose internal surfaces or outer surfaces were made semi-transparent.

Time lapse movie for real-time detection ofACD-NRCC: To detect ACD- NRCC in real-time, DNAs was labeled with Cy5-dUTP (VWR) by microporation (Harvard Apparatus) and directly visualized with time lapse confocal microscopy. Cell doubling time was first determined. Cells were then arrested in SFM, followed by double-thymidine arrest, washed and microporated with Cy5-dUTP as follows. 5e6 cells were resuspended in 108 μΐ of R-buffer with 12 μΐ of Cy5-dUTP (100 uM), loaded into 100 μΐ tip and the microporation tube containing 3 ml of microporation buffer, microporated at 1400 V for 20 millisecond twice, then transferred

immediately to growth media. After cells passed the first labeling cycle, Cy5 high cells were sorted by FACS (BD FACSARII®), plated in collagen-rV coated 8-well chamber slide at 5e4 cells/ml, stained for nucleic acid with Cyto9 at 0.5uM

(Invitrogen) and imaged. As a control, Cy5+ cells were also plated for DAPI staining to confirm that Cy5-dUTP labels nuclei. Time lapse imaging was performed on the Zeiss LSM 710 NLO confocal equipped with an environmental chamber (Carl Zeiss). Images were acquired every 30 minutes for 65 hours and time lapse movies were made with the ZEN software (8 bit, 512 x 512 pixels).

Isolation of live cells undergoing ACD-NRCC: Cells undergoing ACD- NRCC were isolated with double-labeling and double-sorting. Cells were first arrested with SFM, plated, microporated before S phase with Cy5-labeled dUTP (ΙΟΟμΜ) or unlabeled dUTP (ΙΟΟμΜ), and cultured to allow completion of the first cell cycle. Then cells were microporated with Alexa555-dUTP (ΙΟΟμΜ, Invitrogen) or unlabeled-dUTP (ΙΟΟμΜ), respectively, and sorted by FACS for Alexa555+ high/Cy5 high cells before the completion of the second cell cycle. The double positive cells were cultured to allow the completion of their second cell cycle and sorted for the Alexa555+/Cy5- cells (cells undergoing ACD-NRCC) and

Alexa555+/Cy5+ cells (cells undergoing symmetric cell division). CFSE staining (5.0uM for 15 minutes, Invitrogen) was used to monitor cell cycles.

Isolation of live LRC: Cells were first arrested with serum free medium (SFM), plated, microporated before S phase with Cy5-dUTP (ΙΟΟμΜ) or unlabeled dUTP (ΙΟΟμΜ). After completing the labeling cell cycle, Cy5 high cells were sorted, propagated in log phase for different cell cycles. Then LRC (Cy5+) and non- LRC (Cy5-) were gated based on the unlabeled dUTP-microporated cells and sorted.

Gene expression analysis: Sorted live cells undergoing ACD-NRCC and symmetric cell division, and sorted live LRC and non-LRC were used to isolate total RNAs using miRNeasy Mini kit and RNase-Free DNase Set (QIAGEN). RNA quantification (Nanodrop), reverse-transcription, pre-amplification, real-time qPCR and Ct value analysis were done for Human Stem Cell and Wnt Pathway in triplicates using 384 well plates with ABI 7900 HT system (Applied Biosystems) according to the manufacturer's protocol (SABiosciences).

Ki67 and pHH3 detection by FACS analysis: Cells were microporated with

Cy5-dUTP or dUTP, cultured for 8 cell cycles, stained for Ki67 (Ki67-FrfZ, Dako) or pHH3 (pHH3-Alexa-488, S-10, Cell Signaling), acquired on BD FACS aria Π and analyzed with FLOWJO® in triplicate.

Cell cycle analysis of LRC and non-LRC: After sorted into LRC and non- LRC, cells were fixed, washed, stained with Vindelov's PI, acquired on BD FACS aria Π and analyzed with FlowJo in triplicates.

It has been described that the minimum numbers of putative solid organ CSC required to generate tumors are 100 cells with only one exception using more immune-deficient IL-2R-/- mice (Quintana et al., Nature 456, 593-598, 2008;

Visvader and Lindeman, Nature reviews 8, 755-768, 2008). Tumors were generated with as few as 10 LRC. The results from the HCC cell line were confirmed by using fresh primary human pancreatic cancer cells. Cancer derived LRC have superior tumor initiating capacity in-vivo.

Mouse xenogeneic transplantation: Live LRC and non-LRC were isolated after 6 or 8 cell cycles for the human HCC cell line (PLC/PRF/5) or fresh primary pancreatic cancer cells (CSCL-04-Ke), respectively, and injected into Nude/SCID mice (SHO, Jackson Lab) subcutaneously with 25% of Matrigel (10 cells/injection, two sites/mouse). Mice were blinded with transponders (Bio Medic Data Systems, Inc) for PLC/PRF/5 or manually for CSCL-04-Ke and monitored weekly.

Statistics: All data are presented as the means + s.e.m. and statistical differences were evaluated as follows. (A) The statistical significance of observing ACD-NRCC was calculated with the two-tailed p-value by the exact binomial test. (B) To test for significance of the relative proportions of cells undergoing ACD- NRCC between tested groups, the Poisson method was used. (C) The data with respect to the observed effect of the niche on ACD-NRCC was analyzed by two- tailed test using Mehta's modification to Fisher's exact test. (D) Fisher's exact test was used to test for significance of tumor initiating capacity. In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for identifying a stem cell, comprising:

a) synchronizing the cell cycle of cells in a population of cells;

b) labeling the population of cells undergoing cell cycle division by exposing the cells to a first nucleotide or an analogue thereof labeled with a first detectable marker for one cell cycle, wherein the first nucleotide or analogue thereof is incorporated into DNA of the cells during a first DNA replication, and wherein the first detectable marker can be detected in a viable cell;

c) removing from the population of cells the first nucleotide or analogue thereof labeled with the first detectable marker and allowing the population of cells to undergo cytokinesis;

d) isolating cells that include the first nucleotide analogue incorporated into the DNA;

e) labeling the cells that include the first nucleotide analogue incorporated into the DNA by exposing the cells to a second nucleotide or analogue thereof labeled with a second detectable marker for the duration of one cell cycle, wherein the first detectable marker and the second detectable marker are different, wherein the second nucleotide or analogue thereof is incorporated into the DNA of the cells during a second DNA replication, and wherein the second detectable marker can be detected in a viable cell;

f) removing the second nucleotide or analogue thereof and allowing the cells to undergo cytokinesis; and

e) identifying at least one viable cell that comprises only the second nucleotide or analogue thereof incorporated into the DNA of the cell, in the absence of the first nucleotide or analogue thereof incorporated into the DNA of the cell, by detecting the presence of the second detectable marker and the absence of the first detectable marker in the viable cell,

thereby identifying the stem cell.

2. The method of claim 1, wherein the population of cells is a population of cancer cells, and wherein the method identifies a cancer stem cell.

3. The method of claim 1 or claim 2, further comprising labeling the population of cells with a membrane permeable cell proliferation dye.

4. The method of claim 3, wherein the cell proliferation dye is fluorescent and is detectable by FACS.

5. The method of claim 4, wherein the cell proliferation dye is

carboxyfluorescein diacetate, succinimidyl ester or another membrane dye.

6. The method of any one of claim 1-5, wherein the first detectable marker and the second detectable marker are fluorescent, and wherein identifying the at least one viable cell comprises the use of fluorescence activated cell sorting (FACS).

7. The method of any one of claims 2-6, wherein the population of cells is a population of cancer cells, and wherein the cancer is a breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer.

8. The method of any one of claims 1-7, wherein the first and the second nucleotide are the same nucleotide, and wherein the first and the second nucleotide are labeled with different fluorescent markers.

9. The method of claim 8, wherein the nucleotide is dUTP.

10. The method of any one of claims 1-9, wherein the first detectable marker or the second detectable marker is Cy5-dUTP.

11. The method of any one of claims 1-10, wherein the first detectable marker or the second detectable marker is Alexa-555-dUTP.

12. The method of any one of claims claim 1-11, further comprising isolating at least one viable stem cell.

13. The method of claim 12, wherein isolating the at least one viable stem cell comprises the use of fluorescence activated cell sorting (FACS).

14. The method of claim 13, wherein the stem cell is a cancer stem cell, and wherein the population of cells is a population of breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer cells.

15. A method for identifying a stem cell, comprising

labeling a population of synchronized cells undergoing cell cycle division by exposing the cells to a nucleotide or analogue thereof labeled with a detectable marker prior to a first round of DNA replication and allowing the cells to undergo a single cell division, wherein the nucleotide or analogue thereof is incorporated into chromosomes of the cells, and wherein the detectable marker can be detected in a viable cell;

removing the nucleotide or analogue thereof labeled with the detectable marker that is not incorporated into the chromosomes of the cells;

allowing the cells to divide for at least five additional cell divisions in the absence of the nucleotide or analogue thereof labeled with a detectable marker; and identifying viable cells comprising the nucleotide or analogue thereof incorporated into the chromosomes of the cells by detecting the detectable marker, thereby detecting the stem cell.

16. The method of claim 15, wherein the population of synchronized cells is a population of synchronized cancer cells, and wherein the method identifies a cancer stem cell.

17. The method of claim 15 or claims 16, further comprising labeling the population of cells with a membrane permeable cell proliferation dye.

18. The method of claim 17, wherein the cell proliferation dye is fluorescent and is detectable by FACS.

19. The method of any one of claims 15-18, wherein the cell proliferation dye is carboxyfluorescein diacetate succinimidyl ester.

20. The method of any one of claims 15-19, wherein the detectable nucleotide or analogue thereof is labeled with a fluorescent marker, and wherein identifying the at least one viable cell comprises the use of fluorescence activated cell sorting (FACS).

21. The method of any one of claims 15-20, wherein the population of synchronized cells is a population of synchronized cancer cells, and wherein the method identifies a cancer stem cell, and wherein the cancer is a breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer.

22. The method of any one of claims 15-21, wherein the nucleotide is dUTP.

23. The method of any one of claims 15-22, wherein the detectable marker is Cy5 or Alexa-555.

24. The method of any one of claims 15-23, further comprising isolating at least one viable stem cell.

25. The method of claim 24, wherein isolating the at least one viable cell comprising the use of fluorescence activated cell sorting (FACS).

26. The method of claims 24-25, wherein the at least one viable stem cell is is a breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer stem cell.

27. A method of identifying a chemotherapeutic agent of use in treating a subject with a cancer, comprising:

isolating cancer stem cells from a sample of cancer cells from the subject, wherein the cancer stem cells are isolated using the method of any one of claims 1- 26; and

contacting the isolated cancer stem cells with a chemotherapeutic agent of interest;

wherein death of the cancer stem cells following treatment with the chemotherapeutic agent of interest as compared lo a control indicates that the chemotherapeutic agent is of use in treating the subject.

28. The method of claim 27, wherein the control is a standard value or the death of the cancer stem cells in the absence of the chemotherapeutic agent.

29. The method of claim 27 or claim 28, wherein the cancer is a breast cancer, leukemia, lymphoma, colorectal cancer, pancreatic cancer, lung cancer, melanoma, gastric, mesothelioma, or liver cancer.

30. The method of any one of claims 27-29, wherein the chemotherapeutic agent is an alkylating agent.

31. A method of treating a subject with a lymphoma or a leukemia, comprising

isolating bone marrow from the subject; and

isolating cancer stem cells from the bone marrow using the method of claim 2 or 15, thereby producing bone marrow depicted of cancer stem cells; and reintroducing the bone marrow depleted of cancer stem cells into the subject, thereby treating the subject.

32. The method of any of claims 1-26, further comprising measuring the expression of one or more of sex determining region Y-box 2 (Sox2) and neurogenin (Neurog) 2.

33. The method of claim 32, comprising measuring Sox2 or Neurog2 protein.

34. The method of claim 32, comprising measuring Sox2 or Neurog2 mR A.