WO2007124125A2 - Methods of identifying stem cells in normal and cancerous tissues and related progeny cells - Google Patents

Abstract

The present invention relates to methods and compositions for the identification of active cells, such as stem cells or cancer stem cells, and the cells differentiated from them. The invention may be useful in the identification of progenitor cells from a wide variety of tissues including pre-cancerous or cancerous tissue.

Description

METHODS OF IDENTIFYING STEM CELLS IN NORMAL AND CANCEROUS TISSUES AND RELATED PROGENY CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims benefit to U.S. Provisional Application

Serial No. 60/745,377 filed on April 21 , 2006, which is incorporated by reference as if recited in full herein.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and kits for the identification and treatment of stem cells and their progeny associated with tumor formation and their recurrence. The present invention also relates to a rapid DAL method for identifying and locating stem cells in normal and cancerous tissues.

BACKGROUND OF THE INVENTION

[0003] Stem cells are primal undifferentiated cells that can differentiate into other cells and are consequently known to have a role in normal tissue homeostasis and the pathogenesis of disease. Adult stem cells, including those of self-renewing tissues and non-self renewing tissues, are pluripotent and multipotent cells that provide the foundation for every organ of the body. As such, stem cells are unique from other cells because they have a high capacity of self- renewal and are ultimately responsible for the homeostasis of steady-state tissues. These primitive cells are long-lived, give rise to daughter cells that have the capacity to generate an entire adult cell component within their indigenous tissue, and regenerate tissue after injury.

[0004] Most adult stem cells are slow-cycling and remain quiescent during normal tissue homeostasis, residing in a niche where they receive feedback signals to regulate their state of quiescence or activation. Injury to a tissue through mechanical stress, abrasion, wound, low dose irradiation or unexpected cell loss results in a feedback signal to stem cells for replenishment activity. Upon injury to or death of surrounding cells, the stem cells actively respond with rapid cell division to thereby replenish lost cells or damaged tissue (Potten, C. S. and Morris, R. J., J. CeII ScL Suppl. 10:45-62, 1988).

[0005] Generally, it is thought that stem cells are located in well-protected, highly vascularized and innervated areas; however, identification of stem cells at a precise location has been difficult to determine due to the lack of immunological or biochemical markers. The purported location of the cells in several tissue types including skin, intestine and bone marrow, has been determined largely through long-term label-retaining cell (LRC) assays.

[0006] Under standard conditions currently used in LRC assays, labeling putative stem cells requires repeated administration of a cell-marking label for a prolonged period of time (Kameda, T. et al., Genes to Cells 7, 2002 923-931; Hong, K. U., et al., Am. J. of Respir. Cell and MoI. Biol. 24, March 20, 2001, 671- 681; Potten, CS. , et al., Cell 15, November 1978, 899-906; Lavker, R. M., et al. Cell 57(2): 201-209, April 21, 1989; US Patent 5,279,969 filed November 4, 1992). Weeks to months after exposure to the cell-marking label, cells that cycle slowly retain the marking label for an extended period of time in their nuclei; whereas, the initial incorporated label is relatively quickly diluted from most cells due to rapid expansion to give rise to differentiated cells. While LRCs are thought to be stem cells because of their long lifespan and anatomic location, their ability to regenerate a tissue with all appropriate cell types has remained questionable. Attempts at defining the regenerative capacity of epidermal, lung and intestinal LRCs have included different approaches. For instance, stem cells of the epidermis are thought to reside in the bulge region of the hair follicle where LRCs also reside. Thus, one experiment targeted elimination of these LRCs by photo- induced cell killing, which resulted in a defect of follicular regeneration suggesting that the LRCs of the epidermis are stem cells as predicted (Kameda, T. et al.. Genes to Cells 7, 2002 923-931).

[0007] Attempts in the lung (see Hong, K. U., et al.. Am. J. of Respir. Cell and MoL Biol. 24, March 20, 2001, 671-681) and intestine (see Potten, CS. , et al., Cell 15, November 1978, 899-906) have employed a method of eliminating all cycling cells followed by continuously administered label. While the tissue in question was regenerated with labeled cells, the ultimate source of the labeled differentiated cells was questionable. It was believed that all of the cells dividing during the continuous labeling period incorporated the label resulting in all cycling cells of the regenerated tissue, regardless of source, having the label. Problematically the stem cells could not be specifically identified. To date, there is not a reliable system for identifying stem cells within a population of cells. [0008] Such a system would have tremendous clinical benefit. For example, the theory that cancer is initiated and driven by cancer stem cells (CSCs) is gaining appreciation in many areas of cancer research (Reya et al., 2001). Existence of CSCs has been demonstrated in variety of tissues including blood, breast, brain, lung and gut (Al-Hajj et al., 2003; He et al., 2007; Kim et al., 2005; Lapidot et al., 1994; O'Brien C et al., 2006; Ricci-Vitiani et al., 2006; Singh et al., 2004; Yilmaz et al., 2006; Zhang et al., 2006). Identification of CSCs and their tumor microenvironment (or niche) (Li and Neaves, 2006) has tremendous clinical implications; however, lack of an effective way of identifying CSCs presents a major hurdle in this effort.

[0009] Currently, there are basically six different approaches for identification of stem cells or their locations in adults: 1) isolation by surface markers and functional tests through transplantation — isolation of candidate stem cell populations using a combination of markers and demonstration of stem cell properties by transplantation experiment. This approach is exemplified by the pioneering work in the hematopoietic system (Spangrude et al., 1988), in which a combination of mature lineage markers are required to remove differentiated lineages first and then use a few stem cell markers to isolate HSCs, a strategy of 'draining the pond to catch the fish'; 2) identification of long-term retention cells (LRCs) using DNA labeling — identification of cells in a given tissue that undergo slow cycling as measured by their ability to retain the labeled DNA for a much longer period than the rapid cycling progenitor cells. This approach has been used to identify the location of skin, intestinal, and hematopoietic stem cells (Cotsarelis et al., 1990; Potten et al., 1997; Zhang et al., 2003). However, whether LRCs are stem cells varies in different tissues and requires further functional characterization. At least in skin it has been demonstrated that LRCs can give rise to the entire hair follicle upon transplantation into recipient animals (Blanpain et al., 2004); 3) In vivo lineage tracing to search for cells that give rise to the downstream lineages— Using this approach, neural stem cells and Drosophila gut stem cells were identified. (Doetsch et al., 1999; Ohlstein and Spradling, 2006; Palmer et al., 1997; Sanai et al., 2004); 4) identification of_multi potent cells - as revealed by their co-expression of multiple downstream lineage markers of stem cells. Lung stem cells were identified as bronchioalveolar stem cells (BASCs) based on their co-expression of two downstream Clara and Alveolar lineage markers, CCA and SP-C, and their ability to give rise to Clara and Alveolar lineages (Kim et al., 2005); 5) Identification of side population (SP) — based on a feature of stem cells to express certain types of multiple drug-resistant genes and display a unique pattern in flow cytometry assay. SP has been shown to be enriched with HSCs (Goodell et al., 1997) and stem cells in other non- hematopoietic tissues (Goodell et al., 2005). 6) based on functional characteristics of stem cells (Berardi et al., 1995; Gordon et al., 1985; Lerner and Harrison, 1990; Reisner et al., 1982; Sharkis et al., 1997) — These methods are based on the functional features of stem cells including binding to soybean agglutinin (Reisner et al., 1982), resistance to the treatment of either 5-fluorouracil (5-FU ) (Gordon et al., 1985; Berardi et al., 1995) or alkylating agent (Lerner and Harrison; Sharkis et al., 1997) and density-gradient (Juopperi et al., 2007). The methods can only achieve the goal of enrichment but are not very pure (Berardi et al., 1995). There are possibly other approaches for identification of stem cells (e.g. in vitro colony assay) that are not included here.

[0010] Each of the approaches described above has its advantages and disadvantages and is based on limited aspects of stem cell properties. For example, often the lack of appropriate surface markers and/or a reliable transplantation system limit the ability to identify, isolate, and further functionally test stem cells in solid tissues. It is difficult to functionally demonstrate that LRCs have stem cell properties, since BrdU is not a cell surface marker, unless sophisticated genetic models are used (Blanpain et al., 2004). Multipotentiality is a recognized stem cell property. However, whether stem cells in all the adult tissues co-express down-stream lineage markers varies, depending on the type of tissue. An in vivo lineage chasing experiment has offered a convincing approach for stem/progenitor cell identification, however, it can not be readily applied to many mammalian systems. Unless appropriate genetic models are available allowing a stem-cell's marking and lineage tracing to be performed, the limited ability to obtain viable cells after sorting the SP cells from solid tissue may affect transplantation experiments to test the repopulation capacity of the candidate cells. Thus, establishing a simple and effective methodology for determining stem cell location and identity in different tissues could not only facilitate studies in the less defined systems, but also help clarify some important issues in the more established ones.

[0011] For example, in the hematopoietic system, HSCs have been prospectively purified in the Thy-1¹⁰ or Flk2^" Lineage^"Sca-1 ⁺c-Kit⁺ population for quite some time (Christensen and Weissman, 2001; Uchida et al., 1996). However, since the identification of HSCs requires an array of multiple surface markers, only recently were HSCs revealed to reside in either an osteoblastic niche or a vascular niche in bone marrow (Arai et al., 2004; Calvi et al., 2003; Kiel et al., 2005; Kopp et al., 2005; Zhang et al., 2003). Since HSCs undergo routine mobilization, circulation, and homing (Lapidot et al., 2005; Wright et al., 2001 ), the differential functions of osteoblastic and vascular niches during this dynamic process remain to be determined. In the pancreas, a variety of cells have been shown to be potential sources for pancreatic stem/progenitor cells (Bonner-Weir et al., 1993a; Guz et al., 2001a; Lardon et al., 2004; Sarvetnick and Gu, 1992; Zulewski et al., 2001b). To date, the existence, location, and identity of pancreatic stem/progenitor cells are still debatable (Dor et al., 2004). In the intestine, the stem cell location was revealed by LRCs, which, however, have not been shown to be able to give rise to the entire-crypt-villus structure. The ability to readily label and further trace stem cell activity during the process of regeneration would be very helpful in identification of stem/progenitor cells, and could further delineate the relationship between stem cells and their niches.

SUMMARY OF THE INVENTION

[0012] Based on the functional role of stem cells in repairing or regeneration of damaged tissues, we have developed a simple procedure to identify stem cells and their location. Using this approach we have demonstrated the efficiency and reliability of this simple method in the well-defined hematopoietic system, we have identified the locations of stem/progenitor cells in pancreatic tissue, and we have revealed and confirmed the function of leukemia initiating cells in the PTEN mutant animal model.

[0013] Thus, one embodiment of the present invention provides a method to identify stem cells, as well as the cells differentiated from such identified stem cells located within a population of cells. The invention advantageously utilizes the characteristic that stem cells are activated to proliferate in response to injury. The invention thus provides new methods, kits and uses for identifying activated stem cells and their progeny.

[0014] The methods provided by the invention comprise damaging one or more cells within a population of cells such that the damage results in stimulating one or more quiescent cells to become activated. Optimally, all cycling, or activated, cells within a population of cells are damaged within the targeted cell population. These damaged cycling cells are unable to be labeled or reproduce and ultimately die or undergo apoptosis. Damage to the cycling cells elicits a signal to quiescent stem cells to become activated or enter the cell cycle to replenish lost cells.

[0015] The term "cycling" refers to any cell that is in a state of reproduction or doubling. Such a cell includes a cell in the cell cycle, cell division, mitosis or that is active. A cycling cell can be a stem cell, a cancer stem cell, a pluripotent cell, a totipotent cell, a unipotent cell, a non-stem cell, a precursor cell, a progenitor cell, a differentiated cell, or a progeny of a stem cell or cancer stem cell.

[0016] The term "activated" refers to any cell triggered to enter a state of reproduction or doubling and can include a cell entering the cell cycle, cell division, or mitosis. An activated cell can be a pluripotent cell, a totipotent cell, a unipotent cell, a stem cell or a cancer stem cell.

[0017] Damage to cycling cells within a population of cells can be elicited by treating the target population with mitotic killing agents. A suitable mitotic killing agent comprises any agent that targets cycling cells for destruction and does not eliminate non-cycling cells. The agent may be applied at about 100,

125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 mg/10 kg of cell population weight or other dose known in the art.

[0018] A suitable agent may include 5-fluoruracil (5-FU), or a derivative thereof, or comprise any of the agents listed in Table 1 or derivatives thereof or combinations of such agents. It is expected that the time period during which to administer the mitotic killing agent will vary with the type of tissue from which the population of cells is derived, as this period is dependent upon the number of cells cycling within a population, as well as the purpose for which the cells are being damaged. [0019] Self-renewing tissues, including the epidermis, intestine, and testes, continuously turnover throughout the life of an organism and thus have an adequate number of cycling cells at any given time. All other tissues are considered non-self renewing and do not have a vast number of cells within them cycling except during development. It is envisioned that administering mitotic killing agents to non-self renewing tissues will generally be optimal during the development stage. In contrast, the timing of the administration of mitotic killing agents to self-renewing tissues, precancerous, cancerous, or tumorigenic tissues is not limited to the development stage and can occur at anytime. [0020] Methods of the invention also comprise labeling an activated cell such that the label is incorporated into DNA of the activated cell. By exposing the population of cells to a cell-marking label after treatment with a damaging agent, activated cells, such as stem cells and their progeny may be labeled. It is expected that the time period during which to apply the cell-marking label will vary with the type of tissue from which the population of cells is derived, as this period is dependent upon the tissue regeneration time. For example, it is envisioned that the optimal time period during which to apply label to cells derived from intestinal tissue ranges from about 0.5, 1 , 2, 3, 4, 5, 6, or 7 days after a mitotic killing agent has been applied. Further, the most optimal time period for labeling is expected to be at or about three days after application of the mitotic killing agent. It is envisioned that the optimal time period during which to apply label to cells derived from hematopoietic tissue ranges from about 0.5, 1, 2, 3, or 4 days after application of a mitotic killing agent, and for cells derived from the pancreas, the optimal time period ranges from about 0.25, 0.5, 0.75, 1 , 1.5, 2, 2.5, 3, 3.5 or 4 days after application of a mitotic killing agent. It is envisioned that the most optimal time period to apply a label is at or about three days after the mitotic killing agent is applied in all tissues or populations of cells. Those of skill in the art will recognize that the time period chosen will depend upon not only the tissue from which the population of cells is derived, but also the objective of the labeling. Hence, shorter or longer periods may be desirable. In general, one of skill in the art would recognize that the time period for dosing a population of cells with a label would be less than the time period required to fully regenerate the population. Further, the skilled artisan would also recognize that this time period may vary, depending upon the organism and the purpose for which the invention was used.

[0021] It is envisioned that the cell-marking label will be selected so that it incorporates into activated cells at sufficient levels to allow external detection. For example, the cell-marking label may be a halogenated deoxyribonucleotide, such as bromodeoxyuridine (BrdU) that is provided to the population of cells at about 0.5, 1 , 2, 3, 4, 5, 6, or 7 mg/kg of population of cell weight. The cell-marking label includes any cell-marking label that incorporates into DNA or other cell components of an activated cell and does not turn over or degrade in the absence of cell division. Thus, a label may include biotinylated, radiolabeled, fluorescent, colorimetric molecules or other suitable labels known in the art. [0022] The invention provides that once a label is incorporated into a cell, the label is reduced, or diluted, in that cell by its division. With each cell division, a fraction of the label is transferred to a progeny cell. This sharing of the label allows the identification of the progeny cell, indicating the regenerative capability of the activated stem cell, and allows for the tracing of the progeny to and from the active cell from which it was derived. [0023] Further, the methods of the invention comprise identifying labeled cells. Identification may be through any method that recognizes the difference between labeled and unlabeled cells, including but not limited to those methods that result in visualization or separation. Exemplary identification techniques include immunodetection, autoradiographic, flow cytometry, and fluorescence activated cell sorting (FACS) techniques. A skilled artisan would recognize that the identification of a specific label depends upon the label and the purpose for which the invention was used.

[0024] Visualization of identified, labeled cells may be through the use of any agent that renders the labeled cell visible to the eye or other means of detection. Exemplary agents include radiolabels, fluorescent tags, enzymatic tags, and fluorogenic or chromogenic substrate tags.

[0025] The invention can be practiced to allow the labeling and identification of activated cancer stem cells. Such a cell is responsible for the development and reoccurrence of cancer. To identify an activated cancer stem cell, the invention comprises damaging cycling cells within a population of cells, labeling activated cells, and identifying the labeled activated cell. It is envisioned that treatment of a cancer with a therapeutic agent, such as a mitotic killing agent, for example those agents provided in Table 1 , will eliminate cycling cancer cells and activate quiescent cancer stem cells. As such, it is envisioned that the invention may be used to subsequently identify an activated quiescent cancer stem cell, and thereby, determine the prognosis for recurrence of a tumor and the optimal time period for treatment with an appropriate agent to eliminate the activated cancer stem cell and its progeny. This determination of the presence of an activated cancer stem cell or its progeny may occur either in vivo or in vitro. If in vivo, the label is applied to the subject. If in vitro, a biopsy may be obtained by using standard methods and the label applied thereafter as described. Label may be applied immediately to cells obtained by biopsy, or the cells may be cultured an the label applied at a later time period. Those of skill in the art will appreciate that the timing of the label's application will depend upon the source of the cells, the cell type(s), and the type of cancer(s) involved.

[0026] Further, the invention provides for the labeling and identification of activated stem cell progeny. To identify stem cell progeny, the invention comprises damaging cycling cells within a population of cells, labeling the activated cells, and identifying the labeled activated cells over a period of time. Over time, the labeled cycling cells will produce progeny cells. As a progeny cell will have incorporated some label from its parent cell, the progeny cell may also be visualized. It is envisioned that tracking the progress of cell reproduction will lead to a greater understanding of cell and tissue regeneration, as well as, a better understanding of aging and a wide variety of disease states. [0027] A "progeny" referred to in the present invention include any cell type derived from a stem cell. These cell types may include stem cells, less potent progenitor cells, transit amplifying cells, precursor cells, differentiated cells and non-stem cells. Any cell that is a descendant of the labeled stem cell may be considered a progeny of that stem cell.

[0028] A "population of cells" includes any cell or group of cells. It is envisioned that the population of cells includes one or more stem cells and/or one or more progeny cells of a stem cell. Such population of cells can comprise a cell in culture, comprise in vitro tissue, or comprise a tissue within a living organism. The population of cells may be mammalian and includes, but is not limited to, murine, human, bovine, porcine, equine, ovine, or canine.

[0029] The population of cells may include normal, precancerous, cancerous, and tumorigenic tissues. These tissues include, and are not limited to, the intestine (small or large), pancreas, skin, breast, liver, heart, kidney, bone marrow, blood, cartilage, adipose, stomach, oral mucosa, skeletal muscle, thymus, thyroid gland, bladder, lung, bilary track, ovary, testes, brain, lymphoid tissue, prostate gland, bone, uterine cervix, epithelial, endothelial, mesenchymal, and other stem cell-containing tissues.

[0030] The invention provides kits comprising an effective amount of at least one mitotic killing agent, a sufficient amount of at least one DNA incorporating label, at least one label-detecting agent that detects the label, and instructions for use of each component. It is envisioned that combinations of more than one killing agent and/or more than one DNA incorporating label may be provided in a kit or used in practicing the invention. Exemplary mitotic killing agents include agents that target cycling cells for destruction and do not eliminate non-cycling cells. Exemplary DNA incorporating labels include any cell-marking label that incorporates into an activated cell.

[0031] Such kits may be combined with at least one visualizing agent to visualize the DNA incorporating label. Other biological agents or components may be included, such as those for making and using the agents. It is also envisioned that combinations of more than one label-detecting agent or more than one visualizing agent may be provided in the kit or used in practicing the invention. In such kits, the diagnostic agents are maintained separately in containers within a container that holds all components. [0032] It is envisioned that the provided kits will be used to practice the methods of identifying an activated cell and progeny thereof. As such, the kits will be used on a population of cells as described above. The mitotic killing agent, or damaging agent, within the kit will include at least one of the following: an alkylating agent, nitrosurea, antitumor antibiotic, mitotic inhibitor, or antimetabolite. The included label or cell-marking label will comprise either BrdU or other halogenated deoxyribonucleotides, labeled deoxyribonucleotides, halogenated deoxyribonucleotides, radio-labeled nucleotides, deuterium labeled nucleotides, or deuterium labeled DNA synthesis precursors.

[0033] The label-detecting agent included in the kit is dependent upon the label used. The label-detecting agent may include monoclonal or polyclonal antibodies that specifically identify the label, tagged antibodies, or photographic emulsion to detect radiolabels. Tagged antibodies are antibodies conjugated to radiolabels, fluorescent tags, enzymatic tags, and fluorogenic or chromogenic substrate tags, which allows the label-detecting agent to also be a visualizing agent.

[0034] The kit may also contain visualizing agent which may include a radiolabel, a fluorescent tag, an enzymatic tag, a fluorogenic substrate tag, or a chromogenic substrate tag.

[0035] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The application contains at least one drawing executed in color.

Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0037] FIG. 1 schematically shows normal hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) cycling prior to 5FU treatment or labeling by BrdU pulse injection. Long-term HSCs are self-renewing cells switching between quiescent and activated states. When necessary, for example after tissue injury, quiescent long-term HSCs become activated to yield highly proliferative short-term HSCs and MPPs. Short-term HSCs and MPPs give rise to cells that differentiate into differentiated hematopoeitic cell types.

[0038] FIG. 2 schematically shows that by Day 2 after 5FU treatment all common lymphoid progenitor cells were killed, and no cells, including long-term HSCs, can be labeled by BrdU. Neither activated long-term HSCs or short-term HSCs or MPPs are present.

[0039] FIG. 3 schematically shows that on Day 3 after 5FU treatment quiescent long-term HSCs have activated and entered the cell cycle to reproduce. This is the optimal time to label the activated long-term HSCs. Proliferating cells can be labeled with BrdU by administering BrdU 1 to 3 hours prior to tissue sampling. Activated stem cells can be labeled with BrdU by administering BrdU after 5FU treatment. [0040] FIG. 4 schematically shows that on Days 4-6 after 5FU treatment long-term HSCs are active and yield highly proliferative short-term HSCs and MPPs.

[0041] FIG. 5 shows cells surrounding bone and blood vessels, 2 days after

5FU treatment, without BrdU incorporation. The data indicates that all cell proliferation activity has ceased and no active HSCs are present. The slight brown cells are red blood cells within a blood vessel.

[0042] FIGs. 6A-F shows cells with incorporated BrdU (red) three days following 5FU treatment. Specifically, FIG. 6A shows cells with incorporated BrdU (red) on the bone surface, indicating cells lining the bone surface are long-term HSCs capable of activation to replenish lost cells. FIG. 6B shows cells with incoφorated BrdU (red) on the surface of blood vessels, indicating activated long- term HSCs also line blood vessels. FIG. 6C shows cells with incorporated BrdU (red) on the surface of blood vessels, indicating activated long-term HSCs line blood vessels. FIG. 6D shows activated long-term HSCs (white) actively dividing on the bone surface (dashed line). FIG. 6E shows that BrdU-labeled cells (red) were mainly observed on the bone surface (shown by N-cadherin staining in green). FIG. 6F shows that BrdU-labeled cells (red) were also observed on the surface of blood vessels (shown by N-cadherin staining in green). [0043] FIG. 7 shows that on Day 3 after 5FU treatment, BrdU-labeled cells

(pink) were mainly observed on the bone surface (shown by N-cadherin staining in green) as well as next to blood vessels (red).

[0044] FIG. 8A shows activated HSCs labeled with BrdU (red) 3 days after

5FU treatment. FIG. 8B shows cells that are positive for the hematopoietic stem cell marker c-kit (green). FIG. 8C shows the merge of FIG. 8A and FIG. 8B, in which the BrdU-labeled cells (red) are also positive for c-Kit staining (green), indicating that BrdU-labeled cells are indeed HSCs. The absence of BrdU in a few of the c-Kit positive cells, indicates that there are non-activated HSCs present. [0045] FIG. 9 shows that on Day 3 after 5FU treatment, BrdU-labeled cells

(red) are cell of the hematopoietic system since they are also positive for C045 (green), a hematopoietic system specific marker.

[0046] FIG. 10A shows that BrdU-labeled cells (red) cluster together on the bone surface (green), suggesting the occurrence of cell division in activated HSCs 5 days after 5FU treatment. FIG. 10B shows at a higher magnification, the clustering of BrdU-labeled cells (red).

[0047] FIG. 11A shows BrdU-labeled cells (red) in clusters on the bone surface (green) 6 days following 5FU treatment, suggesting activated HSCs proliferate to replenish lost cells following damage. FIG. 11 B shows a few clusters of BrdU-labeled cells (red) on the bone surface (green), indicating activated HSCs. FIG. 11C shows actively dividing BrdU-labeled cells (red) on sinusoid surfaces (green), which are blood vessels within bone marrow and the true niche of bone marrow stem cells. This suggests that stem cells residing in the niche can be activated to proliferate in response to injury.

[0048] FIG. 12A shows BrdU-labeled cells (red) at the base of intestinal crypts 1.5 days following 5FU treatment, indicating complete depletion of proliferative intestinal cells. FIG. 12B shows a decrease in BrdU-labeled cells (red) at the base of intestinal crypts 2 days after 5FU treatment, indicating activated stem cells. FIG. 12C shows a significant increase in BrdU-labeled cells (red) 3 days after 5FU treatment, indicating replenishment of lost cells by activated stem cells and progeny. FIG. 12D shows a sustained increase in BrdU- labeled cells (red) four days following 5FU treatment, indicating continued proliferation of activated stem cells and progeny. FIG. 12E shows a slight decrease in proliferating cells (red) five days after 5FLJ treatment, indicating the lost cells have been replenished.

[0049] FIG. 13A shows a few Ki67 positive cells (red) two days following

5FU treatment, indicating a few proliferating cells at the base of the intestinal crypts. FIG. 13B shows an increase in number of Ki67 positive cells (red) 51 hours after 5FU treatment, indicating an increase in proliferating cell number from 48 hours after 5FU treatment. FIG. 13C shows a further increase in Ki67 positive cell (red) number 57 hours after 5FU treatment compared to 48 hours after 5FU treatment. FIG. 13D shows a continued increase in number of Ki67 positive cells (red) at the bottom of intestinal crypts 3 days after 5FU treatment compared to 48 hours after treatment. FIG. 13E shows a significant increase in number of proliferating Ki67 positive cells (red) that have started to line the intestinal villi 4 days after 5FU treatment compared to 48 hours after treatment. FIG. 13F shows a profound increase in the number of proliferating Ki67 positive cells (red) five days following 5FU treatment compared to 48 hours after treatment. [0050] FIG. 14A shows a BrdU positive cell (red) along a pancreatic duct 3 days following 5FU treatment, indicating the location of an activated stem cell. An activated stem cell (red) was also observed within a pancreatic islet (FIGs. 14B and 14C). Four days following 5FU treatment, multiple BrdU positive cells (red) were observed within a pancreatic islet (FIG. 14D), indicating the progeny of the activated stem cells. Six days following 5FU treatment, multiple BrdU positive cells (red) were observed within both pancreatic duct and islets (FIG. 14E). [0051] FIG 15 shows bone marrow cells were analyzed by flow cytometry for the incorporation of BrdU. In a sample population, 16.6% of cells were proliferating cells in the normal sample (FIG. 15A) compared to 23.4% in the leukemia sample (Fig. 15B). Four days following 5FU treatment, FIG. 15C shows a decrease in the percentage of BrdU positive leukemia cells (1.32%) identified by flow cytometry. These cells are cancer stem cells remaining and activated after 5FU treatment. Fourteen days following 5FU treatment, the percentage of remaining activated cancer stem cells increased to 13.3% (FIG. 15D). [0052] FIG. 16 shows BrdU positive cells (red) in bone marrow 4 days following 5FU treatment. These cells are leukemia cancer stem cells activated by 5FU treatment.

[0053] FIG. 17 is a schematic that shows that the DAL method locates HSC activity and reveals dynamic HSC niche interactions.

[0054] FIG. 18 shows bone marrow RICs are enriched in LSK with long- term repopulation activity. FIGs. 18A-P demonstrate that the DAL method can pinpoint HSC locations and reveal interactions of HSCs with osteoblastic and vascular niches.

[0055] FIGs. 19A-I show that the regeneration initiating cells in response to bone marrow damage are enriched with HSCs.

[0056] FIGs. 20A-O show that the DAL method is able to identify regeneration initiating cells in response to injury in the pancreas. [0057] FIGs. 21A-F shows: that the DAL method is effective to identify pancreatic regeneration activity in the duct, islet and acinus. [0058] FIGs. 22A-L shows: DAL chases pancreatic RICs to undergo different lineage commitments and unveils ductal derivation of endocrine and exocrine stem/progenitor cells.

[0059] Figs. 23A-E show that the DAL method is effective to identify leukemia stem cells in a Pten deficient mouse model.

[0060] FIGs. 24A-D shows MCZEG mice plPC induction day 1.5.

[0061] FIG. 25 shows a 3-D whole mount analysis of a single GFP⁺ cell.

[0062] FIG. 26 shows a schematic illustrating the strategy of tracing a single labeled GFP⁺ cell.

[0063] FIG. 27 shows a lineage trace of a single GFP+ cell at day 28 after plPC induction.

[0064] FIG. 28 shows four types of cells that were labeled at day 28 after plpC induction.

[0065] FIGs. 29A-F show that labeled GFP cells dominantly proliferate in vitro.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention relates to methods and kits for identification of activated stem cells and the resultant progeny, as well as, to the identification and treatment of cells giving rise to tumors or cancers. The invention allows the identification of the location of stem cells when they are activated. Further, the invention allows the resultant progeny to be traced to determine the regenerative capacity of activated stem cells.

[0067] The present invention includes damaging all cycling cells to result in one or more quiescent stem cells becoming activated, which is followed by labeling the activated stem cells with a single exposure to a label. The progeny of the activated stem cells are then traced if desired. Beneficially, labeled stem cells are provided as well as methods for analyzing the regenerative property of stem cells.

A. Damaging Cycling Cells Triggers Quiescent Stem Cells to Become Activated

[0068] The present method starts by damaging cycling cells within a population of cells prior to labeling to ensure that the cells dividing during exposure to the label are activated stem cells and not any other type of cycling cell. Quiescent stem cells in a niche are resistant to pharmacological chemotherapy and become activated in response to injury. As such, any of a variety of compositions, including mechanisms and compositions causing either chemical or physical injury, can be used to damage cells and cause an injury response. For example, tumor therapeutic agents can be used to damage cycling cells and concomitantly elicit an injury response. The loss of numerous cells due to treatment with a tumor therapeutic agent induces an injury response resulting in feedback signals to the quiescent stem cells. These feedback signals entice only the quiescent stem cells to enter the cell cycle and become activated to replenish the lost cells. Therefore, the only cells cycling immediately after tumor therapeutic treatment will be the newly activated quiescent stem cells.

[0069] A range of chemotherapy drugs that target cycling cells for apoptosis

(cell death) may be used with the present invention. The five main chemotherapy drug categories include alkylating agents, nitrosureas, antitumor antibiotics, mitotic inhibitors, and antimetabolites. Alkylating agents attach alkyl groups to DNA bases that results in crosslinking of the DNA or DNA fragmentation, both of which prevent DNA synthesis and result in apoptosis of the cell. Nitrosureas interfere with enzymes needed for DNA repair resulting in apoptosis. Antitumor antibiotics bind to DNA and interfere with enzymes necessary for cell division resulting in apoptosis of the cell. Mitotic inhibitors stop mitosis or inhibit enzymes thus preventing cells from making proteins needed for cell growth. Antimetabolites incorporate into DNA or RNA and prevent correct processing resulting in apoptosis. Any of these may be used in practicing the invention. As shown in Example 1 and 2, the antimetabolite 5-fiorouracil (5FU) was used. Additionally, a number of well-known chemotherapy drugs are listed in Table 1 below. It is envisioned that any of the drugs listed in Table 1 may be used in the practice of the invention. Each of the agents listed are exemplary and not limiting. One skilled in the art will recognize that any agent that results in the targeted elimination of cycling cells may be used in the practice of the invention. A candidate agent will fall into one of the five main chemotherapy drug categories described above or exhibit cell cycle dependent toxicity resulting in apoptosis of only cycling cells. An appropriate agent will not target quiescent cells or cells not undergoing cell division. Table 1^s. Commonly used chemotherapeutic agents.

. Information was compiled from httpJ/www.paralleljourneys- cancer.com/poc/drugs.html and http://www.cancer.gov/drugdictionary January 2006.

[0070] Doses and modes of administration for each agent listed in Table 1 can be found in Remington: The Science and Practice of Pharmacy, pub. Lippincott Williams & Wilkins; 21st edition (2005) or at http://www.bccancer.bc.ca/HPI/DrugDatabase/DruglndexPro/default.htm, each of which, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0071] For example, 5FU was intravenously injected, about

250-300mg/10kg body weight, into C57/B6 mice to kill the cycling cells in the bone marrow, gut, pancreas, and other tissues. The tumor therapeutic agent 5FU was incorporated into the RNA of cycling cells to prevent RNA processing, thereby resulting in apoptosis. After exposure to 5FU, all cycling cells were eliminated (see FIG. 5). Since quiescent stem cells were not cycling, they were retained in the injured tissue.

[0072] Alternatively, other chemotherapeutic agents can be used to damage cycling cells (see Table 1). For example, Busulfan may be selected. The preferred dosage and administration route for Busulfan is about 3.2mg/kg body weight injected intravenously

(http://www.bccancer.bc.ca/HPI/DrugDatabase/DruglndexPro/default.htm). [0073] The methods of this invention are particularly useful in animals, especially mammals such as mice, rats, dogs, non-human primates, cattle, swine, and other animals. Also, the methods of this invention could be useful in humans. [0074] It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. Range finding studies may be conducted to determine appropriate dosage as described in Current Protocols in Pharmacology, Unit 10, pub. John Wiley & Sons, 2003 and incorporated herein by reference. The therapeutically effective amount for each active compound can vary with factors including, but not limited to, the activity of the compound used, stability of the active compound in the recipient's body, the total weight of the recipient treated, the route of administration, the ease of absorption, distribution, and excretion of the active compound by the recipient, the age and sensitivity of the recipient to be treated, the type of tissue, and the like, as will be apparent to a skilled artisan.

[0075] In general, there are two types of tissue, self-renewing and non-self renewing, which will affect when to administer an agent that will activate the desired cells. Self-renewing tissues are those tissues that continue to rapidly renew throughout most of the life of an animal and include the intestine, epidermis, and testes. All other tissues are considered non-self renewing tissues and do not have a vast number of cells within them cycling at any given time except during development. Since tumor therapeutic agents only target cycling cells, analyzing activated stem cells and their progeny in non-self renewing tissues may be optimized during the development stages. Otherwise, there may not be a sufficient number of cycling cells to be targeted by the tumor therapeutic agent in order to elicit activation of quiescent stem cells. Non-self renewing tissues develop during gestation and continue developing after birth. This development period is dependent on the organism the invention is being practiced with. For example, in non-self renewing tissues of the mouse, the method should be practiced within in the first 3 weeks after birth when the tissues are still developing. Thus, if the invention is being used to study cell and tissue regenerative pathways, application during the development phase is likely necessary. But, if self-renewing tissues, precancerous, cancerous, or tumorigenic tissues are being treated, then the optimal period for treatment with a mitotic killing agent is not limited and the method can be practiced regardless of the age of the organism.

[0076] Alternatively, damage that results in activating quiescent stem cells may result from other types of chemical treatments for other disease states, from the disease state itself (e.g. Acid reflux disease, Barret's esophagus), or even physical injury. In each case, the damage to a population of cells occurs and results in the activation of previously quiescent stem cells. In some cases, the type of damage may result in an increased risk of cancer due to activation of a cancer stem cell. For example, it is widely presumed in the art that Barrett's esophagus increases the risk of throat cancer. B. Labeling Stem Cells

[0077] When an injury causes a stem cell to become activated and enter the cell cycle, less label exposure is required, compared to a quiescent cell, for a label to be incorporated into the activated stem cell and to allow its progeny to be identified and traced. While standard conditions used in a LRC assay require repeated administration of a cell-marking label for a prolonged period of time, the present invention requires only a single administration of a cell-marking label for a short period of time. Once the stem cells are activated, only a single pulse of a cell-marking label labels the activated stem cells. As such, a single, properly timed pulse of cell-marking label results in labeling activated stem cells. Further, as the activated labeled stem cells give rise to progeny cells, the progeny cells also become labeled and can be traced by their retention of some marking label shared by the activated stem cell.

[0078] Those of skill in the art will appreciate the advantage provided by the present invention of requiring only a single short-term labeling event. Present methods in the art require either prolonged labeling or multiple labeling events. In both instances, the utility of the methods are limited compared to the present invention due to toxicity, stress, imprecise timing, inability to accurately and precisely trace cell lineage, or a combination thereof. These limitations are resolved by the present invention.

[0079] The timing of administering the label following treatment with an activation agent is important in the present invention and precise timing is needed for optimal effect. In most tissues, administration of the label can follow therapeutic treatment by approximately 1 , 2, 3, or 4 days, or even as many as 5, 6, or 7 days. The optimal time for administration of the label is about 3 days following 5FU treatment, which is when stem cells become activated. Nevertheless, those of skill in the art will appreciate that the time for labeling both activated stem cells and progeny will likely need to be adjusted for each tissue and individual to reach optimal labeling.

[0080] For example, by using the proliferation marker Ki67, the proliferation profile following 5FU treatment can be determined. Proliferation should be monitored each day following 5FU treatment to identify the time it takes for proliferation to begin or lag in proliferation. Once the lag in proliferation is identified, BrdU can be administered at this time point to label activated stem cells. At the time proliferation increases, BrdU can be administered to label progeny.

[0081] Only cycling cells are able to incorporate a cell-lineage marker.

Damaging the cycling cells by treatment with a tumor therapeutic or other agent provides that the activated stem cells are the only cells able to incorporate the cell-lineage marker immediately following the treatment. After a period of time, all cycling cells will effectively be progeny of activated stem cells. The resultant progeny can be traced using a label shared by stem cells with progeny cells or by administering the cell-marking label such that all cycling cells can be identified, of which all would be progeny of the activated stem cells. Cycling cells can be labeled by administering a cell-marking label 1 , 2, or 3 hours prior to tissue sampling.

[0082] Furthermore, a combination of cell-marking label can be used to identify both the activated stem cells and the progeny of the activated stem cells. For example, a cell-marking label can be administered during the lag in proliferation after tumor therapeutic treatment to label the activated stem cells. A second cell-marking label, other than the one used to label activated stem cells, can be administered 1, 2, or 3 hours prior to tissue sampling to label cycling progeny of the activated stem cells.

[0083] It will be appreciated by those of skill in the art, that while only a single labeling event is required to practice the invention, second, third or even more labeling events using different labels are also encompassed by the invention. For example, a first labeling event would label the activated previously quiescent stem cell. A second labeling event would label the same stem cell and its first generation progeny. A third labeling event would label the originally labeled stem cell, the first generation progeny, and the second generation. The number of labeling events, as well as the number and type of different labels used, would be determined by the artisan's purpose and desire to trace the ancestry of a population of cells. C. Administering Label

[0084] A cell-marking label is a label capable of incorporating into cycling or activated cells to permit their identification. Cell-marking labels may be incorporated into DNA or other components of cells that do not turn over in the absence of cell division. For example, cell-marking labels include labeled deoxyribonucleotides, halogenated deoxyribonucleotides, radio-labeled nucleotides, deuterium labeled nucleotides, or deuterium labeled DNA synthesis precursors known in the art as described, for example, in US patent application 20030224420, filed April 4, 2003 and incorporated herein by reference to the extent that it provides exemplary procedures or other details supplementary to those set forth herein. Commonly used cell-marking labels include tritiated thymidine, bromodeoxyuridine (BrdU) and iododeoxyuridine (IdU), which are incoφorated into DNA during cell division.

[0085] The cell-marking label may be a labeled deoxyribonucleotide (dN).

Labeled deoxyribonucleotides include any labeled deoxyribonucleotides known in the art (Huijzer, J. C. and Smerdon, M. J. Biochemistry 31(21 ): 5077-5084, June 2, 1992). The deoxyribonucleotides include any known nucleic acids, including deoxythymidine (dT), deoxyadenosine (dA), deoxycytosine (dC), deoxyguanosine (dG), and deoxyuridine (dU). [0086] Cell-marking labels may also be halogenated deoxyribonucleotides.

Halogenated deoxyribonucleotides may include any halogenated deoxyribonucleotide, including, but not limited to dT, dA, dC, dG, and dU (Li, X. and Darzynkiewicz, Z. Cell Prolif. 28(11): 571-579, November, 1995). Specific examples of halogenated cell-marking labels are halogenated deoxyribonucleotides such as bromodeoxyuridine (BrdU), iododeoxyuridine (IdU), and bromodeoxycytidine (BrdC).

[0087] The cell-marking label may be a radiolabeled nucleotide. The radiolabei may be any radioisotope known in the art (Hume, W. J. and Potten, C. S. Cell Tissue Kinet. 75(1): 49-58, January 15, 1982). These include halogen radioisotopes, such as Br∞-BrdC, Br∞-BrdU, Br∞-BrdA, Br∞-BrdT, and Br∞-BrdG. Other radiolabeled nucleotides include tritiated nucleotides, such as ³H-dC, ³H- dG, ³H-dA, ³H-dT, and ³H-dU.

[0088] Cell-marking labels may also be deuterium labels. Specific examples include deuterium labeled DNA synthesis precursors such as glucose, and deuterium labeled nucleotides such as ²H-dT, ²H-dA, ²H-dG, ²H-dC, and ²H- dU (Macallan, D. C. et al. Blood 105(9): 3633-3640, epub. January 11, 2005). [0089] Cell-marking labels suitable for use in vivo are prepared in accordance with conventional methods in the art using a physiologically and clinically acceptable solution as described in Current Protocols in Pharmacology, Chapter 7.3 Supplement 15, pub. John Wiley & Sons, Inc., 2001 and incorporated herein by reference. Proper solution is dependent upon the route of administration chosen. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transcutaneous, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections as described in Current Protocols in Pharmacology, Chapter 7.3 Supplement 15, pub. John Wiley & Sons, Inc., 2001 and incorporated herein by reference. For example, mice can be intravenously injected with BrdU approximately 1 to 3 hours prior to tissue collection in order to label cycling cells. D. Identification Of Labeled Cells

[0090] Identification of incorporated labels may be achieved using monoclonal or polyclonal antibodies that specifically identify the marking labels. An antibody is an immunoglobulin molecule capable of specific binding to a target, such as a label, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact antibodies, but also fragments thereof (such as Fab, Fab", f(ab'), Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, fully or partially humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

[0091] For example, identification of BrdU, IdLJ, or other cellular markers may be conducted using anti-BrdU and anti-ldU monoclonal antibodies. The labeling of activated or cycling cells with BrdU or IdU and the subsequent detection of incorporated BrdU or IdU with specific anti-BrdU or anti-ldU monoclonal antibodies, respectively, may be accomplished by immunodetection methods. The steps of various useful immunodetection methods have been described in Current Protocols in Molecular Biology, Unit 14, pub. John Wiley & Sons, Inc., 2004 and incorporated herein by reference. The immunobinding methods include methods for detecting or quantifying the amount of cell-marking label in a sample, which methods require the detection or quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing the cell-marking label and contact the sample with an antibody specific for the cell-marking label and then detect or quantify the amount of immune complexes formed.

[0092] The biological sample analyzed may be any sample that is suspected of containing the cell-marking label. The samples may be a tissue section or specimen, a biopsy, a swab or smear test sample, a group of cells, a homogenized tissue extract or separated or purified forms of such. [0093] Contacting the chosen biological sample with the cell-marking label specific antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of adding an antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibodies to form immune complexes with, i.e., to bind to, any cell-marking label present. After this time, the sample-antibody composition will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. [0094] The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels known in the art as described in Current Protocols in Molecular Biology, Unit 14, pub. John Wiley & Sons, Inc., 2004 and incorporated herein by reference. The use of enzymes that generate a colored product upon contact with a chromogenic substrate are generally used. A secondary binding ligand, such as a second antibody or a biotin/avidin ligand binding arrangement, may also be used, as is known in the art. [0095] The identification of radiolabels may be conducted using autoradiographic histology, a procedure well known by those skilled in the art (see Current Protocols in Molecular Biology, Unit 14, pub. John Wiley & Sons, Inc., 2004, incorporated by reference). Once a population of cells is labeled, they are removed and fixed for standard histological examination using fixatives such as formalin or paraformaldehyde and embedded in paraffin. When the population of cells are sectioned and applied to a glass slide they will contain radioactive nuclei, but only those nuclei that were in S-phase during exposure to the radiolabel. Since radioactive sources cannot be detected directly, a photographic emulsion is applied directly over the section that becomes exposed by the radioactive sources. The photographic emulsion is developed using a developer and the exposed portions of the emulsion will contain reduced silver grains in direct proportion to the amount of radiation in the nuclei of the sample. Developed slides can be examined with a microscope to detect radiolabeled nuclei. The number of silver grains can be counted to give a quantitative measure of radiolabel incorporation.

E. Identification of Stem Cell Specific Cell-Surface Markers [0096] In addition to the above-described use of the present invention, the method can also be utilized to identify stem cell specific cell-surface markers and the presence of a cancer stem cell within a population of cells. It is envisioned that once an activated stem cell is labeled, it can be isolated from a population of cells for other applications and uses. Activated stem cells can be isolated from a population of cells by using fluorescence-activated cell sorting (FACS), flow cytometry, or other cell separation techniques compatible with intracellular markers known in the art. A skilled artisan will recognize that isolation via intracellular markers is not compatible with living cells, therefore an external marker, such as a cell-surface marker, is desirable for isolation of live cells from a population of cells.

[0097] Once isolated, cell-surface markers specific to the activated stem cell type can be identified using DNA-microarray technology as described in Current Protocols in Molecular Biology, Unit 22, pub. John Wiley & Sons, Inc., 2000 and incorporated herein by reference. RNA extracted from activated stem cells and non-stem cells of the same population of cells or tissue type is transcribed into labeled-cDNA and hybridized to microarrays. It is envisioned that the microarrays will consist of cell-surface marker probes or a set of probes representing a substantial amount of an organism's genome, respective to the organism from which the cells were isolated. The difference in expression of cell- surface markers between non-stem cells and the activated stem cells can be determined by comparing the respective expression profiles. Analysis of microarray expression profile data is described in Current Protocols in Bioinfoπnatics, Unit 7, pub. John Wiley & Sons, Inc., 2003 and incorporated herein by reference.

[0098] It is envisioned that newly identified stem cell surface markers can be used for a variety of applications including isolating live stem cells for subsequent culturing, further characterizing stem cell location within a population of cells, and isolating live stem cells for therapeutic treatments. Living stem cells can be isolated using an antibody or combination of antibodies that recognize specific cell-surface markers present on the surface of a stem cell in conjunction with a cell sorting technique known in the art such as, for example, flow cytometry, FACS, or magnetic cell sorting. It is envisioned that once stem cells are isolated from a population of cells, they can be cultured by methods known in the art. A skilled artisan will recognize that culturing conditions will depend on the tissue from which the cells were derived and may include the addition of growth factors, special substrates (poly-L-lysine, collagen, fibronectin, feeder layer cells, etc.), differentiation inhibitors, or proliferation activators. It is also envisioned that stem cells can be used to treat diseases caused by genetic mutations. Stem cells can be isolated from a host using the cell-surface markers, manipulated in culture to produce a specific phenotype, and replaced into the host correcting deleterious genetic mutations. Antibodies specific for the cell-surface markers identified can also be used to locate stem cells within a population of cells through immunodetection methods as described in described in Current Protocols in Molecular Biology, Unit 14, pub. John Wiley & Sons, Inc., 2004 and incorporated herein by reference.

F. Identification of Cancer Stem Cell Specific Cell-Surface Markers [0099] The method can be practiced to identify activated cancer stem cells by administering a mitotic killing agent to a pre-cancerous, cancerous, or tumorigenic population of cells, labeling the activated cancer stem cells, and identifying the labeled cells. It is envisioned that once activated cancer stem cells are labeled they can be isolated from a population of cells by using fluorescence- activated cell sorting (FACS), flow cytometry, or other cell separation techniques compatible with intracellular markers known in the art. [0100] Once isolated, cell-surface markers specific to the activated cancer stem cell type can be identified using DNA-microarray technology as described in Current Protocols in Molecular Biology, Unit 22, pub. John Wiley & Sons, Inc., 2000 and incorporated herein by reference. RNA extracted from activated cancer stem cells and non-cancer stem cells or non-stem cells of the same population of cells or tissue type is transcribed into labeled-cDNA and hybridized to microarrays. It is envisioned that the microarrays will consist of cell-surface marker probes or a set of probes representing a substantial amount of an organism's genome, respective to the organism from which the cells were isolated. The difference in expression of cell-surface markers between non-stem cells and the activated cancer stem cells can be determined by comparing the respective expression profiles. Analysis of microarray expression profile data is described in Current Protocols in Bioinformatics, Unit 7, pub. John Wiley & Sons, Inc., 2003 and incorporated herein by reference.

[0101] It is envisioned that newly identified cancer stem cell surface markers can be used for the applications described above as well as for targeting cancer therapies to the cancer stem cell and optimizing cancer therapy treatment regimens. Cell-surface markers found specifically on cancer stem cells may provide a solution for distinguishing between normal stem cells and cancer stem cells. The ability to distinguish between the two cell types can provide a foundation for targeting cancer therapeutics to the cell responsible for cancer development and recurrence. Such therapeutics include tumor-directed monoclonal antibody immunotherapies, chemical therapies, irradiation modalities, and other therapies that could be targeted using cell-surface markers. [0102] Cell-surface markers can be used to optimize cancer treatment regimens through in vitro experimentation. It is envisioned that a pre-cancerous, cancerous, or tumorigenic population of cells sampled from a patient can be cultured to mimic the in vivo cancer. Such a population of cells can be treated with a mitotic killing agent followed by subsequent labeling with intracellular markers (i.e. BrdU) or immunodetection of cancer stem cell specific cell-surface markers to determine the time point at which cancer stem cells become activated. The time at which the cancer stem cells become activated in vitro, will likely be the most beneficial time at which, following an initial cancer therapeutic treatment, a second treatment should be administered to target the cancer stem cell and any progeny for elimination. G. Therapeutic Kits

[0103] The present invention provides utility kits with reagents for use with the above described methods. Accordingly, a tumor therapeutic agent, a cell- marking label, a label-detecting agent, an appropriate visualizing agent, and optionally a protocol describing use are provided in the kit, generally comprised within a suitable container.

[0104] The preferred tumor therapeutic agent (mitotic killing agent) would have properties consistent with targeting cycling cells and inducing apoptosis, such as 5FU or any of the agents listed in Table 1 or derivatives thereof. Other tumor therapeutic agents with similar properties to alkylating agents, nitrosureas, antitumor antibiotics, mitotic inhibitors or antimetabolites and not listed in Table 1 may also be used. Alkylating agents attach alkyl groups to DNA bases and result in crosslinking of the DNA or DNA fragmentation, both of which prevent DNA synthesis and result in apoptosis of the cell. Nitrosureas interfere with enzymes needed for DNA repair resulting in apoptosis. Antitumor antibiotics bind to DNA and interfere with enzymes necessary for cell division resulting in apoptosis of the cell. Mitotic inhibitors stop mitosis or inhibit enzymes thus preventing cells from making proteins needed for cell growth. Antimetabolites incorporate into DNA or RNA and prevent correct processing resulting in apoptosis. One skilled in the art will recognize that any agent that results in the targeted elimination of cycling cells may be used. A candidate agent will exhibit cell cycle dependent toxicity resulting in apoptosis of only cycling cells. However, an appropriate agent would not target elimination of quiescent cells or cells not undergoing cell division. The provided tumor therapeutic agent may be supplied as a solution or powder for recoπstitution.

[0105] An exemplary cell-marking label is a halogenated deoxyribonucleotide, such as BrdU. Other cell-marking labels described above or those not currently recognized that incorporate into DNA of cycling cells may also be used. The cell-marking label may be supplied as a solution or powder for reconstitution.

[0106] A preferred label-detecting agent is one that recognizes exclusively the cell-marking label, such as an antibody specific for BrdU. Other label- detecting agents described above or those not currently recognized that exclusively recognize the cell-marking label may also be used. The provided label-detecting agent may be supplied as a solution or powder for reconstitution. [0107] A visualizing agent is one that recognizes exclusively the label- detecting agent and allows visual detection. The visualization agent may take any one of a variety of forms, including detectable labels that are associated with or linked to the label-detecting agent. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody specific for the cell-marking label.

[0108] Further suitable visualization agents for use in the present kits include a two-component reagent that comprises a secondary antibody that has binding affinity for a first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. These kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. However, the label and attachment means could be separately supplied. Exemplary labels include, but are not limited to, radiolabels, fluorescent tags, enzymatic tags, and fluorogenic or chromogenic substrate tags that allow visualization.

[0109] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other vessel, into which the described reagents may be placed, and preferably, suitably aliquoted. Where a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed.

[0110] The kits of the present invention will also typically include a means for containing the reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

H. The DAL Method Pinpointed HSC Locations and Revealed Dynamic Interactions of HSCs with Osteoblastic and Vascular Niches [0111] More particularly, to establish a methodology for identification of stem cells in diverse systems we took advantage of two widely used techniques: applying chemotherapeutic agent 5-Fluorouracil (5FU), which targets cycling cells but not cells in quiescent state and (Lemer and Harrison, 1990; Longley et al., 2003), to spare and subsequently activate primitive stem cells by eliminating proliferating cells (Harrison and Lerner, 1991), and to incorporate Bromodeoxyuridine (BrdU) into the DNA of the activated cells that initiate the process of regeneration (defined as regeneration initiating cells, or RICs). This meanwhile also allows tracing of the lineage potential of the BrdU-labeled RICs. We have named this approach the "Destroying proliferating cells, Activating and Labeling stem cells (DAL)" method (Figure 17). The method includes the following three steps: 1 ) enforced activation (transition from G₀ into cell cycle) of stem cells by chemically eliminating proliferating cells; 2) labeling the activated stem cells with, e.g., BrdU in an appropriate time window; and 3) further tracing the BrdU- labeled cells to determine if they can give rise to downstream lineages in that given tissue.

[0112] We first tested this approach in the hematopoietic system, in which the stem cell identity and location have been well-defined. Our strategy was to inject 5FU intravenously (IV) and examine the proliferating cells in bone marrow using a 3-hour BrdU-pulse labeling assay, thus looking for a time frame within which the proliferating cells would have been largely destroyed and the stem cells that survived from this injury are activated and ready to be incorporated with BrdU. Proliferating cells can be readily detected in normal bone marrow (Figure 18A). As shown in Figure 18B and N, IV injection of 5FLJ effectively eliminated proliferating cells on day 2 (D2 hereafter) post 5FU treatment (P5FT) as demonstrated by the absence of BrdU-positive cells throughout the major area of the bone marrow sections. We monitored the regeneration process in response to the bone marrow damage induced by 5FU treatment by focusing on proliferation initiation sites. This was carried out by a BrdU-chasing assay. BrdU was injected into a group of mice 3 hours (hr) prior to D3 P5FT. The mice were then analyzed in pairs at each time point following BrdU labeling: D3, D3+12hr, D3+24hr, and D3+36hr (Figure 18N). Surprisingly, sporadically distributed single BrdU⁺ cells were detected on D3 P5FT, indicating that the putative stem cells that had survived 5FU insult were activated for regeneration in response to the injury (Figure 18C, E). At the beginning of this regeneration, single BrdU-incorporated cells were sporadic and concentrated mainly in the trabecular bone area and at a relatively lower number in the long bone region (not shown). Notably, in the trabecular bone area these RICs were overwhelmingly located on or close to the bone surface (72±4%) or in proximity to blood vessels (26±4%) (Figures 18C, E). The single BrdU⁺ cells were found in locations consistent with the notion that HSCs reside in the osteoblastic or the vascular niche and this was confirmed by their co-localization with either N- cadherin^* osteoblastic cells (Figure 18D) (Zhang et al., 2003) or CD31⁺ endothelial cells (Figure 18F) (Kiel et al., 2005). It is notable that the asymmetric distribution pattern of N-cadherin (arrow) on two distal sides of the dividing cells is consistent with the role of the adherent junction (composed of cadherin plus β- catenin) in determination of the orientation during stem cell division (Yamashita et al., 2003). Strikingly, on D3+12hr multiple pairs of BrdU-labeled cells were found frequently lined up proximally to the bone surface (Figure 18G) or blood vessel surface (Figure 18H), indicating that the RICs at both niches indeed undergo replication during initiation of regeneration. The orientation of the dividing cells parallel to the interface between the replicating cells and the bone or blood vessel surface suggested that the regenerating cells were likely undergoing symmetric division (Figure 18G and H). Starting from D4 P5FT and onward, clusters of BrdlT cells were found to gradually move away from the bone surface and migrate to the blood vessel surface (Figures 181-K), indicating their continuing mobilization and proliferation. Figure 18L and M, respectively, show 3hr BrdU-pulse labeling of cells on D3 and D6 P5FT, confirming that proliferating cells were mainly clustered onto the blood vessel surface at these time points. To further confirm that the regenerating cells were mobilized from the bone surface to the blood vessel surface in response to 5FU treatment we compared the number of BrdlT cells on the bone surface to the number of BrdLT cells in the marrow during the time window from D3 to D6 P5FT. The results showed that there were about two times more BrdU^* cells on the trabecular bone surface than in the marrow on D3 P5FT, but the ratio gradually decreased on D3+36hr and onward (Figure 18O), supporting the finding that the putative stem cells were first activated in response to bone marrow damage (as indicated by ready incorporation of BrdU⁺), followed by mobilization and proliferation from the osteoblastic niche to the vascular niche (Figure 18P). The cluster of proliferating cells with the heterogeneous moφhologies also reflects ongoing lineage commitment concomitant with their expansion (Figure 18L). Meanwhile, there were also BrdlT cells egressing from bone marrow into the peripheral blood, indicating some HSCs located on the vascular surface migrated into circulation (not shown). This is consistent with a previously proposed model (Heissig et al., 2002).

[0113] We would like to point out that there were basically two types of single BrdlT cells detected on D3 P5FT judged by their morphologies: round/oval shaped cells (majority) and spindle shaped cells (minority). Only the former showed the phenomena of mobilization as described above. We would also like to point out that some BrdU+ cells may be candidates for mesenchymal stem cells or other non-hematopoietic progenitor cells.

/. The Regeneration Initiating Cells in Response to Bone Marrow Damage Were Enriched with Hematopoietic Stem Cells

[0114] To confirm that the RICs in response to bone marrow damage were enriched hematopoietic stem cells (HSCs), we first performed immunofluorescent assays. The results showed that a large portion (round/oval-shaped) of the BrdlT cells detected on D3 P5FT located on the bone surface or in proximity to blood vessels were enriched with HSCs as revealed by their co-staining with hematopoietic stem cell markers Sca-1 , c-Kit, CD45, and CD201 (Figure 19A-D). Co-staining with CD201 is of significance as CD201⁺ cells were shown to be overlapped with the Lineage Sca-1⁺c-Kit⁺ (LSK) cell population, and also enriched in the SP cell population (Balazs et al., 2006). This raises the possibility that CD201 could be used to isolate the regeneration initiating cells, recognized as BrdlT and round/oval-shaped cells on D3 P5FT. Indeed, the immunofluorescent staining results showed that a significant portion, but not all, of the BrdlT oval- shaped cells were still CD201⁺ on D4 P5FT (Figure 19G). Flow cytometry analysis showed that only fewer than 50% of the LSK cells were CD201⁺ without 5FU treatment (Figure 19E), whereas more than 90% of the LSK cells were CD201^* on D3 P5FT (Figure 19F). Furthermore, approximately 30% (not shown) or 65% (Figure 19H) of the LSK cells analyzed on D3 P5FT were positive for BrdlT if cells were labeled by single (3 hours pulse) or 3 times (20, 12, and 3 hours) Brdu injection respectively prior to harvesting the bone marrow. Thus, the BrdlT RICs were enriched in CD201⁺ LSK cell population.

[0115] To characterize the functionality of the RICs located at the putative

HSC niches, we isolated CD201⁺LSK cells using fluorescence-activated cell sorting (FACS) (Figure 19E and F) from C57/B6 mice (expressing CD45.2 as a donor cell marker) on D3 P5FT and transplanted a single CD201*LSK cell into each of the lethally irradiated Ptprc recipient mouse (expressing CD45.1 as a recipient cell marker) (Figure 191). Repopulation assay showed that 6 out 7 transplanted mice receiving a single CD201⁺LSK cell had engrafted (the CD45.2⁺ population). These engrafted donor cells reconstituted long term (> 3 months) myeloid and lymphoid lineages (Figure 181). Taken together, both immunophenotypical analysis and functional repopulation assays demonstrated that a significant portion of BrdU⁺ RICs induced by bone marrow damage were enriched with HSCs.

J. Identification of Regeneration Initiating Cells in Response to Injury in Pancreas

[0116] After validating the DAL method was able to identify stem cells and their locations in the well-established and fast turnover hematopoietic system, we next asked whether the DAL approach could also be applied to a slow turnover tissue such as, for example, the adult pancreas. Because the slow turn over system in adults may not respond well to 5FU treatment, we chose 3-week-old mice, an age at which the pancreatic tissue is still developing and therefore would be sensitive to 5FU treatment. Similar to the hematopoietic system, no proliferating cells were detected on D2 P5FT, indicating effective elimination of the proliferating cells (Figure 20A). The pancreatic RICs, revealed as single BrdU⁺ cells, appeared on D3 P5FT and were found in different regions including the islet, acinus, and duct (Figure 2OB, C, D). To distinguish these RICs according to their location, we named them as ductal (D-) RICs, islet (I-) RlCs, and acinar (A-) RICs, respectively. Consistent with previous reports, D-RICs were found in both large and small ducts (Figures 2OD, G, J); However, early ductal regeneration was more frequently seen in small ducts, especially near islets. In islet, most l-RICs were detected in the peripheral area (Figure 20B), and were very often, but not always, found to be next to ductal or duct-derived cells recognized by CK19 staining (Bouwens et al., 1994) (Figure 20E). The same was true for the A-RICs which were often found in the centroacinar cell position but were also found next to ductal structures derived from ductal cells as evident by Dolichos Biflorus Agglutinin (DBA) detection (Kobayashi et al., 2002) (Figure 20F). These observations suggest a possible hierarchical relationship among the three types of RICs. As both l-RICs and A-RICs were either adjacent to or even derived from ductal cells, they were most possibly derived from D-RICs.

[0117] We noticed that the BrdU incorporated RICs were sometimes found to reside in a pocket-like niche structure in duct, islet, and acinus, implicating that the structure formed by columnar cells in duct (blue arrow, Figure 20G) or stromal- like cells in the centroacinar position (Figure 4I) were the putative niche cells (Figure 20G-I). The identity of these supporting cells was not clear, but they were most likely composed of duct-derived cells (arrows in Figure 20D.E.F) and other non-duct derived cells (yellow arrow in Figure 20G). On D3+12hr P5FT multiple paired Brdll+ cells were seen in the ductal, islet, and centroacinar regions respectively, indicating these RICs as putative stem/progenitor cells were indeed initiating regeneration in response to injury (Figure 2OJ, K, L). [0118] Using BrdU-pulse labeling on subsequent days P5FT to monitor ongoing regeneration, most active proliferation occurred in the smaller ducts especially near the islet region (Figure 2OM, O). In the islet, proliferating cells were more dispersed than clustered, indicating that cells replicated from the original I- RICs tended to migrate out from the initial regeneration center. This is consistent with observations during development (Figure 20N). In the acinar region, a small group of dividing cells formed a cluster surrounding the centroacinar position, implicating they were generated from A-RICs (Figure 20O) and resembling exocrine "clonal" formation during embryonic development. These proliferating cells were very often found next to small ducts, further supporting the idea that the l-RICs and A-RICs that seed transit amplifying cells are derived from the ducts (Figures 20F.H).

K. The BrdU-incorporated RICs Give Rise to Downstream Endocrine or Exocrine Lineages in vivo

[0119] To determine whether the BrdU-incorporated RICs are pancreatic stem/progenitor cells, we used BrdU-chasing assays following the DAL procedure. The results indicated that D-RICs located in ducts may initiate de novo formation of the islet or acinar structures by giving rise to subsequent I- or A-RICs located in the peripheral region of islet or in the centroacinar region, which respectively produce insulin/glucagon-expressing cells in islet or amylase-expressing cells in the acinar region. [0120] As Figure 21A shows, within the active regenerating ducts, we detected some BrdU^* cells (D-RICs) expressing the /S-cell lineage marker insulin (white arrow Figure 21A). A typical BrdU* and insulin-expression D-RIC appears to be initiating budding (Figure 21A). This observation suggests that lnsulin*:BrdU* D-RICs can initiate de novo islet formation by giving rise to committed l-RICs, or l-progenitor cells for endocrine cell lineages. Indeed, in the newly formed islet, pairs of newly divided l-RICs could undergo asymmetric division resulting in two daughter cells with different fates: one expressed pancreatic stem/progenitor cell marker Pdx1 , the other did not (Figure 21 B) or, in other cases, one expressed insulin but the other did not (Figure 21C). We also detected that some l-RICs were able to co-express downstream lineage markers, insulin and glucagons, with polarized distribution patterns of these two markers, indicating their bipotentiality (Figure 21D), a phenomenon often found during pancreatic organogenesis. Furthermore, these insulin⁺:glucagon⁺ bipotential I- RICs subsequently gave rise to β-ce\\ lineages (insulin*, white arrow in Figure 21 D) and /?-cell lineages (glucagon^*, green arrow Figure 21D). [0121] As Figure 21 E shows, in the centroacinar region, we found that the

A-RICs that express amylase (white arrow, Figure 21E) were often adjacent to BrdlT-amylase^" cells (green arrows, Figure 21 E) that were part of a small duct extension (green arrow. Figure 21F). This observation further supports the idea that A-RICs are derived from D-RICs. In the acinar region, the A-RICs at the centroacinar position (green arrow, Figure 21 F) were seen to undergo clonal expansion, giving rise to a small group of cells committed to amylase^* exocrine fate (white arrows, Figures 21 F). [0122] Taken together, using the combined approaches of DAL and BrdU chasing we have shown that D-RICs (pancreatic stem cells) located in ducts initiate new islet or acinar structures in response to 5FU induced pancreatic damage by giving rise to l-RICs (or l-progenitors) in islet and A-RICs (or A- progenitors) in acinus respectively, which in turn generated both σ-cell and /?-cell lineages in islet, and exocrine cells in acinus. Thus pancreatic stem/progenitor cells and their locations were revealed by the DAL plus BrdU-chasing method in duct, islet, and acinus.

L. Identification of leukemia (or cancer) stem cells

[0123] We and others have reported recently that HSCs with PTEN deficiency undergo uncontrolled proliferation resulting in myeloid proliferative disorder (MPD) and bone marrow cells bearing a PTEN mutation develop acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) in wild type (Wt) recipient animals upon transplantation (Yilmaz et al., 2006; Zhang et al., 2006). Intriguingly, it is difficult to determine which population of cells was enriched with leukemia initiating cells (LICs) in the PTEN mutant animals because the sorted HSCs, myeloid progenitor cells (Mac-1+Gr1+), and T lymphoid progenitor cells were all able to form leukemia in Wt recipient animals (Yilmaz et al., 2006). We thus wished to test whether the DAL method could effectively identify LICs in the PTEN leukemia model.

[0124] As we reported previously, the leukemia mouse model was generated by transplanting PTEN-deficient bone marrow into Wt recipient mice. These animals first showed signs of MPD, which then transformed into AML or ALL in a time window of weeks to months (Yilmaz et al., 2006; Zhang et al., 2006). We applied 5FU to a group of mice one month after they received PTEN-deficient BM, at a time when they showed signs of leukemia as revealed by an abnormally high number of myeloid cells in peripheral blood when compared to the Wt bone marrow transplanted control group (Zhang et al., 2006). This was also evident by dramatically increased proliferating cells (detected by BrdU-pulse labeling) in the leukemic bone marrow measured by both flow cytometry and immunohistochemical assays. (Figures 22A-B, 22E-F). After injecting 5-FU, BrdU incorporating cells were markedly (100 X) reduced (Figure 22C). We found that it took one day longer to completely eliminate the proliferating cells in PTEN leukemia animals than in the control group. By D4 P5FT, single BrdU labeled cells were detected at much lower numbers (Figure 22C.G) and were sporadically distributed in marrow (Figure 22G). When we measured the proliferating cells again two weeks P5FT, it seemed that the remaining single BrdU labeled cells had survived the injury, activated, and redeveloped the leukemia phenotype as revealed by rapidly increased number of proliferating cells throughout the bone marrow, and at a much higher than normal number compared to the control animals (compare, Figure 22D with A, 22H with E).

[0125] To verify whether the sporadically distributed BrdU^* cells detected on day 4 P5FT were surviving (5FU-resistant) LICs, we performed a transplantation experiment. We first confirmed the costaining of the single BrdU⁺ cells with CD201 (not shown). We then sorted out the CD201⁺LSK population from the PTEN-leukemia animals on day 4 P5FT and transplanted 100 cells per mouse into lethally irradiated normal recipient mice; 60 times more (6000) CD201^" Lin^* cells were transplanted into the control group (Figure 22I). Three months later, peripheral blood was collected and the cells were subjected to flow cytometry analysis. The results showed that 4 out 6 recipient mice receiving CD201⁺ϋn^" cells showed dominant myeloid progenitor cells, a sign of AML

(Figures 22K.L) , whereas 6 out of 6 mice receiving CD201 Lin⁺ cells had basically

no sign of leukemia (Figure 22J.L). Thus, we functionally demonstrated that the 5FU-resistant BrdlT cells in the PTEN leukemia animals detected by the DAL method on day 4 P5FT were LICs, which were able to redevelop leukemia in primary and secondary transplanted animals.

DEFINITIONS

[0126] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton ef a/., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger ef a/, (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0127] A quiescent or dormant cell becomes "activated" when it is triggered to enter into the cell cycle. The term "activated" refers to any cell triggered to enter a state of reproduction or doubling and can include a cell entering the cell cycle, cell division, or mitosis. An activated cell can be a pluripotent cell, a totipotent cell, a unipotent cell, a cancer stem cell, a stem cell, or a progeny of a stem cell.

[0128] An "active" cell is a cell undergoing cell division and can be at any point in the cell cycle. An "active" cell also includes "activated" or "cycling" cells. [0129] The term "cycling" refers to any cell that is in a state of reproduction or doubling. Such a cell includes a cell in the cell cycle, cell division, or mitosis and a cell that is active, dividing, or proliferating. A cycling cell can be a stem cell, a cancer stem cell, a pluripotent cell, a totipotent cell, a unipotent cell, a non-stem cell, a precursor.cell, a progenitor cell, a differentiated cell, or a progeny of a stem cell or a cancer stem cell.

[0130] A "stem cell" refers to any cell capable of giving rise to all cell types within a given tissue with a long lifespan and capacity for self-renewal and includes pluripotent, totipotent, and unipotent cells as well as cancer stem cells. [0131] A "non-stem cell" refers to any cell not capable of giving rise to all cell types within a given tissue or capable of self-renewal. A "non-stem cell" can also described as transient amplifying, differentiated or terminally differentiated. [0132] A "post-mitotic cell" refers to any cell type not undergoing cell division including any cell in GO or cell cycle arrest and differentiated cells. [0133] A "quiescent cell" is any cell in a state of inactivity, repose, or tranquility. The quiescent cell may be resting at the time or dormant, but can be stimulated to enter the cell cycle.

[0134] The term "population of cells" refers to one or more cells arising from at least one stem cell. This includes intact tissue, fractionated/homogenized tissue, cells derived from a tissue, and stem cell cultures isolated from a tissue. [0135] The term "less potent progenitor cells" refers to any cell capable of giving rise to two or more different cell types within a tissue. [0136] The term "transit amplifying cells" refers to any cell capable of giving rise to two or more different cell types within a tissue that has lost the capacity to self-renew and proliferates at a high rate. [0137] The term "differentiated cells" refers to any cell not capable of giving rise to different eel! types within a tissue (without genetic alterations).

[0138] The term "terminally differentiated cells" refers to any cell not capable of giving rise to different cell types within a tissue and that has lost the capacity to proliferate (without genetic alterations).

[0139] The term "damage" refers to any injury or insult to a cell or population of cells that results in cell death of cycling cells or inability of cycling cells to be labeled. Damage to cycling cells can be accomplished by using a mitotic killing agent such as alkylating agents, nitrosureas, antitumor antibiotics, mitotic inhibitors, or antimetabolites. Damage or injury to a population of cells also results in quiescent cells becoming activated, including stem cells.

[0140] The term "labeling" refers to using a cell-marking label that incorporates into the DNA or other cell components of cycling cells and does not turn over in the absence of cell division. The cell-marking label, labels or marks a cell for identification.

[0141] The term "identifying" refers to the detection of a label or marker, recognizing the difference between labeled and unlabeled cells. Identifying the label or marker is not limited to visual identity. It also includes separation without visual identity.

[0142] The "visualizing agent" recognizes exclusively the label-detecting agent and allows visual detection and separation. The visualization agent may take any one of a variety of forms, including detectable labels that are associated with or linked to the label-detecting agent. Detectable visualizing agents that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody specific for the cell-marking label. [0143] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1. Identification of Hematopoietic Stem Cells

[0144] To identify the location of activated stem cells within the hematopoietic system, 6-10 week old C57/B6 mice were subcutaneously injected with 250-300 μg/10g body weight of 5FU in phosphate buffer solution (PBS). Treatment with 5FU was followed by a single injection of 2 mg/kg body weight of BrdU in PBS 1 to 3 hours prior to sample collection. Two days following 5FU treatment, no actively proliferating cells could be detected (FIG. 5). Three days after 5FU treatment, a few BrdU positive cells could be detected on the surface of the bone (FIG. 6A) and surrounding blood vessels (FIGs. 6B and 6C). A few of these BrdU positive cells were found actively dividing (FIG. 6D). The location was further verified by staining tissue taken 3 days following 5FU treatment with N- cadherin, which labels the surface of the bone. BrdU positive cells could be observed on the edge of N-cadherin staining (FIGs. 6E, 6F, and 7). BrdU positive cells were identified as stem cells since they were positive for c-Kit, a well-known hematopoeitic stem cell marker (FIGs. 8A, 8B, and 8C). Further, BrdU positive cells were confirmed to be cells of the hematopoeitic system by staining positively for CD45 (FIG. 9).

[0145] Two or more BrdU positive cells could be observed in the same location 5 days following 5FU treatment (FIGs. 10A and 10B), thus suggesting the occurrence of cell division. Clusters of activated stem cells could be observed 6 days following 5FU treatment (FIGs. 11A and 11B). Also, clusters of activated stem cells could be observed on the sinusoid surface, blood vessels within bone marrow and the true niche of bone marrow stem cells (FIG. 11 C). [0146] The data suggests that quiescent stem cells residing on the surface of bone can be activated by an injury response to replenish lost cells of the hematopoeitic system. Furthermore, stem cells residing in the true niche of bone marrow, on the sinusoid surface, are also activated in response to injury. These results thereby confirm the location of stem cells on the sinusoid surface as well as uncover the location of additional responsive stem cells on the surface of bone. Example 2. Identification of Intestinal Stem Cells

[0147] To identify the location of activated stem cells within the intestine, 6-

10 week old C57/B6 mice were subcutaneously injected with 250-300 μg/10g body weight of 5FU in PBS. Treatment with 5FU was followed 1.5, 2, 3, 4, and 5 days by a single injection of 2 mg/kg body weight of BrdU in PBS 1 to 3 hours prior to sample collection, unless otherwise stated. By 1.5 days after 5FU treatment, complete depletion of proliferative intestinal cells was observed (FIG. 12A). Two days after 5FU treatment, the activated stem cells could be identified (FIG. 12B). Three days after 5FU treatment, there was a significant increase in proliferating cells (FIG. 12C) that persisted for 4 (FIG. 12D) and 5 days (FiG. 12E). Since BrdU only allows the identification of cells in the S-phase of the cell cycle, additional proliferating cells were observed by staining with the proliferation marker Ki67. The significant increase in proliferation observed between 2 and 3 days after 5FU treatment was verified with the proliferation marker Ki67. Minimal cell proliferation was observed 48 hours following 5FU treatment (FIG. 13A). Cell proliferation increased 51 hours after treatment (FIG. 13B) and continued increasing 57 hours (FIG. 13C), 72 hours (FIG. 13D), 4 days (FIG. 13E) and 5 days (FIG. 13F) after treatment. These proliferating cells are progeny of the activated stem cells replenishing lost tissue. Example 3. Identification of Pancreatic Stem Cells

[0148] To identify the location of activated stem cells within the pancreas,

2-4 week old C57/B6 mice were subcutaneously injected with 250-300 μg/10g body weight of 5FU in PBS. Treatment with 5FU was followed 3, 4, and 6 days with a single injection of 2 mg/kg body weight of BrdU in PBS 1 to 3 hours prior to sample collection. Three days following 5FU treatment, single BrdU positive cells were observed along pancreatic duct structures (FIG. 14A) and islet structures (FIGs. 14B and 14C), confirming the putative location of stem cells. Four and six days following 5FU treatment, multiple BrdU positive cells were observed in islets and ductal structures (FIGs. 14D and 14E). Once multiple BrdU positive cells were present, a few were positive for differentiation markers such as insulin, amylase, and Dolichos Biflorus Agglutinin (DBA). The presence of multiple BrdU positive cells expressing different differentiation markers indicates that activated stem cells were capable of regenerating lost pancreatic tissue. Example 4. Identification of Cancer Stem Cells [0149] Cancer stem cells are responsible for the initial development and reoccurrence of cancers. Using a mouse model of leukemia, cancer stem cells that remained following therapeutic treatment that may result in cancer reoccurrence were identified. The leukemia mouse model used consists of an irradiated mouse transplanted with a Phosphatase and tensin homolog (PTEN) knockout in bone marrow. These mice exhibit increased populations of proliferative monocytes and granulocytes in multiple tissues. Furthermore, the tissues become infiltrated by myeloid cells and develop a blast crisis stage of acute myeloid leukemia (AML) or acute myeloid and lymphoid leukemia (AML/ALL).

[0150] To identify the presence of cancer stem cells following therapeutic treatment, PTEN mutant mice were subcutaneously injected with 250-300 μg/10g body weight of 5FU in PBS. Treatment with 5FU was followed by a single injection of 2 mg/kg body weight of BrdU in PBS 1 to 3 hours prior to sample collection. Prior to 5FU treatment, the leukemia model exhibited 23.4% stem cells in a population of cells (FIG. 15A) compared to 16.6% observed in normal bone marrow sampling (FIG. 15B). Four days following 5FU treatment, 1.1% of cells analyzed by flow cytometry were BrdU positive and thus activated stem cells (FIG. 15C). The number of activated cells increased to 13.3% 14 days following 5FU treatment (FIG. 15D). The activated stem cells were found in the osteoblastic niche (FIG. 16).

[0151] The data suggests that quiescent cancer stem cells can be activated by chemotherapeutic treatments of cancer. The presence of cancer stem cells following treatment indicates a high likelihood of cancer reoccurrence. Additional treatment during the activation of the remaining cancer stem cells is an ideal time period for treatment to eliminate these culprit cells and therefore eliminate cancer. Example 5. DAL Method

[0152] Identification of stem cells in either normal or cancerous tissues is the essential first step in isolation, functional testing, and molecular and cellular characterization of stem cells, and for successful targeting on cancer stem cells as well. A variety of methods have been developed for this purpose thus far, however, a simple and straightforward approach is still required to effectively identify stem cells and their locations in a variety of normal and cancerous tissues. To this end, we have developed the DAL methodology for identification of stem cells based on the functional role of stem cells to regenerate damaged tissue in response to injury (Figure 17A). We further demonstrated the effectiveness of the DAL method in identifying stem cells, and their locations in the hematopoietic system. We have also determined the hierarchical relationship between stem and progenitor cells by tracing stem cell activity and their lineage differentiation potential in the pancreatic system. Thus, the DAL method can be applied to both fast and slow turnover systems, as well as cancerous tissues. Comparison of the LRC, SP₁ and DAL methods

[0153] Similar to the LRC method, the DAL approach is to first identify the putative stem cell location. The LRC method is to distinguish stem cells from cycling progenitor cells based on the feature that stem cells are either slow cycling or are very often in the quiescent state. However, the disadvantage of this approach is that LRCs may also include post-mitotic somatic cells and it is difficult to demonstrate the function of the quiescent LRCs in terms of their ability to give rise to corresponding downstream lineages. In addition, with the LRC approach there is a great variation between fast and slow turnover tissues (timing to obtain LRCs varies dramatically in different types of tissues) and this approach only provides static "endpoint" information. The advantages of the DAL approach are that it distinguishes proliferating progenitor cells from stem cells by destroying the former and sparing the latter and meanwhile activates stem cells to regenerate tissue in response to the injury imposed by this method. Further, it readily labels the activated stem cells, which are normally difficult to label in the prolonged quiescent state, and most importantly, it makes it possible to trace the BrdU- labeled RICs to reveal their lineage potential (Figure 17A).

[0154] Since BrdU was incorporated into the stem cell DNA, BrdU⁺ cannot be used as a marker to isolate viable cells as the preparation process for fluorescence activated cell sorting (FACS) involves permeabilization, which is lethal to the cells. However, BrdLT can be used as an internal stem cell marker using the DAL approach, facilitating sorting of the stem cell (BrdlT) population. Stem cell specific surface markers can then be identified after microarray analysis to compare the BrdlT and BrdU^" populations of cells. Actually, the SP approach for identifying populations of cells serves the same purpose, particularly in the solid tissues. The difference is that the SP cells are heterogeneous in both the cellular components and their corresponding functional roles while the BrdU^* RICs can be functionally tested for their lineage potential in vivo by the DAL method. Finally, we would like to emphasize that the BrdU⁺ cells labeled by the DAL method can pinpoint the putative stem cell position, however, not all the BrdU+ cells are stem cells. Therefore, lineage tracing following BrdU-chasing is a necessary step to distinguish stem from non-stem cells. A. Asymmetric segregation of parental versus daughter DNA strands and possibly biased BrdU incorporation

[0155] Both the LRC and OAL approaches are based on the assumption that stem cells can readily be incorporated with BrdU during DNA synthesis. However, whether BrdU labeled cells are the parental stem cells, the daughter cells, or both may vary depending on the type of tissue and the stage of development (Cairns, 1975; Merok et al., 2002a; Potten et al., 2002). If an adult animal is injected with BrdU under normal conditions, very often the BrdU would be favorably labeled in the daughter cells derived from stem cell division and would then be diluted during further division or sometimes partially retained in post-mitotic cells (Shinin et al., 2006). It is therefore worthwhile to point out that LRCs that represent primitive stem cells can only be obtained by incorporation of BrdU either at the neonatal stage or in response to injury. Only under these two conditions is the stem cell population expanding and therefore able to be labeled and to further retain the incorporated BrdU (Potten et al., 2002). This phenomenon can be partially explained by the theory of "immortal DNA strand" (Cairns, 1975). This theory proposed that DNA strands are selectively segregated with parental strands remaining in the parental stem cell during stem cell division to protect stem cells from accumulating genetic mutations. Although the molecular mechanism is not clear, accumulated evidence appears to support this theory (Karpowicz et al., 2005; Merok et al., 2002b; Rambhatla et al., 2005; Shinin et al., 2006). For example, during muscle stem cell division only the daughter cell with the newly synthesized DNA strand can incorporate the BrdU, or satellite LRCs labeled with BrdU at an early stage fail to distribute BrdU to the daughter cells (Shinin et al., 2006). Thus, further verification is needed to determine if the RICs revealed by BrdU incorporation are parental, daughter, or both stem cells. We have observed that LRCs could be detected in bone marrow at least one month P5FT, indicating that BrdU can be labeled either in parental stem cells, or the daughter stem cells that may reoccupy the niche to become a new parental stem cells. Nevertheless, identification of RICs using the DAL approach is still very helpful for tracing the stem cell position (in most cases, parental and daughter cells are adjacent to each other) and parental and daughter stem cells may also share similar markers.

B. Combination of the DAL method and GFP transgenic animal models [0156] Given that BrdU may be in cases favorably labeling daughter cells and that BrdU labeling is not retained after more than four divisions (not shown), it will be very useful to test the DAL method using GFP transgenic animals as outlined in Figures 24-29. Ideally, GFP expression driven by a constitutively active promoter can be induced in cells, including stem cells. Thus, proliferating progenitor cells should be eliminated following 5FU treatment. Here we would like to emphasize that completely eliminating proliferating cells is a key for the successful use of this approach because the efficiency of IV injection of 5FU varies depending on dosages of, injection efficiency of, the ages of mice injected with, and the sensitivity of the tissue to 5FU. Consequently, any surviving stem cells with GFP expression would be activated to initiate regeneration. Because the GFP expression would continue in all the downstream lineages, it would greatly facilitate lineage tracing. In addition, combining BrdU incorporation with genetic GFP marking should help to distinguish between parental and daughter stem cells if the latter is favorably labeled by BrdU, and to distinguish between stem cells and differentiated cells (which are not sensitive to 5FU treatment and therefore

could be GFP⁺ but BrdU^").

C. Pancreatic regeneration in response to damage can occur in at least two ways.

Duplication of existing /?-cells

[0157] It has been demonstrated that jff-cells can regenerate as a fast response following chemical, surgical, and genetic (Finegood et al., 1999; Meier et al., 2001 ; Sarvetnick and Gu, 1992) damage. A direct lineage tracing study in the mouse demonstrated that adult /?-cell replenishment under normal conditions or partial pancreatectomy is achieved mostly by duplication of preexisting >S-cells rather than differentiation of progenitor/stem cells (Dor et al., 2004). Stem/progenitor cell-driven regeneration

[0158] It is also possible that pancreatic regeneration can be driven by stem/progenitor cells when existing /?-cells with replication potential are eliminated. Yet to date, the identity and even the existence of pancreatic stem cells are still in dispute. Previously, in vivo and in vitro studies suggested that β- cell regeneration could be derived from duct cells (Bonner-Weir et al., 1993b; Sarvetnick and Gu, 1992), islet cells (Guz et al., 2001b), acinar cells during prolonged hyperglycemia (Lipsett and Finegood, 2002), nestin-positive cells (Hao et al., 2006; Zulewski et al., 2001a). Most of these studies, however, did not trace the in vivo stem cell properties: generating downstream multi-lineages cells Therefore, the location and identity of pancreatic stem/progenitor cells remains controversial.

[0159] Using the DAL method, we located regeneration initiating cells in duct, islet, and acinus that were located at niche-like structures (Figure 23). We also determined the hierarchical relationship among the RICs located in duct, islet, and acinus using co-staining analysis and lineage tracing (Figure 23E). In the duct, D-RICs located in the hyper-proliferative ducts (Figure 23B) initiated either islet or acinar structures by giving rise to either l-RICs (Figure 23C) or A-RICs (Figure 23D). In the islet, l-RICs generate insulin- and glucagon-expressing cells. In the acinus, A-RICs located at the centroacinar cell position give rise to acinar (amylase-expressing) cells (Figure 23E). Thus, the D-RICs function as stem cells that give rise to l-RICs or l-progenitor cells and A-RICs or A-progeriitor cells, respectively. In supporting this observation, both l-RICs and A-RICs are derived from duct cells. DAL chasing further revealed clonal exocrine regeneration that resembled the pattern seen during embryonic development (Castaing et al., 2005). In the islet, l-RICs that co-express at least a- and β- lineage markers, glucagons and insulin, were shown to commit to different endocrine lineages (Insulin^* £-cell versus glucagons⁺ αr-cell lineages).

[0160] Taken together, we have demonstrated the existence of stem/progenitor cells that initiate regeneration of pancreatic tissue in response to damage (Figure 23B), and proposed existence of two ways of pancreatic regeneration.

D. Identification of leukemia initiating cells

[0161] To test whether the DAL method can also be used to identify cancer initiating/stem cells we tested its effectiveness using the PTEN mutant animal model, which developed acute leukemia (Yilmaz et al., 2006; Zhang et al., 2006). Both immunohistochemistry and flow cytometry analyses of PTEN mutant animals revealed overt leukemia by overwhelming BrdU staining throughout the bone marrow. Administration of 5-FU drastically reduced proliferating cells as revealed by dramatically reduced BrdU incorporation. On day 4 post 5-FU treatment only sporadically distributed single BrdlT cells were detected, presumably a surviving leukemia initiating/stem cell. Indeed, 12 days after 5-FU treatment leukemia had been re-initiated, as exemplified by the recurrence of overwhelming BrdU staining. On D4 P5FT, a CD201 marker was used to sort cells that include sporadically distributed single BrdU^* cells. These cells were then transplanted into recipient mice, resulting in transplantable leukemia in most of the recipients. In contrast, up to 60 times more CD201^" cells failed to do so. Hence, the DAL method effectively identified the leukemia initiating cells in the PTEN leukemia model. Thus, the DAL method can be used on primary cells derived from human cancer tissue to identify the corresponding CSCs in tissue culture.

[0162] In summary, the DAL method can be used to effectively identify and locate the position of putative stem cells in a variety of tissues. This method can facilitate further investigation of stem cell properties in well-established systems and can also be used to identify stem cells in systems in which stem cells have not yet been defined. And, perhaps more importantly, the DAL method shows great promise as an effective and straightforward method for identifying cancer stem cells.

E. Animals

[0163] All mice used in this study were housed at the animal facility at

Stowers Institute for Medical Research and handled according to institutional and NIH guidelines.

F. Stem Cell Activation, Labeling and Chasing

[0164] Mice aged 3 to 4 weeks were given a single dose of 5-florouracil (5-

FU) intravenously. To examine tissue turnover, a single dose of 5-Bromo-2- deoxyuridine (BrdU) was injected subcutaneously and the mice were sacrificed 3 hours after injection. For activated stem cell labeling, a single dose of BrdU was given subcutaneously at each targeted day after 5-FU treatment and animals were terminated 3 hours thereafter. For activated stem cell chasing after 5-FU treatment, a single dose of BrdU was injected at each targeted day to a group of mice and a pair of animals were sacrificed for examination at each chasing point thereafter.

G. lmmunohistostaining of Tissue Sections

[0165] Before tissue collection, anesthetized animals were perfused with

PBS and 4% paraformaldehyde respectively. Femur & tibia, intestine and pancreas were fixed in Zn²⁺-Formalin or 4% paraformaldehyde (pancreas) and processed for paraffin and frozen sections respectively.

H. Flow Cytometry Analysis of Hematopoietic Cells

[0166] Bone marrow cells were flushed from femur & tibia with PBS supplemented with 2% fetal bovine serum, and dispersed into single cells by repeatedly vigorous aspiration through 22G11/2 syringe followed by meshing through a 40 μm cell strainer. Peripheral blood was obtained by submandibular bleeding. Red blood cells were lysed before immunofluorescent staining.

[0167] HSCs were sorted in the LSK(Lineage^'Sca-1⁺c-Kit⁺)CD201 ⁺ population while Lineage^'CD201^" or Lineage⁺CD201^" population was isolated for comparison. For the analysis of BrdU⁺CD201⁺ population, cells were subjected to permeablized BrdU staining according to kit instructions followed by LSKCD201 staining as above.

I. Long-Term Competitive Bone Marrow Engraftment Assays [0168] For HSC long-term repopulation assays, donor hematopoietic cells were isolated from C57BL/6J-CD45.2:Thy-1.1 mice or Mx-1-Cre-Ren^fbt/flx (C57BL/6J background) while Ptprc-CD45.1 :Thy1.2 mice were used as recipients. 6-8 weeks old recipient mice were lethally irradiated and received intravenously 100 of LSKCD201⁺ cells or 500 of lineage^'CD201^" cells from B6 or Ren mutant mouse bone marrow with 2x10⁵ supporting cells isolated from Ptprc mouse bone marrow. 4, 8 & 12 weeks after transplantation, peripheral blood of the recipient mice were collected by submandibular bleeding, subjected to red blood cell lysis with ammonium chloride and potassium buffer, and stained with a battery of conjugated antibodies for the detection of reconstitution.

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[0272] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

What is claimed is:

1. A method of identifying an activated cell wherein the method comprises: a. damaging one or more cells within a population of cells wherein the damaging results in a quiescent cell becoming an activated cell; b. labeling the activated cell such that the label is incorporated into DNA of the activated cell; and c. identifying the labeled activated cell.

2. The method of claim 1 wherein the activated cell is a pluripotent cell, a totipotent cell, a unipotent cell or a stem cell.

3. The method of claim 1 wherein the population of cells are mammalian.

4. The method of claim 1 wherein the population of cells are murine.

5. The method of claim 1 wherein the population of cells are human.

6. The method of claim 1 wherein damaging results in cell death or cell injury such that the injured cell is incapable of being labeled.

7. The method of claim 1 wherein the label is incorporated into DNA of a cell progeny of a labeled activated cell.

8. The method of claim 7 wherein the progeny cell comprises a less potent progenitor cell, a transit amplifying cell, a differentiated cell, a precursor cell, or a non-stem cell.

9. The method of claim 1 wherein the population of cells comprises a stem cell, a progeny cell differentiated from a stem cell or a combination thereof.

10. The composition of claim 9 wherein the population of cells comprises intestine, pancreas, bone marrow, skin, brain, bone, cartilage, adipose, heart, kidney, liver, stomach, oral mucosa, muscle, epithelial tissue, endothelial tissue, mesenchymal tissue, endoderm, mesoderm or ectoderm.

11. The composition of claim 9 wherein the population of cells comprises normal, pre-cancerous, cancerous, or tumorigenic tissue.

12. A method of identifying an activated cell or its progeny cell in a population of cells wherein the method comprises: a. damaging one or more cells within a population of cells, wherein the damaging results in a quiescent cell becoming an activated cell; b. labeling the activated cell such that label is incorporated into DNA of the activated cell; c. allowing the activated cell to differentiate such that label is incorporated into DNA of a progeny cell; and d. identifying the labeled progeny cell, the labeled activated cell, or a combination thereof.

13. The method of claim 11 wherein the population of cells are mammalian.

14. The method of claim 11 wherein the population of cells are murine.

15. The method of claim 11 wherein the population of cells are human.

16. The method of claim 11 wherein damaging results in cell death or injury such that a cell is incapable of being labeled.

17. The method of claim 11 wherein the progeny cell is a less potent progenitor cell, a transit amplifying cell, a differentiated cell, a precursor cell, or a non-stem cell.

18. The method of claim 11 wherein the population of cells comprises a stem cell or a cell differentiated from a stem cell or a combination thereof.

19. The method of claim 17 wherein the population of cells comprises intestine, pancreas, bone marrow, skin, brain, bone, cartilage, adipose, heart, kidney, liver, stomach, oral mucosa, muscle, epithelial tissue, endothelial tissue, or mesenchymal tissue.

20. The method of claim 11 wherein the population of cells comprises normal, pre-cancerous, cancerous, or tumorigenic tissue.

21. The method of claim 11 wherein the activated cell is a pluripotent cell, a totipotent cell, a unipotent cell, or a stem cell.

22. A kit for identifying an activated cell or its progeny wherein the kit comprises: a. a mitotic killing agent; b. a label capable of being incorporated into DNA of a cell; c. a label detecting agent; d. a protocol; and e. a container.

23. The kit of claim 20 wherein the kit identifies a mammalian cell.

24. The kit of claim 20 wherein the kit identifies a murine cell.

25. The kit of claim 20 wherein the kit identifies a human cell.

26. The kit of claim 20 wherein the mitotic killing agent comprises alkylating agents, nitrosureas, antitumor antibiotics, mitotic inhibitors, or antimetabolites.

27. The kit of claim 20 wherein the label comprises Brdll, a halogenated deoxyribonucleotide, a labeled deoxyribonucleotide, a radiolabeled nucleotide, a deuterium labeled nucleotide, or a deuterium labeled DNA synthesis precursor.

28. The kit of claim 20 wherein the label detecting agent comprises a visualizing agent.

29. The kit of claim 26 wherein the visualizing agent comprises a radiolabel, a fluorescent tag, an enzymatic tag, a fluorogenic substrate tag, or a chromogenic substrate tag.

30. The kit of claim 20 wherein the kit also comprises a visualizing agent.

31. The kit of claim 28 wherein the visualizing agent comprises a radiolabel, a fluorescent tag, an enzymatic tag, a fluorogenic substrate tag, or a chromogenic substrate tag.

32. The kit of claim 20 wherein the kit is used for the method comprising: a. damaging one or more cells within a population of cells wherein the damaging results in a quiescent cell becoming an activated cell; b. labeling the activated cell such that the label is incorporated into DNA of the activated cell; and c. identifying the labeled activated cell.

33. A method for identifying and locating a stem cell in a tissue comprising a. contacting proliferating cells in a tissue with an agent that destroys the cells and activates quiescent stem cells; b. labeling the activated stem cells; and c. detecting the labeled stem cells.

34. The method according to claim 33, wherein the tissue is normal or cancerous tissue.

35. The method according to claim 33, wherein the detecting step is selected from the group consisting of locating the labeled stem cell, identifying the labeled stem, and locating and identifying the labeled stem cell.

36. A method of identifying cancer stem cells comprising: a. contacting proliferating cells in a tissue with an agent that destroys the cells and activates quiescent stem cells; b. labeling the activated stem cells; c. transplanting at least one labeled stem cell into a lethally irradiated recipient host; and d. determining whether the transplanted cells developed into cancer.

37. The method according to claim 36, wherein the cancer is selected from the group consisting of a myeloid proliferative disorder, acute myeloid leukemia, and acute lymphoid leukemia.