WO2007124125A2 - Méthodes d'identification de cellules souches dans des tissus normaux et cancéreux et cellules de descendance apparentées - Google Patents

Méthodes d'identification de cellules souches dans des tissus normaux et cancéreux et cellules de descendance apparentées Download PDF

Info

Publication number
WO2007124125A2
WO2007124125A2 PCT/US2007/009775 US2007009775W WO2007124125A2 WO 2007124125 A2 WO2007124125 A2 WO 2007124125A2 US 2007009775 W US2007009775 W US 2007009775W WO 2007124125 A2 WO2007124125 A2 WO 2007124125A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
stem
activated
population
Prior art date
Application number
PCT/US2007/009775
Other languages
English (en)
Other versions
WO2007124125A3 (fr
Inventor
Linheng Li
Xi He
Zhixin Li
Jiwang Zhang
Original Assignee
Stowers Institute For Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stowers Institute For Medical Research filed Critical Stowers Institute For Medical Research
Publication of WO2007124125A2 publication Critical patent/WO2007124125A2/fr
Publication of WO2007124125A3 publication Critical patent/WO2007124125A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • CSCs cancer 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.
  • BASCs bronchioalveolar stem cells
  • SP-C side population
  • HSCs have been prospectively purified in the Thy-1 10 or Flk2 " Lineage " Sca-1 + c-Kit + population for quite some time (Christensen and Weissman, 2001; Uchida et al., 1996).
  • 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).
  • 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).
  • stem cells 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell-marking label will be selected so that it incorporates into activated cells at sufficient levels to allow external detection.
  • 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.
  • a label may include biotinylated, radiolabeled, fluorescent, colorimetric molecules or other suitable labels known in the art.
  • 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.
  • 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.
  • 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.
  • agents include radiolabels, fluorescent tags, enzymatic tags, and fluorogenic or chromogenic substrate tags.
  • the invention can be practiced to allow the labeling and identification of activated cancer stem cells.
  • a cell is responsible for the development and reoccurrence of cancer.
  • 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.
  • 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.
  • the invention provides for the labeling and identification of activated 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the diagnostic agents are maintained separately in containers within a container that holds all components.
  • 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.
  • 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.
  • 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.
  • visualizing agent may include a radiolabel, a fluorescent tag, an enzymatic tag, a fluorogenic substrate tag, or a chromogenic substrate tag.
  • the application contains at least one drawing executed in color.
  • FIG. 1 schematically shows normal hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) cycling prior to 5FU treatment or labeling by BrdU pulse injection.
  • HSCs normal hematopoietic stem cells
  • MPPs multipotent progenitors
  • Short-term HSCs and MPPs give rise to cells that differentiate into differentiated hematopoeitic cell types.
  • 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.
  • 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.
  • 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.
  • FIG. 5 shows cells surrounding bone and blood vessels, 2 days after
  • FIGs. 6A-F shows cells with incorporated BrdU (red) three days following 5FU treatment.
  • 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 inco ⁇ orated 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).
  • 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).
  • FIG. 8A shows activated HSCs labeled with BrdU (red) 3 days after
  • 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.
  • FIG. 9 shows that on Day 3 after 5FU treatment, BrdU-labeled cells
  • 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).
  • 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.
  • 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.
  • FIG. 13A shows a few Ki67 positive cells (red) two days following
  • 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. 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).
  • FIG. 14D shows the progeny of the activated stem cells.
  • FIG. 14E shows the progeny of the activated stem cells.
  • FIG 15 shows bone marrow cells were analyzed by flow cytometry for the incorporation of BrdU.
  • FIG. 15A 16.6% of cells were proliferating cells in the normal sample
  • FIG. 15B 14 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.
  • 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.
  • FIG. 17 is a schematic that shows that the DAL method locates HSC activity and reveals dynamic HSC niche interactions.
  • 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.
  • FIGs. 19A-I show that the regeneration initiating cells in response to bone marrow damage are enriched with HSCs.
  • FIGs. 20A-O show that the DAL method is able to identify regeneration initiating cells in response to injury in the pancreas.
  • FIGs. 21A-F shows: that the DAL method is effective to identify pancreatic regeneration activity in the duct, islet and acinus.
  • FIGs. 22A-L shows: DAL chases pancreatic RICs to undergo different lineage commitments and unveils ductal derivation of endocrine and exocrine stem/progenitor cells.
  • Figs. 23A-E show that the DAL method is effective to identify leukemia stem cells in a Pten deficient mouse model.
  • FIGs. 24A-D shows MCZEG mice plPC induction day 1.5.
  • FIG. 25 shows a 3-D whole mount analysis of a single GFP + cell.
  • FIG. 26 shows a schematic illustrating the strategy of tracing a single labeled GFP + cell.
  • FIG. 27 shows a lineage trace of a single GFP+ cell at day 28 after plPC induction.
  • FIG. 28 shows four types of cells that were labeled at day 28 after plpC induction.
  • FIGs. 29A-F show that labeled GFP cells dominantly proliferate in vitro.
  • 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.
  • 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.
  • labeled stem cells are provided as well as methods for analyzing the regenerative property of stem cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Busulfan may be selected.
  • the preferred dosage and administration route for Busulfan is about 3.2mg/kg body weight injected intravenously
  • 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.
  • 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.
  • 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.
  • the damage to a population of cells occurs and results in the activation of previously quiescent stem cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a combination of cell-marking label can be used to identify both the activated stem cells and the progeny of the activated stem cells.
  • 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.
  • first labeling event would label the activated previously quiescent stem cell.
  • second labeling event would label the same stem cell and its first generation progeny.
  • 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.
  • 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.
  • 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 inco ⁇ orated into DNA during cell division.
  • the cell-marking label may be a labeled deoxyribonucleotide (dN).
  • dN deoxyribonucleotide
  • 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).
  • 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).
  • 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 3 H-dC, 3 H- dG, 3 H-dA, 3 H-dT, and 3 H-dU.
  • 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 2 H-dT, 2 H-dA, 2 H-dG, 2 H-dC, and 2 H- 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.
  • 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.
  • mice can be intravenously injected with BrdU approximately 1 to 3 hours prior to tissue collection in order to label cycling cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the population of cells 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.
  • 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.
  • FACS fluorescence-activated cell sorting
  • 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 Bioinfo ⁇ natics, Unit 7, pub. John Wiley & Sons, Inc., 2003 and incorporated herein by reference.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Cell-surface markers can be used to optimize cancer treatment regimens through in vitro experimentation.
  • 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.
  • intracellular markers i.e. BrdU
  • 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.
  • 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.
  • the preferred tumor therapeutic 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.
  • 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 reco ⁇ stitution.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed.
  • 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.
  • the method includes the following three steps: 1 ) enforced activation (transition from G 0 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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 ⁇ -cell and /?-cell lineages in islet, and exocrine cells in acinus.
  • pancreatic stem/progenitor cells and their locations were revealed by the DAL plus BrdU-chasing method in duct, islet, and acinus.
  • 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).
  • MPD myeloid proliferative disorder
  • AML acute myeloid leukemia
  • ALL acute lymphoid leukemia
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term “differentiated cells” refers to any cell not capable of giving rise to different eel! types within a tissue (without genetic alterations).
  • terminal 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • mice 2-4 week old C57/B6 mice were subcutaneously injected with 250-300 ⁇ g/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).
  • 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.
  • PTEN Phosphatase and tensin homolog
  • 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).
  • AML acute myeloid leukemia
  • AML/ALL acute myeloid and lymphoid leukemia
  • the DAL method can be applied to both fast and slow turnover systems, as well as cancerous tissues. Comparison of the LRC, SP 1 and DAL methods
  • 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.
  • 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.
  • 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).
  • 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.
  • FACS fluorescence activated cell sorting
  • 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.
  • the SP approach for identifying populations of cells serves the same purpose, particularly in the solid tissues.
  • 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.
  • Pancreatic regeneration in response to damage can occur in at least two ways.
  • 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.
  • ⁇ - 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.
  • l-RICs that co-express at least a- and ⁇ - lineage markers, glucagons and insulin, were shown to commit to different endocrine lineages (Insulin * £-cell versus glucagons + ⁇ r-cell lineages).
  • the DAL method effectively identified the leukemia initiating cells in the PTEN leukemia model.
  • the DAL method can be used on primary cells derived from human cancer tissue to identify the corresponding CSCs in tissue culture.
  • 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.
  • mice used in this study were housed at the animal facility at
  • FU intravenously.
  • a single dose of 5-Bromo-2- deoxyuridine (BrdU) was injected subcutaneously and the mice were sacrificed 3 hours after injection.
  • a single dose of BrdU was given subcutaneously at each targeted day after 5-FU treatment and animals were terminated 3 hours thereafter.
  • 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.
  • 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 ⁇ m cell strainer. Peripheral blood was obtained by submandibular bleeding. Red blood cells were lysed before immunofluorescent staining.
  • HSCs were sorted in the LSK(Lineage ' Sca-1 + c-Kit + )CD201 + population while Lineage ' CD201 " or Lineage + CD201 " population was isolated for comparison.
  • BrdU + CD201 + population cells were subjected to permeablized BrdU staining according to kit instructions followed by LSKCD201 staining as above.
  • 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 2x10 5 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.
  • Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68, 245-253.
  • Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 707, 2317-2321.
  • Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proceedings of the National Academy of Sciences of the United States of
  • Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703-716.
  • Hydroperoxycyclophosphamide inhibits proliferation by human granulocyte- macrophage colony-forming cells (GM-CFC) but spares more primitive progenitor cells.
  • GM-CFC granulocyte- macrophage colony-forming cells
  • SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109-1121.
  • Lectin as a marker for staining and purification of embryonic pancreatic epithelium.
  • PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 441, 518-522.
  • Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50, 521-
  • Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phe ⁇ otypes. Diabetes 50, 521-

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention porte sur des méthodes et sur des compositions utiles dans l'identification de cellules actives telles que des cellules souches ou des cellules souches cancéreuses, et les cellules différenciées de celles-ci. L'invention peut être utile dans l'identification de cellules progénitrices issues d'une grande variété de tissus tels que des tissus précancéreux ou cancéreux.
PCT/US2007/009775 2006-04-21 2007-04-21 Méthodes d'identification de cellules souches dans des tissus normaux et cancéreux et cellules de descendance apparentées WO2007124125A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74537706P 2006-04-21 2006-04-21
US60/745,377 2006-04-21

Publications (2)

Publication Number Publication Date
WO2007124125A2 true WO2007124125A2 (fr) 2007-11-01
WO2007124125A3 WO2007124125A3 (fr) 2008-11-20

Family

ID=38625651

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/009775 WO2007124125A2 (fr) 2006-04-21 2007-04-21 Méthodes d'identification de cellules souches dans des tissus normaux et cancéreux et cellules de descendance apparentées

Country Status (1)

Country Link
WO (1) WO2007124125A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111024624A (zh) * 2019-12-20 2020-04-17 东南大学 基于暗场散射成像的parp-1单粒子检测方法
US20200325222A1 (en) * 2011-10-28 2020-10-15 Chugai Seiyaku Kabushiki Kaisha Cancer stem cell-specific molecule
CN112051321A (zh) * 2020-08-24 2020-12-08 复旦大学 结合氘水培养和基质辅助激光解吸电离飞行时间质谱分析的快速抗生素敏感性测试方法
US11965180B2 (en) 2010-10-06 2024-04-23 Chugai Seiyaku Kabushiki Kaisha Cancer stem cell population and method for production thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AIZAWA K. ET AL.: 'Augmentation of 5-Fluorouracil cytotoxicity by epidermal growth factor in a newly established human signet-ring cell carcinoma of the stomach in culture' SURGERY TODAY vol. 24, May 1995, pages 420 - 428 *
KHOW ET AL.: 'The long term effects of 5-Fluorouracil and Sodium butyrate on human tenon's fibroblasts' INVEST. OPHTHALMOL. VISION SCI. vol. 33, 1992, pages 2043 - 2052 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11965180B2 (en) 2010-10-06 2024-04-23 Chugai Seiyaku Kabushiki Kaisha Cancer stem cell population and method for production thereof
US20200325222A1 (en) * 2011-10-28 2020-10-15 Chugai Seiyaku Kabushiki Kaisha Cancer stem cell-specific molecule
US11858987B2 (en) * 2011-10-28 2024-01-02 Chugai Seiyaku Kabushiki Kaisha Cancer stem cell-specific molecule
CN111024624A (zh) * 2019-12-20 2020-04-17 东南大学 基于暗场散射成像的parp-1单粒子检测方法
CN112051321A (zh) * 2020-08-24 2020-12-08 复旦大学 结合氘水培养和基质辅助激光解吸电离飞行时间质谱分析的快速抗生素敏感性测试方法
CN112051321B (zh) * 2020-08-24 2022-02-15 复旦大学 结合氘水培养和基质辅助激光解吸电离飞行时间质谱分析的快速抗生素敏感性测试方法

Also Published As

Publication number Publication date
WO2007124125A3 (fr) 2008-11-20

Similar Documents

Publication Publication Date Title
Asada et al. Differential cytokine contributions of perivascular haematopoietic stem cell niches
Sullivan et al. Brain tumor cells in circulation are enriched for mesenchymal gene expression
Ratajczak et al. Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ‘hide out’in the bone marrow
US7767410B2 (en) Identification and isolation of acute myeloid leukemia stem cells
US20010049139A1 (en) Hepatic regeneration from hematopoietic stem cells
TWI635177B (zh) 體幹細胞
JP6382408B2 (ja) 体性幹細胞
WO2002088335A1 (fr) Cellules souches et procede d'extraction de ces cellules
TWI439276B (zh) 肝袓細胞及其使用
CN111713450B (zh) 一种慢性粒细胞白血病的pdx模型的建立方法
Lee et al. Hepatic stellate and endothelial cells maintain hematopoietic stem cells in the developing liver
US20230126485A1 (en) Pancreatic stem cells and uses thereof
Kang et al. Multipotent, dedifferentiated cancer stem-like cells from brain gliomas
Nygren et al. A novel assay to trace proliferation history in vivo reveals that enhanced divisional kinetics accompany loss of hematopoietic stem cell self-renewal
WO2007124125A2 (fr) Méthodes d'identification de cellules souches dans des tissus normaux et cancéreux et cellules de descendance apparentées
Eisenwort et al. Phenotypic characterization of leukemia-initiating stem cells in chronic myelomonocytic leukemia
Liu et al. A patient tumor-derived orthotopic xenograft mouse model replicating the group 3 supratentorial primitive neuroectodermal tumor in children
WO2001071016A1 (fr) Cellules souches pluripotentielles
JP2007105037A (ja) キメリズムを利用した幹細胞移植のための検査
Warner et al. Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors
Yoshimoto et al. Two Different Roles of Purified CD45+ c‐Kit+ Sca‐1+ Lin− Cells After Transplantation in Muscles
Kobayashi et al. In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii)
US20230090462A1 (en) Isolation, enrichment and expansion of cone progenitor cells and uses thereof
Wang et al. Chlorotoxin redirects chimeric antigen receptor t cells for specific and effective targeting of glioblastoma
Drazer Defining Leukemogenic Mechanisms in Hereditary Hematopoietic Malignancies

Legal Events

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

Ref document number: 07794358

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07794358

Country of ref document: EP

Kind code of ref document: A2