Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. All references cited herein are specifically incorporated herein by reference in their entirety.
Neural Stem Cells
Traditionally, stem cells were thought to be located only in tissues where differentiated cells were most susceptible to loss and the need for replacement great, such as the skin (Huelsken et al., Cell 105: 533-45, 2001), intestinal epithelia (Potten et al., Development 110: 1001-20, 1990) and the blood (Morrison et al., Annu Rev Cell Dev Biol 11: 35-71, 1995). Indeed, the best-known example of an adult stem cell is the hematopoietic stem cell (HSC), which is found in the bone marrow and is ultimately responsible for the generation of all blood cell types throughout the life of the animal (Morrison et al., supra.; Weissman, Cell 100: 157-68, 2000; Weissman, Science 287: 1442-6, 2000). Since the adult central nervous system (CNS) was thought not to exhibit a significant amount of neuronal death, and have no regenerative capacity, the existence of neural stem cells seemed both unlikely, and unnecessary. However, in 1992 two independent groups successfully demonstrated the existence of precursor cells within the adult mammalian CNS with the ability to give rise to new neurons (Reynolds and Weiss, Science 255: 1707-10, 1992; Richards et al., Proc Natl Acad Sci USA 89: 8591-5, 1992).
The source of the new neurons was identified as stem cells that line the entire ventricular neuroaxis of the adult mammalian CNS (Reynolds and Weiss, 1992). Like stem cells found in other tissues, CNS stem cells (or neural stem cells (NSCs)) have been shown to demonstrate the defining in vitro stem cell characteristics (Hall et al., Development 106: 619-33, 1989; Potten et al, supra.) of proliferation, extensive self-renewal, generation of a large number of progeny, multi-lineage differentiation potential and the in vivo characteristic of regenerating tissue after injury.
One of the roles of a stem cell is to divide and give rise to more committed precursor cells with the ability to proliferate and generate a large number of undifferentiated cells. Ultimately it is the progeny of these more committed precursor cell types that give rise to differentiated progeny. Thus, stem cells can be thought of as a relatively quiescent reservoir of uncommitted cells with the ability to divide throughout the lifespan of the animal and hence with an extensive proliferation potential, while progenitor cells are more committed and divide more frequently but have a more limited proliferation potential over time. Both during development, and in the adult, the proliferation of stem and progenitor cells underpins cell genesis.
Due to the lack of any specific morphological, molecular or antigenic signature stem cells are identified based on a functional criterion. Hence, to study the regulation of stem cells in vitro a tissue culture methodology must be developed that induces stem cell division. Few such assays exist, however, in the nervous system a culture methodology referred to as the Neurosphere Assay (NA) (Reynolds and Weiss, supra.) is commonly used to identify, propagate and enumerate NSCs in vitro. Briefly, the NA involves the microdissection of embryonic through to adult CNS tissue followed by the disruption of cell to cell contacts and the generation of a suspension of single cells. Cells are plated (typically at a low density) in tissue cultureware in a defined serum-free medium in the presence of at least one proliferation-inducing growth factor (ie. Epidermal Growth Factor [EGF], basic Fibroblastic Growth Factor [bFGF] etc.). Under these conditions within 2-5 days a multipotent NSC begins to divide giving rise to a clonally derived cluster of undifferentiated cells referred to as a neurosphere. In the continued presence of the proliferation inducing factor the cells in the neurosphere continue to divide resulting in an increase in the number of cells comprising the neurosphere and consequently the size of the neurosphere. Neurospheres can be collected, disrupted in to a single cell suspension, and the cells replated in culture to generate new neurospheres. Passaging of NSC in this manner results in an arithmetic increase in viable CNS precursor cells. The NA assay allows for NSCs to be isolated and expanded in defined conditions so the behavior of the putative stem cells can be studied under different experimental conditions. The NA has become the standard assay for the isolation of mammalian NSC and forms the core of many assays used to understand the cellular and molecular biology of stem cells in the nervous system.
The concept of tumors arising from a small population of cells with stem cell characteristics that contribute to the growth and propagation of the tumor is not new to the cancer biology field. The idea was proposed in early 1970's and experimentally confirmed in studies on acute myelogenousleukaemia (AML) where low frequency tumor initiating cells were demonstrated to resemble normal haematopoietic stem cells (HSCs). These studies suggested that leukemia stem cells were the direct descendents of HSC or the produce of a more differentiated cell that had acquired HSC features. Discovery of stem cells outside of the blood system raised the possibility that cancers of solid tissues may also contain stem like cells. The existence and isolation of tumor initiating stem-like cells in solid tumors was first demonstrated in human breast cancer tissue, an approach that has also been applied to tumors of the CNS.
Several groups have recently reported on the ability of cells derived from human glioma tissue to generate neurosphere-like cells in culture, suggesting the presence of NSCs within CNS tumors. Interestingly, it has been demonstrated, based on fluorescence activated cell sorting (FACS) isolation of “side-population” cells, that the well-established C6 glioma cell line contains a minor population of neurosphere-forming cells that retain in vivo malignancy. Galli and colleagues (Galli et al., Cancer Research (2004) 64: 7011-7021) reported on the isolation, propagation and serial transplantation of tumor neural stem cells (tNSCs) from human glioblastoma multiforme (GBM) that exhibit near identical functional properties as NSC derived from the embryonic and adult CNS. These GBM tNSCs are prominin positive precursors, which display the critical neural stem cell features in vitro, can be expanded in a stable fashion and, throughout serial transplantation-culturing cycles reproduce the original tumor-initiating characteristics. Together, these studies strongly support the hypothesis that CNS tumors contain a population of stem cells that may be responsible for tumor initiation and malignancy. The tNSCs can be sorted from other GBM cells using FACS by virtue of the expression on the tNSCs of CD133 (Singh et al., Nature (2004) 432:396-401).
GBM is the most common adult malignant brain tumor, with a median survival time of 9-12 months. The vast majority of patients die by two years from diagnosis. There is essentially no cure, and management therapy is commonly based on the combination of surgery, radiotherapy and chemotherapy. Survival rates have changed very little in over thirty years, which has prompted the active search for new treatments such as gene therapy, antiangiogenesis, immunotherapy and small molecule transduction inhibitors.
LIF
Leukemia inhibitory factor (LIF) is a polyfunctional glycoprotein cytokine whose inducible production can occur in many, perhaps all, tissues. LIF is also sometimes referred to as Cholinergic Differentiation Factor (CDF). LIF acts on responding cells by binding to a heterodimeric membrane receptor composed of a low-affinity LIF-specific receptor (LIFR) and the gp130 receptor chain also used as the receptor for interleukin-6, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. LIF is essential for blastocyst implantation and the normal development of hippocampal and olfactory receptor neurons. LIF is used extensively in experimental biology because of its key ability to induce embryonic stem cells to retain their totipotentiality. LIF has a wide array of actions, including acting as a stimulus for platelet formation, proliferation of some hematopoietic cells, bone formation, adipocyte lipid transport, adrenocorticotropic hormone production, neuronal survival and formation, muscle satellite cell proliferation, and acute phase production by hepatocytes (for review see Metacalf, Stem Cells 2003; 21:5-14).
BMP
Bone morphogenetic proteins (BMPs) are members of the TGF-h superfamily (Hoodless et al., Cell 85:489-500, 1996). There are more than 20 members known that can be subgrouped according to the homology in their sequence (Hoodless et al., supra, Wozney et al. J Cell Sci, Suppl 13:149-156, 1990). BMPs play crucial roles during the embryonic development. For example, they influence gastrulation, neurogenesis, apoptosis and hematopoiesis (see Nohe et al., Cellular Signalling 16, 291-299 (2004) for review). BMP receptors are hereinafter referred to as BMPRs. BMPRs from humans include BMPR1a, BMPR1b, and BMPR2.
In accordance with the present disclosure, it has now been determined that LIF and BMPs regulate progenitor and stem cell survival, self-renewal, proliferation and/or differentiation and in particular can reduce the numbers of proliferating cells in cancerous tissues.
USPTO Backgrounds
USPTO Backgrounds