Overview
Manipulation of stem cells (both normal and transformed) has great therapeutic potential for treating neurological disorders and cancer. We are interested in understanding the molecular mechanisms that regulate self-renewal, proliferation and differentiation of stem cells. A major area of interest in my laboratory is to use the mouse model system to test the cancer stem cell hypothesis, and to develop novel reagents that will target cancer stem cells specifically. Our other major area of focus lies in understanding the genetic pathways that regulate maintenance and regulation of normal stem cells from the brain and other tissues. By combining these two areas of interest, we seek to identify and understand molecular differences between normal and transformed stem cells.
Research details
Normal and cancer stem cells of the brain
Manipulation of stem cells (both normal and transformed) has great therapeutic potential for many human diseases. We are interested in understanding the molecular mechanisms that regulate self-renewal, proliferation and differentiation of stem cells. A major area of interest in my laboratory is to use the mouse model system to test the cancer stem cell hypothesis, and to develop novel reagents that will target cancer stem cells specifically. Our other major area of interest lies in understanding the genetic pathways that regulate maintenance and regulation of normal stem cells from the brain and other tissues. By combining these two areas of interests, we seek to identify and understand molecular differences between normal and transformed stem cells.
Targeting brain cancer stem cells
The discovery of cancer stem cells in human brain and other solid tumors opens the possibility for novel, targeted therapeutic agents aimed at eradicating these special cells which are responsible for tumor initiation and growth. Cancer stem cells are multi-potential, self-renewing cells within a tumor that can initiate a tumor when transplanted in a host mouse. Since even a small number of stem cells can re-initiate tumor formation, the presence of cancer stem cells may explain the high recurrence rate in brain cancer patients. We hypothesize that ablation of these cells is both necessary and sufficient to block existing tumor growth and inhibit recurrence, a concept that has not yet been tested. We are using a mouse model of medulloblastoma and oligodendrogliomas to test this hypothesis.
To test the efficacy of stem cell ablation in vivo, we first need to demonstrate that at the cellular level, mouse models accurately replicate the pathobiology of human brain cancer. We recently isolated and characterized cancer stem cells from a transgenic mouse model of oligodendroglioma (Tg(S100b-v-erbB)4496Waw), providing the initial proof of principle that mouse brain tumors also contain cancer stem cells. A number of features of these mouse cells corresponded to those of human cancer stem cells. When tumor cells from transgenic mice are dissociated and cultured in a serum-free, defined medium, a small percentage of cells could form spheres, similar to normal neurospheres. They formed secondary spheres upon multiple (>16) passages (self-renewal), and some cells within spheres expressed markers of normal stem cells such as CD133 (currently PROM1), BCRP1 (currently ABCG2), SSEA1 (currently FUT4), CD44, CD34, NES, GFAP and SOX2. When induced to differentiate, most cells express markers of premature oligodendrocytes, consistent with their tumor origin, though a small percentage of the cells expressed GFAP and beta 3 tubulin (TUBB3), markers of astrocytes and neurons, respectively. Furthermore, when transplanted in immune deficient mice, these tumor spheres initiated new tumors, even when a single sphere or 100 dissociated cells were injected. We have injected and re-isolated these cells through four rounds of serial transplantation without the loss of stem cell characteristics. Hence, the cells isolated from this mouse brain tumor model indicate that at the cellular level, the pathobiology of tumorigenesis between mouse and human is similar.
To determine which cells within the tumor stem cell culture are stem cells, we isolated subpopulations of cells using FACS sorting, and showed that the "side-population" (SP) is enriched for stem cells. As few as 50 SP cells can initiate a tumor, while 5,000 non-SP cells will not initiate tumorigenesis. Since SP cells constitute only approximately 1 percent of the total culture, it was necessary to isolate this pure population of cells to examine molecular differences between normal and cancer stem cells. From genetically identical animals, we isolated normal stem cells and cancer stem cells from the brain and purified SP cells. We then used combined gene expression analysis and aCGH analysis to identify genetic lesions that are associated with the transformed stem cell phenotype. We identified 41 candidate genes through this approach, and we are in the process of testing whether they are tumor suppressors or oncogenes with the ability to transform normal stem cells.
We are also isolating cancer stem cells from a mouse model of medulloblastoma to compare and contrast to oligodendroglioma cancer stem cells to examine the molecular signature of cancer-initiating cells from different brain tumor models and human brain tumor initiating cells. In parallel, we are generating new transgenic animals that will allow selective ablation of different populations of cells expressing specific stem cell markers in vivo. We will use these animals to test the efficacy of ablating cancer stem cells in vivo.
Id gene function in neural stem cells
Abnormal brain development is a common underlying cause of many psychiatric and neurological disorders affecting children. For example, 2 to 6 children per 1,000 in the U.S. have autism, and the rate is increasing. While the cause for autism is not known, it is evident that the condition arises from the disruption of normal brain development. Our long-term goal is to understand the molecular mechanisms that control mammalian brain development by focusing on pathways that regulate neural stem cell maturation.
Id genes encode helix-loop-helix (HLH) transcription factors that are thought to inhibit differentiation and promote proliferation. Mutational analyses of Id genes clearly demonstrate their essential functions in many tissues during mammalian development. Recently, we have found that two members of this gene family, Id2 and Id4, can either stimulate or inhibit proliferation of neural cells, depending on the cell type in which they are expressed. Id4 is required for cell cycle progression and expansion of the early telencephalic neuroepithelium. However, in postmitotic neurons of the cortex, Id4 is required to block aberrant cell cycle re-entry (Yun et al., 2004). Similarly, although there are many reports that indicate that Id2 promotes proliferation, we have recently observed that overexpression of Id2 in the neuroepithelium has the surprising effect of reducing neuroepithelial expansion, leading to a smaller brain size, the opposite of what one might expect from the traditional view of Id2 gene function. Consistently, neurospheres (neural stem cells) derived from Id4-/- or Id2-overexpressing mice do not proliferate or form secondary spheres (self-renewal) as robustly as those derived from littermate controls.
To begin to explore the possible mechanisms of Id gene function in the developing brain, we have performed microarray analysis of gene expression in Id4-/- cortex and Id2-gain-of-function mutant cortex and identified many potential downstream target genes. Among the differentially expressed genes are known targets of the Wnt signaling pathway and genes involved in chromosome condensation. We are currently validating these genes and testing their function in vitro.
Stem cell-based therapy for MPS VII mouse model
The neurodevelopmental and neurodegenerative problems in the central nervous system (CNS) of patients with MPS remain a major challenge for treatment. Our goal in this project is to isolate and manipulate neural stem cell-like cells from the bone marrow or cord-blood and use them for gene therapy and autologous transplantation. We are using the MPS VII mouse (a model for Sly syndrome in humans in which the GUS gene is mutated) to test the efficacy of expressing GUS in neural stem cell-like cells prior to their transplantation in the brain.
We found that cells isolated from mouse bone marrow or fetal liver tissue, which serve as a model for human cord-blood cells, can be manipulated to form neurospheres in culture. In particular, we found that addition of Noggin, a BMP inhibitor, significantly increased the number of neurospheres that form from fetal liver tissue. When induced to differentiate in vitro, fetal liver-derived neural stem cells (FLNSC) differentiated along the neural lineage, expressing markers of neurons, astrocytes, and oligodendrocytes.
Currently, we are testing the efficiency of lenti-viral transduction of FLNSC as well as their survival and cell fate when transplanted into neonatal MPS VII mutant brains.
Lab staff
Principal Investigator:
Kyuson Yun, Ph.D.Research Assistant: Benjamin Low, B.S., Meng Xie
Postdoctoral Fellow: Xiaoyang (Tony) Luo
Predoctoral Student: Molly Harris, B.S.
Visiting Investigator: Yaochen Li, M.D., Ph.D.
Co-Op Student: Emily Crotteau
Research Administrative Assistant: Norma D. Buckley
Publication listings
Nam JS, Park E, Turcotte TJ, Palencia S, Zhan X, Lee J, Yun K, Funk WD, Yoon JK. 2007. Mouse R-spondin2 is requred for apical ectodermal ridge maintenance in the hindlimb. Dev Biol 311:124-135.
Yun K, Tennent B. 2007. Cancer stem cells. Drug Discovery Today: Disease Models, doi:10.1016/j.ddmod.2007.10.001.
Yun K, Mantani A, Garel S, Rubenstein J, Israel M. 2004. Id4 regulates neural progenitor proliferation and differentiation in vivo. Development 131(21):5441-5448.
Yun K, Garel S, Fischman S, Rubenstein JLR. 2003. Patterning of the lateral ganglionic eminence by the Gsh1 and Gsh2 homeobox genes regulates striatal and olfactory bulb histogenesis and the growth of axons through the basal ganglia. J Comp Neruol 461(2):151-165.
Andrews G, Yun K, Rubenstein J, Mastick G. 2003. Dlx transcription factors regulate differentiation of dopaminergic neurons of the ventral thalamus. Mol Cell Neurosci 23(1):107-120.
Garel S, Yun K, Grosschedl R, Rubenstein JLR. 2002. The early topography of thalamocortical projection is shifted in Ebf1 and Dlx 1/2 mutant mice. Development 129:5621-5634.
Yun K, Fischman S, Johnson J, Hrabe de Angelis M, Weinmaster G, Rubenstein JLR. 2002. Modulation of the Notch signaling pathway by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon. Development 129:5029-5040.
Yun K, Potter S, Rubenstein JLR. 2001. Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128:193-205.
