Studies on adult stem cells

While our bodies are aging, in quite a few tissues, new cells are born every day, till the day we die. As you are reading this sentence, there is perhaps a new neuron born in your brain (and some are dying). Where does it come from, why does it come now and will it do any good? The field of adult stem cell biology deals with the basic functions of stem cells as found in most species and with their therapeutic potential. It is the stem cells found in each tissue that possess that almost magical regenerative ability. Having endogenous cell replacement is not without risks. Most of us will at some point develop tumors; is there a connection between aging, stem cells and cancer? It is likely, but we don’t know. Accumulating evidence these days, including research presented in this thesis, points to the common molecular pathways that govern behavior of stem cells and cancer development. There are several ways one can study stem cells of the adult mammalian brain; our efforts have focused on employing unbiased searches for stem cell signatures, testing candidate genes for their importance in regulating adult stem cell characteristics and creating transgenic mice for the study of the stem cell progeny during health and injury. The first two studies presented in this thesis aim at developing methods for the analysis of gene expression profiles of stem and progenitor cells. Based on a collaborative effort, we constructed cDNA libraries representing transcripts present in adult neural stem cells in vivo and in vitro as well as genes expressed by cells in the neurogenic microenvironment of the lateral ventricle. The cDNA libraries have further been used for the assembly of cDNA arrays for the quantitative analysis of gene expression in stem cells. The collected sequencing data and gene expression profiles have been used in a bioinformatic search for genes that are preferentially expressed in neural stem cells. We have found several interesting targets that could serve as new stem and progenitor cell markers. Studies III and IV are based on the same investigative principle: we chose two candidate pathways for the analysis of their influence on adult neural stem cell biology. A clinically important issue is the cellular origin of tumors. There is an emerging view in tumor biology that only a subset of the cells in a tumor supports the expansive cancer growth. Consequently, study III describes the functions of a tumor suppressor, p53, in adult neural stem/progenitor cells as a way of finding common mechanisms between stem cells and tumor formation. p53 is established as an important inhibitor of tumorigenesis and p53 mutations are found in most human cancers. We have found that p53 is an important negative regulator of self-renewal and proliferation of neural stem cells. Study IV continues with a signaling pathway involved in stem cell identity and selfmaintenance. We focused on elucidating the importance of RBP-J/Notch signaling in adult neural stem cells. Using viral approaches in vivo by ventricular injections of adenoviruses and lentiviruses expressing Cre recombinase in a conditional floxed RBP-J mouse line we have been able to pinpoint the effects of deleting RBP-J in a specific cell population, ependymal cells. The deletion of RBP-J in ependymal cells has uncovered their differentiation potential along the neurogenic pathway and the importance of Notch signaling in maintenance of ependymal cell quiescence. Studies V and VI are based on transgenic mice for genetic tracing of candidate stem cell populations. We have constructed two mouse lines: a Nestin-CreER line and a FoxJ1-CreER line. Nestin-CreER expresses CreER in stem and progenitor cells of the central nervous system providing a lineage tool by temporal induction of recombination and thereby visualization of neurogenesis as well as temporal gene deletion. FoxJ1-CreER expresses CreER in ependymal cells of the brain and spinal cord and is used in study six for delineating the functions of ependyma-derived progeny in response to spinal cord injury. In conclusion, by defining the molecular events that are instrumental in neural stem cell biology and following cell fates of candidate stem cell populations, I have contributed to our understanding of stem cell behavior. List of Thesis Publications I. Sievertzon M, Wirta V, Mercer A, Meletis K, Erlandsson R, Wikstrom L, Frisén J, Lundeberg J. Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method. BMC Neurosci. 2005 Apr 15;6(1):28. II. Williams C*, Wirta V*, Meletis K, Wikström L, Carlsson L, Frisén J, Lundeberg J. Catalog of gene expression in adult neural stem cells and their in vivo microenvironment. Experimental Cell Research. 2006 Mar 16; [Epub ahead of print] III. Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisén J. p53 suppresses the self-renewal of adult neural stem cells. Development. 2006 Jan;133(2):363-9. IV. Carlén M, Meletis K, Cassidy R, Evergren E, Tanigaki K, Almendola M, Naldini L, Honjo T, Shupliakov O, Frisén J. RBP-J maintains ependymal cell quiescence and inhibits neurogenesis in the adult brain. Manuscript V. Carlén M*, Meletis K*, Barnabé-Heider F, Frisén J. Genetic visualization of neurogenesis. Submitted Manuscript VI. Meletis K, Carlén M, Barnabé-Heider F, Evergren E, Shupliakov O, Frisén J. Fate mapping of candidate stem cells in the adult spinal cord. Manuscript