Sean J. Morrison, Ph.D. - US grants

DESCRIPTION (Applicant's abstract): Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time (Morrison et al. Cell 88:287). There is great interest in neural stem cells because of their importance in neural development and their therapeutic potential. Although neural stem cells have been extensively characterized at the cellular level little is known about how stem cell function is regulated at a molecular level. The recent isolation of uncultured neural crest stem cells (NCSCs) by flow-cytometry (Morrison et al. Cell 96:737) should greatly facilitate such studies by allowing us to focus gene-profiling experiments on a population of cells that is nearly homogeneous with respect to multipotency and self-renewal; furthermore, these cells are known to function as stem cells in vivo. By profiling gene expression in purified, freshly isolated stem cells, we are much more likely to detect patterns of gene expression that regulate important stem cell properties. Specific Aim 1 will be to screen for genes that regulate NCSC self-renewal by analyzing microarrays for genes that hybridize to probe from self-renewing NCSCs but not to probe from NCSCs undergoing lineage commitment. Specific Aim 2 will be to screen for genes that regulate glial lineage specification by analyzing microarrays for genes that hybridize to probe from immature glial progenitors derived from NCSCs but not to probe from self-renewing NCSCs. Finally, although stem cells from different tissues have long been hypothesized to share common regulatory mechanisms this hypothesis has not been systematically tested. Specific Aim 3 will be to test whether there are "stem cell specific" genes by hybridizing probe made from purified NCSCs or purified hematopoietic stem cells (HSCs) to microarrays of ESTs and "HSC-specific" genes. By identifying genes that are expressed by different types of stem cells but not by restricted progenitors, we may identify elements of genetic programs that are conserved between stem cells. By combining the precise tools that are available to study NCSC function at a cellular level with microarray analysis, it will be possible to address fundamental questions about the genetic regulation of neural stem cells.

DESCRIPTION (provided by applicant): Stem cells persist throughout life in many tissues including the central nervous system (CNS) and regenerate mature cells that are lost due to turnover, injury, and disease. However, the function of stem cells declines with age in diverse tissues including the hematopoietic system, muscle, and brain. Consistent with this, aging tissues have less repair capacity and an increased incidence of degenerative disease. These observations raise the possibility that declines in stem cell function during aging contribute to age-related morbidity and that by uncovering the mechanisms responsible for these declines we might uncover targets for therapeutic intervention. The forebrain lateral ventricle subventricular zone (SVZ) contains stem cells that engage in neurogenesis throughout adult life. The frequency of stem cells, their self-renewal potential, mitotic activity in vivo, and rate of neurogenesis all decline with age, but the physiological mechanisms responsible for these declines are only beginning to be identified. During the prior funding period we discovered that Ink4a expression increases with age in these stem cells. Ink4a encodes a cyclin-dependent kinase inhibitor, p16Ink4a, that impairs proliferation and promotes cellular senescence by activating Rb. Deletion of Ink4a partially rescues the decline in stem cell frequency, mitotic activity, and neurogenesis in aging mice without affecting young mice. In preliminary studies we have discovered that the Alternative reading frame (Arf) at the Ink4a/Arf locus is not detectable in fetal and young adult stem cells but increases in expression with age and negatively regulates stem cell frequency and function. Arf encodes the p19Arf tumor suppressor, which impairs proliferation and promotes cellular senescence by promoting p53 function. This raises the question of whether Arf contributes to the decline in stem cell function with age. In Aim 1 we will test whether conditional deletion of Arf from aging neural stem cells partially rescues the decline in stem cell frequency and function during aging. A second question is what regulates the increase in Ink4a and Arf expression with age. We have discovered that the high mobility group transcriptional regulator, Hmga2, is expressed by fetal and young adult, but not old adult stem cells. Hmga2 increases the frequency and function of neural stem cells in fetal and young adult mice by negatively regulating Ink4a and Arf expression. In Aim 2 we will test whether Hmga2 negatively regulates Ink4a/Arf expression by repressing the JunB transcription factor and whether changes in JunB expression also regulate neural stem cell aging. Finally, we have discovered that the microRNA let-7b, which inhibits Hmga2 expression, increases its expression with age in neural stem/progenitor cells. In Aim 3 we will test whether the decline in Hmga2 expression during aging is caused by the increase in let-7 function. Our experiments offer the opportunity to unravel a novel pathway in which let-7b, Hmga2, JunB, p16Ink4a, and p19Arf regulate the age-related decline in stem cell function and neurogenesis in the mammalian CNS. PUBLIC HEALTH RELEVANCE: Aging tissues exhibit reduced stem cell function, reduced regenerative capacity, and an increased incidence of degenerative disease. We have identified a series of genes that function as part of a pathway that reduces stem cell function and tissue regenerative capacity during aging. By better understanding these mechanisms we will gain new insights into why aging tissues have reduced regenerative capacity as well as therapeutic strategies to enhance regeneration.

Hirschsprung disease, or aganglionic megacolon, is a congenital defect that affects 1 out of 5,000 live births and is characterized by a failure to form enteric nervous system (ENS) in a variable length of the hindgut ADDIN EN.CITE Newgreen200231143114311417D. NewgreenH.M. YoungEnteric nervous system: development and developmental disturbances - Part 1Pediatric and Developmental Pathology224-24752002[1] . This potentially fatal condition results in an inability to coordinate peristaltic movements of the bowel and is most commonly caused by mutations that reduce signaling through the glial cell line-derived neurotrophic factor (GDNF) or endothelin-3 (EDN3) signaling pathways ADDIN EN.CITE Gariepy200137843784378417Gariepy, C. E.Department of Pediatrics, Pediatric Gastroenterology and Nutrition, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA. Gariepy@utsw.swmed.eduIntestinal motility disorders and development of the enteric nervous systemPediatr Res605-13495AnimalsChildEnteric Nervous System/*growth &development*Gastrointestinal MotilityHumansInfant, NewbornIntestinal Diseases/*physiopathologyResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.2001May11328941http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11328941[2] . Both the GDNF receptor, Ret, and the EDN3 receptor Endothelin receptor B (EDNRB) are expressed by the neural crest stem cells (NCSCs) that give rise to the ENS ADDIN EN.CITE ADDIN EN.CITE.DATA [3] . These signaling pathways interact to regulate the proliferation and migration of NCSCs and other neural crest progenitors that colonize the gut, though questions remain about whether the primary role of EDN3 signaling is to inhibit premature differentiation or to promote migration ADDIN EN.CITE ADDIN EN.CITE.DATA [3-10] . Neural crest cells never migrate into the aganglionic portion of the gut in animals affected by Ret or Ednrb deficiency ADDIN EN.CITE ADDIN EN.CITE.DATA [3,6] . These observations raise the possibility of improving the treatment of Hirschsprung disease by combining traditional surgical approaches with cell therapy in which NCSCs are transplanted directly into the aganglionic portion of the gut to generate enteric ganglia by bypassing the migration/proliferation defects ADDIN EN.CITE ADDIN EN.CITE.DATA [3,6,11,12] . Consistent with this possibility, we and others have shown that NCSCs isolated from the fetal rodent gut can engraft and form enteric neurons after transplantation into the aganglionic region of the gut from rodent models of Hirschsprung disease ADDIN EN.CITE ADDIN EN.CITE.DATA [6,11-13] . Nonetheless, before such a therapy can be contemplated for patients it will be necessary to obtain human NCSCs in quantities adequate for clinical use. Since human fetal tissue is very limited and of inconsistent quality for clinical use ADDIN EN.CITE Bjorklund200036463646364617Bjorklund, A.Lindvall, O.The authors are at the Wallenberg Neuroscience Center, Lund University, Solvegatan 17, S-223 62 Lund, Sweden. anders.bjorklund@mphy.lu.seCell replacement therapies for central nervous system disordersNat NeurosciNat NeurosciNature neuroscience537-4436AnimalsBrain/*cytology/embryologyCell Transplantation/methods/*trendsCentral Nervous System Diseases/*therapyCerebrovascular Accident/therapyClinical TrialsEpilepsy/therapyHumansHuntington Disease/therapyNeurons/*transplantationParkinson Disease/therapyRatsRecovery of FunctionSeizures/prevention &control*Stem Cell TransplantationSwine2000Jun10816308http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10816308[14] , it would be ideal to derive NCSCs with enteric characteristics from human embryonic stem (hES) cells. Having extensively characterized mouse and rat enteric NCSCs, we propose to optimize culture conditions to derive human NCSCs with similar properties from hES cells. We will inject these human NCSCs into the aganglionic hindgut of Ednrb mutant rats to test the ability of these cells to form neurons and glia in vivo. These studies will test whether NCSCs with specific regional characteristics can be derived from hES cells and whether these cells engraft in the gut of an animal model of Hirschsprung disease.

DESCRIPTION (provided by applicant): To sustain hematopoiesis, hematopoietic stem cells (HSCs) must persist throughout life, constantly regenerating hematopoietic cells lost to normal turnover, bleeding, and disease. Much has been learned over the past ten years regarding the mechanisms that regulate HSC maintenance. This work has demonstrated that several aspects of cellular physiology are regulated differently in HSCs as compared to other hematopoietic cells. This raises the fundamental question of whether all aspects of cellular physiology are regulated differently in stem cells as compared to restricted progenitors, or whether certain aspects of cellular physiology are house-keeping functions that are regulated similarly in stem cells and restricted progenitors. Unfortunately, many aspects of cellular physiology are technically difficult to study with existing techniques in small numbers of stem cells and therefore have not yet been addressed, leaving large areas of biology unexplored. One such aspect of cellular physiology is the regulation of protein synthesis. There are almost no data on the regulation of translation in any somatic stem cell population, partly because assays have not yet been developed to study translation in small numbers of cells in vivo. We have recently developed an assay that makes it possible to study the rate at which polypeptides are synthesized by individual cells in vivo. Using this assay we have determined that HSCs have significantly lower rates of protein synthesis than other hematopoietic cells even when we control for differences in cell cycle distribution. Our preliminary functional data suggest that HS maintenance depends upon highly regulated rates of protein synthesis. This discovery may explain previously observed defects in HSC self-renewal that were not understood at a mechanistic level. For example, we have demonstrated previously that deletion of the PTEN tumor suppressor in adult hematopoietic cells increases PI3-kinase pathway signaling in HSCs, leading to leukemogenesis and HSC depletion. Although the depletion of PTEN deficient HSCs is known to depend upon a tumor suppressor response induced by mTORC1 and mTORC2 signaling, it is unknown how elevated mTOR signaling increases tumor suppressor expression. In this application, we propose to extend our preliminary data to test whether PTEN is required in adult HSCs to maintain an unusually low level of protein synthesis and whether increased protein synthesis after PTEN deletion induces the tumor suppressor response that depletes HSCs. This work has the potential to yield new techniques to study protein synthesis in rare cell populations in vivo and to open new areas of inquiry related to the role of regulated protein synthesis in hematopoiesis and stem cell function. Defects in the regulation of protein synthesis could potentially contribute to diverse and poorly understood diseases of the hematopoietic system.

ABSTRACT Most cancer deaths are caused by distant metastasis. Yet the mechanisms that regulate distant metastasis are poorly understood. Metastasis is a very inefficient process in which few disseminated cancer cells survive, and even fewer proliferate, but it is not known why. We developed a patient-derived xenograft (PDX) assay in which melanomas engraft very efficiently and spontaneously metastasize. Using this assay, we discovered intrinsic differences in metastatic potential among melanomas from different patients. Some stage III melanomas are ?efficient metastasizers? that spontaneously form distant metastases in patients and in NSG mice while others are ?inefficient metastasizers? that do not form distant macrometastases in patients or in NSG mice under the same experimental conditions. Using this assay, we discovered that melanoma cells experience a spike in reactive oxygen species (ROS) during metastasis and that distant metastasis is limited by oxidative stress. Successfully metastasizing cells undergo reversible metabolic changes during metastasis that increase their capacity to withstand oxidative stress, including increased folate pathway dependence. However, the mechanisms that confer differences in metastatic potential upon melanomas from different patients have not yet been identified. We hypothesize that efficiently and inefficiently metastasizing melanomas have intrinsic metabolic differences that reduce oxidative stress in efficient metastasizers. One impediment to testing this hypothesis is that melanomas from patients grow poorly at clonal density in known culture conditions, preventing certain approaches for studying cancer metabolism and the use of CRISPR gene editing (because single cell-derived clones could not be screened or expanded). We spent years developing a culture medium in which single melanoma cells from patients form tumor organoids (PDOs). This capability raises the general question of whether metabolism and oxidative stress resistance are regulated similarly in PDXs and in PDOs. To address this question, we will compare, side-by-side, the biological properties and metabolic regulation of efficient and inefficient metastasizers in PDXs and PDOs. We will test if efficiently metastasizing melanomas have lower ROS levels or markers of oxidative stress, or increased use of the folate or pentose phosphate pathways, as compared to inefficient metastasizers, and whether this promotes metastasis in PDXs or migration/invasion in PDOs. We will also test if MCT1, a lactate transporter, promotes metastasis and whether efficient metastasizers reduce oxidative stress partly through lactate exchange. Finally, we will test if there are intrinsic differences in mitochondrial function between efficient and inefficient metastasizers that reduce ROS generation. By comparing the biological properties and metabolic regulation of each melanoma in PDX and PDO assays, we will assess the strengths and weaknesses of PDX and PDO assays for studying cancer metabolism and biological differences among patients. These results have the potential to identify new mechanisms that regulate metastasis, new aspects of cancer metabolism, and strategies to block progression.