Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival‐promoting kinases

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post‐natal rat NPs express low levels of pro‐apoptotic molecules and resist phosphatidylinositol 3′OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage‐restricted precursors as revealed by enrichment for Nestin+/SOX‐2+ cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin+/SOX‐2+ and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3‐times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.

[1]  S. Levison,et al.  Identification of Bax-interacting proteins in oligodendrocyte progenitors during glutamate excitotoxicity and perinatal hypoxia–ischemia , 2013, ASN neuro.

[2]  S. Levison,et al.  Leukemia Inhibitory Factor Is Essential for Subventricular Zone Neural Stem Cell and Progenitor Homeostasis as Revealed by a Novel Flow Cytometric Analysis , 2012, Developmental Neuroscience.

[3]  Chao Chen,et al.  Hypoxia-Ischemia Upregulates TRAIL and TRAIL Receptors in the Immature Rat Brain , 2012, Developmental Neuroscience.

[4]  A. Pyle,et al.  Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. , 2011, Cell stem cell.

[5]  Magdalena Götz,et al.  In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. , 2010, Cell stem cell.

[6]  Erika Pastrana,et al.  Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny , 2009, Proceedings of the National Academy of Sciences.

[7]  K. Blomgren,et al.  Differential Recovery of Neural Stem Cells in the Subventricular Zone and Dentate Gyrus After Ionizing Radiation , 2009, Stem cells.

[8]  Changlian Zhu,et al.  Death effector activation in the subventricular zone subsequent to perinatal hypoxia/ischemia , 2007, Journal of neurochemistry.

[9]  T. Wood,et al.  Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF‐I and FGF‐2 requires differential stimulation of multiple signaling pathways , 2007, Glia.

[10]  Zhengang Yang,et al.  Neural Stem/Progenitor Cells Participate in the Regenerative Response to Perinatal Hypoxia/Ischemia , 2006, The Journal of Neuroscience.

[11]  C. Y. Brazel,et al.  Glutamate enhances survival and proliferation of neural progenitors derived from the subventricular zone , 2005, Neuroscience.

[12]  Jialin C. Zheng,et al.  Cellular IAP1 regulates TRAIL‐induced apoptosis in human fetal cortical neural progenitor cells , 2005, Journal of neuroscience research.

[13]  Brent A Reynolds,et al.  Neural stem cells and neurospheres—re-evaluating the relationship , 2005, Nature Methods.

[14]  S. Levison,et al.  Neural Stem Cells in the Subventricular Zone are Resilient to Hypoxia/Ischemia whereas Progenitors are Vulnerable , 2004, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[15]  S. Orrenius,et al.  Differential regulation of the mitochondrial and death receptor pathways in neural stem cells , 2004, The European journal of neuroscience.

[16]  T. Wood,et al.  IGF‐I prevents glutamate‐mediated bax translocation and cytochrome C release in O4+ oligodendrocyte progenitors , 2004, Glia.

[17]  Mahendra Rao,et al.  SOX2, a Persistent Marker for Multipotential Neural Stem Cells Derived from Embryonic Stem Cells, the Embryo or the Adult , 2004, Developmental Neuroscience.

[18]  S. Noggle,et al.  Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide , 2003, The Journal of cell biology.

[19]  J. Barker,et al.  Prospective Cell Sorting of Embryonic Rat Neural Stem Cells and Neuronal and Glial Progenitors Reveals Selective Effects of Basic Fibroblast Growth Factor and Epidermal Growth Factor on Self-Renewal and Differentiation , 2003, The Journal of Neuroscience.

[20]  S. Levison,et al.  Damage to the Choroid Plexus, Ependyma and Subependyma as a Consequence of Perinatal Hypoxia/Ischemia , 2003, Developmental Neuroscience.

[21]  Y. Hannun,et al.  Ceramide in apoptosis: an overview and current perspectives. , 2002, Biochimica et biophysica acta.

[22]  B. Zhivotovsky,et al.  Cell death in adult neural stem cells , 2002, Cell Death and Differentiation.

[23]  S. Temple,et al.  LeX/ssea-1 Is Expressed by Adult Mouse CNS Stem Cells, Identifying Them as Nonependymal , 2002, Neuron.

[24]  T. Wood,et al.  Insulin-like Growth Factor I, but Not Neurotrophin-3, Sustains Akt Activation and Provides Long-Term Protection of Immature Oligodendrocytes from Glutamate-Mediated Apoptosis , 2002, Molecular and Cellular Neuroscience.

[25]  S. Levison,et al.  Perinatal Hypoxia-Ischemia Induces Apoptotic and Excitotoxic Death of Periventricular White Matter Oligodendrocyte Progenitors , 2001, Developmental Neuroscience.

[26]  Amanda K. Pearce,et al.  Integrating stress-response and cell-cycle checkpoint pathways. , 2001, Trends in cell biology.

[27]  M. Baudry,et al.  Attenuation of Staurosporine-Induced Apoptosis, Oxidative Stress, and Mitochondrial Dysfunction by Synthetic Superoxide Dismutase and Catalase Mimetics, in Cultured Cortical Neurons , 2001, Experimental Neurology.

[28]  Perry F. Bartlett,et al.  Purification of a pluripotent neural stem cell from the adult mouse brain , 2001, Nature.

[29]  Masahiro Yamaguchi,et al.  Generation of Dopaminergic Neurons in the Adult Brain from Mesencephalic Precursor Cells Labeled with a nestin-GFP Transgene , 2001, The Journal of Neuroscience.

[30]  G. Stark,et al.  Regulation of the G2/M transition by p53 , 2001, Oncogene.

[31]  R. Quarles,et al.  Differences in signal transduction pathways by which platelet‐derived and fibroblast growth factors activate extracellular signal‐regulated kinase in differentiating oligodendrocytes , 2001, Journal of neurochemistry.

[32]  H. Okano,et al.  Nestin-EGFP Transgenic Mice: Visualization of the Self-Renewal and Multipotency of CNS Stem Cells , 2001, Molecular and Cellular Neuroscience.

[33]  I. Weissman,et al.  Direct isolation of human central nervous system stem cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Elledge,et al.  The DNA damage response: putting checkpoints in perspective , 2000, Nature.

[35]  M. Luskin,et al.  Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Ebner,et al.  Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells , 2000, Journal of neuroscience research.

[37]  D. Holtzman,et al.  BDNF Protects the Neonatal Brain from Hypoxic-Ischemic InjuryIn Vivo via the ERK Pathway , 2000, The Journal of Neuroscience.

[38]  M. Ljungman.  Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. , 2000, Neoplasia.

[39]  R. Lutz,et al.  Role of the BH3 (Bcl-2 homology 3) domain in the regulation of apoptosis and Bcl-2-related proteins. , 2000, Biochemical Society transactions.

[40]  Z. Xia,et al.  Neuroprotection by Brain-derived Neurotrophic Factor Is Mediated by Extracellular Signal-regulated Kinase and Phosphatidylinositol 3-Kinase* , 1999, The Journal of Biological Chemistry.

[41]  J. Nalbantoglu,et al.  p53 Induction by Tumor Necrosis Factor‐α and Involvement of p53 in Cell Death of Human Oligodendrocytes , 1999, Journal of neurochemistry.

[42]  R. Lutz,et al.  Bak BH3 Peptides Antagonize Bcl-xL Function and Induce Apoptosis through Cytochrome c-independent Activation of Caspases* , 1999, The Journal of Biological Chemistry.

[43]  B. Scurlock,et al.  Differential responses of oligodendrocytes to tumor necrosis factor and other pro‐apoptotic agents: Role of ceramide in apoptosis , 1999, Journal of neuroscience research.

[44]  A. Giaccia,et al.  The complexity of p53 modulation: emerging patterns from divergent signals. , 1998, Genes & development.

[45]  R. Crowder,et al.  Phosphatidylinositol 3-Kinase and Akt Protein Kinase Are Necessary and Sufficient for the Survival of Nerve Growth Factor-Dependent Sympathetic Neurons , 1998, The Journal of Neuroscience.

[46]  S. Levison,et al.  An improved method for propagating oligodendrocyte progenitors in vitro , 1997, Journal of Neuroscience Methods.

[47]  A. Brogi,et al.  Induction of intracellular ceramide by interleukin‐1β in oligodendrocytes , 1997 .

[48]  H. Matsuzaki,et al.  Activation of protein kinase B (Akt/RAC‐protein kinase) by cellular stress and its association with heat shock protein Hsp27 , 1997, FEBS letters.

[49]  John Calvin Reed.  Double identity for proteins of the Bcl-2 family , 1997, Nature.

[50]  E. Van Obberghen,et al.  cAMP stimulates protein kinase B in a Wortmannin‐insensitive manner , 1997, FEBS letters.

[51]  N. J. Edgell,et al.  Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and beta-adrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms. , 1997, The Journal of biological chemistry.

[52]  G. Wahl,et al.  p53 Mediates Permanent Arrest over Multiple Cell Cycles in Response to γ-Irradiation , 1997 .

[53]  David R. Kaplan,et al.  Regulation of Neuronal Survival by the Serine-Threonine Protein Kinase Akt , 1997, Science.

[54]  F. Mcmorris,et al.  Oligodendrocytes and their precursors require phosphatidylinositol 3-kinase signaling for survival. , 1996, Development.

[55]  J. Antel,et al.  Ciliary neurotrophic factor selectively protects human oligodendrocytes from tumor necrosis factor‐mediated injury , 1996, Journal of neuroscience research.

[56]  J. Grinspan,et al.  Platelet‐derived growth factor is a survival factor for PSA‐NCAM + oligodendrocyte pre‐progenitor cells , 1995, Journal of neuroscience research.

[57]  J. Szaflarski,et al.  Cerebral hypoxia-ischemia stimulates cytokine gene expression in perinatal rats. , 1995, Stroke.

[58]  al. et,et al.  Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice , 1995, Science.

[59]  G. Wahl,et al.  DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. , 1994, Genes & development.

[60]  J. Volpe,et al.  Vulnerability of oligodendroglia to glutamate: pharmacology, mechanisms, and prevention , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[61]  B. Vogelstein,et al.  Participation of p53 protein in the cellular response to DNA damage. , 1991, Cancer research.