title: IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis.
abstract: The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability.
  
  
  
  Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot.
  
  
  
  Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells.
  
  
  
  Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells.
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Kamila
authors.initials: K
authors.lastname: Rosiak
authors.affiliation: Department of Research and Development, Celther Polska, Milionowa 23, 93-193, Lodz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Smolarz
authors.affiliation: Department of Research and Development, Celther Polska, Milionowa 23, 93-193, Lodz, Poland.
authors.forename: Wojciech J
authors.initials: WJ
authors.lastname: Stec
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Peciak
authors.affiliation: Department of Research and Development, Celther Polska, Milionowa 23, 93-193, Lodz, Poland.
authors.forename: Dawid
authors.initials: D
authors.lastname: Grzela
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Winiecka-Klimek
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Stoczynska-Fidelus
authors.affiliation: Shriners Hospitals Pediatric Research Center, Center for Neural Repair and Rehabilitation, Temple University School of Medicine, 3500 N. Broad Street, Philadelphia, PA, 19140, United States of America.
authors.forename: Barbara
authors.initials: B
authors.lastname: Krynska
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Sylwester
authors.initials: S
authors.lastname: Piaskowski
authors.affiliation: Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Rieske
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D001253:Astrocytes / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology; D002478:Cells, Cultured; D058732:Embryoid Bodies / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D058953:Neural Stem Cells / Q000378:metabolism*; D055495:Neurogenesis / Q000502:physiology; D009474:Neurons / Q000378:metabolism
chemical_list: D015415:Biomarkers; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D053148:Caspase 3