Mouse and Patient-Derived induced Pluripotent Stem Cell (ipsc) models of

Transcription

1 Mouse and Patient-Derived induced Pluripotent Stem Cell (ipsc) models of Neurogenetic Diseases Tony Wynshaw-Boris, MD, PhD James H. Jewell MD 34 Professor of Genetics Chair, Department of Genetics and Genome Sciences CWRU School of Medicine

2 Precision Medicine: Translation of Genomic Information to Treat Individual Undiagnosed Disease (Neurogenetic Disease) Genetics and Genomics Whole Genome-Exome Sequencing Gene Expression (RNAseq) Epigenetics (ChIPseq) Model Systems Mouse Models Induced Pluripotent Stem Cell (ipsc) Models Drug Screening Drug Libraries Chemical Libraries shrna Libraries Treatment Based on Genomic Alteration(s)/Pathway(s)

3 Precision Medicine: Translation of Genomic Information to Treat Individual Undiagnosed Disease (Neurogenetic Disease) Genetics and Genomics Whole Genome-Exome Sequencing Gene Expression (RNAseq) Epigenetics (ChIPseq) Model Systems Mouse Models Induced Pluripotent Stem Cell (ipsc) Models Drug Screening Drug Libraries Chemical Libraries shrna Libraries Treatment Based on Genomic Alteration(s)/Pathway(s)

4 Normal Type I Lissencephaly (Severe) Severe MR Seizures Early Death Incidence: 1/50,000-1/100,000

5 Type I Lissencephaly Defective neuronal migration, impaired proliferation of neural precursors Severe mental retardation, epilepsy and early death Normal control Isolated Lissencephaly Sequence (ILS) Miller-Dieker Syndrome (MDS) Kato, Dobyns, Hum Mol Genet 2003

6 LIS1: Regulation of Cytoplasmic Dynein Localization and Function Ser198 Thr219 Ser231 Ser251

7 Key insights from mouse models of lissencephaly Dose-dependent decrease of Lis1 protein causes: Disorganized cortical layers and hippocampus (Hirotsune et al., Nat Genet 1998) Defective neuronal migration (Hirotsune et al., Nat Genet 1998; Youn et al., J Neurosci 2009) Hyperexcitability, seizures (Fleck et al., J Neurosci 2000; Greenwood et al., Ann Neurol 2000) Accumulation of excitatory presynaptic vesicles (Greenwood et al., Ann Neurol 2000) Aberrant mitosis and neurogenesis (Faulkner et al., Nat Cell Biol 2000; Yingling et al., Cell 2008) Combined haploinsufficiency of Lis1/14-3-3ε or Lis1/Ndel1 exacerbates defective neural migration (Toyo-oka et al., Nat Genet 2003; Sasaki et al., Mol Cell Biol 2005) Overexpression of Ndel1 can compensate for some of the cellular defects in Lis1 mutants (Yingling et al., Cell 2008; Lam et al., JCS 2009) Hippenmeyer et al., Neuron 2010 Greenwood Greenwood et Fleck al., Ann et et al., al., Neurol Ann J Neurosci Neurol

8 Mouse and Human Neocortical Development Liu et al. Cell 146: 18 (2011)

9 ipsc Models of Neurological Diseases Marcheto et al HMG, 2009

10 ipsc Models of Neurological Diseases Marina Bershteyn, PhD Marcheto et al HMG, 2009

11 Modeling MDS in ipscs Patient fibroblasts Patient ipscs MDS1r(17) MDS2 MDS3 Okita et al. (Nat Meth, 2011) SOX2, KLF4, LIN-28, L-MYC, OCT3/4, shp53 Mesoderm Endoderm Fib ipsc-1 ipsc-2 Fib ipsc-1 ipsc-2 Fib ipsc-1 ipsc-2 Ectoderm SOX2 OCT4 NANOG ACTIN SMA AFP TUJ1

12

13 MDS 9208 MDS 6097 WT BJ Day 26

14 PCNT/pHH3/PARD6b SOX2/PCNT/DAPI

15 WT BJ 1 month MDS month SOX2/PCNT/pHH3/PARD6b

16 Chromosome Therapy in Patient-Derived induced Pluripotent Stem Cells (ipscs) LIs1 Chromosome 17 Bershteyn et al Nature (2014) 507:99

17 Ring chromosome formation Incidence ~1:50,000 Every chromosome Problems: Genetic Structural

18 Unstable behavior of ring chromosomes during mitosis Failure to pair with homologous chromosome Frequent ring loss leading to monosomy Formation of dicentric rings Secondary ring derivatives Formation of anaphase bridges Increased aneuploidy and high cellular death rate

19 Fibroblasts from three MDS patients LIs1 Chromosome 17

20 Restored levels of LIS1 and ε in ipscs from MDS1r(17) fibroblasts

21 Normal karyotype in ipsc clones from MDS1r(17) fibroblasts

22 Potential Mechanisms of Loss of Ring17

23 Replacement of Ring with Duplication of Normal Chromosome 17

24 The same mechanism works with other ring chromosomes

25 Summary of ring chromosome correction in ipscs Non-disjunction, ring loss, compensatory UPD, selection

26 Acknowledgements Wynshaw-Boris Lab (UCSF) o Marina Bershteyn Shinya Yamanaka (UCSF/Kyoto) o Yohei Hayashi Wynshaw-Boris Lab (CWRU) o Taehyun Kim Arnold Kriegstein (UCSF) Bruce Conklin (UCSF/Gladstone) Laurie Weiss (UCSF) o Katie Tsang o Guillaume Desachy Ed Hsiao (UCSF) o Chris Schlieve