Induced Pluripotent Stem Cell Modeling of Dravet Syndrome

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1 Induced Pluripotent Stem Cell Modeling of Dravet Syndrome December 2, 2011 Jack M. Parent, MD, Department of Neurology, University of Michigan Medical Center

2 Disclosures and Acknowledgements Disclosures NINDS: Grant Support CURE: Grant Support DSF: Grant Support; SAB EF: Grant Support; PAB GIF: Medical Advisory Board Cephalon, Inc.: Unpaid consultant Acknowledgements Parent Lab: Yu Liu, Huilin Shi, Helen Zhang Miriam Meisler: Julie Jones, Janelle O Brien Lori Isom: Luis Lopez- Santiago, Yukun Yuan, Heather O Malley, Gustavo Patino, David Auerbach Jose Jalifé, Todd Herron Cleveland Clinic: Marvin Natowicz, Ajay Gupta

3 Learning Objectives Learn what induced pluripotent stem cell (ipsc) reprogramming is and how it is used Describe the abnormalities identified in Dravet Syndrome ipsc-derived neurons What ipscs are Topics Modeling Dravet Syndrome (DS) epilepsy phenotype using patient-derived ipscs Using ipscs to study Sudden Unexplained Death in Epilepsy (SUDEP) mechanisms

4 Pluripotent Stem Cells -Self-renew -Give rise to all cell types (mod. from: 2006 Terese Winslow, NIH Resource for Stem Cell Research) American Epilepsy Society Annual Meeting

5 What is ips? Involves direct reprogramming of somatic cells, including fully differentiated cells, to a stem cell (pluripotent) state self-renew give rise to all tissues of the body Gene (or mrna or protein) transfer of defined transcription factors Yields cells very much like hescs derived from the ICM of blastocysts

6 Use of ipscs 5 ips cells may be differentiated into the relevant tissue type and used to examine disease mechanisms or screen for new therapies

7 The GEFS+ Spectrum

8 Dravet Syndrome (DS) Epileptic encephalopathy with early onset seizures and cognitive impairment Caused by de novo mutations in the neuronal sodium channel gene SCN1A and haploinsufficiency 50% SCN1A is insufficient for normal neuronal function Kearney & Meisler, 2010

9 Sodium Channel Mutation ipscs Transgenic mice NPCs Xenopus oocytes Patientspecific neurons

10 Advantages of ipsc Method Neurons derived from patient fibroblasts via induced pluripotent stem cells Function of the human mutant channel tested directly Includes patient-specific genomic background Tested in neurons, which contain accessory proteins & splice factors that may affect the channel protein Provides cultured cell model in unlimited supply for study of pathogenesis and screening of compounds to identify new therapies

11 ipscs derived from 2 subjects with DS Both seven year old boys with uncontrolled epilepsy and SCN1A mutations One is a SCN1A splice site mutation that causes skipping of exon 14; the other is a c.975t>a SCN1A nonsense mutation (PTC) ipscs from skin biopsy-derived fibroblasts 3 intact human controls (1-25 yrs)

12 ipscs derived from DS patient fibroblasts 4 RV -Sox2 -cmyc -Klf4 -Oct4 Fibroblasts 10d ipscs 30d ipscs H7 hescs Pluripotentcy markers Generate 3 germ layers AES Annual American Epilepsy Society Annual Meeting Meeting Yu Liu

13 DS Patient ipsc-derived Neural Progenitors Cells and Neurons Neural Rosettes Neurospheres Yu Liu Pax 6 and Musashi Neural stem cell markers

14 ipsc-derived neurons show brain sodium currents A) Pyramidal Neuron Sodium Currents 10 ms 50 pa/pf -80 mv -30 mv +TTX A) Bipolar Neuron 3-4 wk neurons Luis Lopez-Santiago American Epilepsy Society Annual Meeting

15 Dravet ipsc neurons show increased sodium current Luis Lopez- Santiago -3-4-week differentiated neurons -peak sodium current density

16 Increased excitability with repetitive firing and spontaneous bursting in DS ipsc neurons 5-6 wk neurons Na v 1.6 overcompensates Human epilepsy in a dish! Yukun Yuan

17 ipscs to study SUDEP Ion channelopathies likely predispose to SUDEP DS and GEFS+ may have high incidence of SUDEP (e.g., multiple SUDEP events in a single family) Fibroblast ipsc -actinin -Ventricular -Atrial -Nodal (Na v 1.1 in SA node) Cardiac Myocyte

18 Cardiac myocytes from DS ipscs

19 Conclusions The ipsc method is providing insight into epilepsy mechanisms ipsc-derived neurons and cardiac myocytes will be useful to screen for new therapies for epilepsy and its co-morbidities The ipsc method offers the potential for individualized treatment of epilepsy