Human Stem Cells & Applications Danielle Feldman July 17, 2013 Danielle Feldman Mriganka Sur MIT

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

Stem Cell Goals Regenerative medicine Renewable source of replacement cells and tissues with which to treat disease Study development of specific cell types in vitro Healthy individuals & those with diseases Alternative to mouse models The “ideal” Non-invasive method of obtaining/generation No rejection from immune system upon transplantation No controversial issues Danielle Feldman Mriganka Sur MIT

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

Embryonic Stem Cells 4-5 days Danielle Feldman Mriganka Sur MIT

Embryonic Stem Cells Pluripotent Regenerative Medicine Can make all 3 germ layers Can replicate indefinitely Regenerative Medicine Tissue Replacement Controversial Destruction of a human embryo Beginning of life issue Wasteful not to use those embryos destined for destruction? Danielle Feldman Mriganka Sur MIT

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

Somatic Cell Nuclear Transfer Danielle Feldman Mriganka Sur MIT

Somatic Cell Nuclear Transfer Danielle Feldman Mriganka Sur MIT

Somatic Cell Nuclear Transfer Cloning controversy Could lead to the cloning of humans? Requires human eggs IVF Donations (requires hormone treatments) Create artificial eggs Not federally funded in the US Danielle Feldman Mriganka Sur MIT

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

Nobel Prize 2012 Danielle Feldman Mriganka Sur MIT

Induced Pluripotent Stem Cells The “ideal” Non-invasive method of obtaining/generation No rejection from immune system upon transplantation No controversial issues Danielle Feldman Mriganka Sur MIT

iPSC Applications Danielle Feldman Mriganka Sur MIT Mercola et al. 2013

iPSCs in Clinical Trials – Macular Degeneration Danielle Feldman Mriganka Sur MIT THE ASAHI SHIMBUN

iPSCs: Summer 2013 Danielle Feldman Mriganka Sur MIT THE ASAHI SHIMBUN

Neurodevelopmental iPSC Models Danielle Feldman Mriganka Sur MIT Chailangkarn et al. 2012

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

Rett Syndrome Autism spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) Affects ~1 in 10,000 girls Patients develop normally up to 6-18 months, at which point they experience developmental stagnation/regression Growth arrest Autistic features Acquired motor abnormalities, stereotypic hand movements, seizures Danielle Feldman Mriganka Sur MIT

Rett Background Neurodevelopmental disorder, Rett syndrome, was first described by Andreas Rett. Bengt Hagberg (Swedish) published Rett cases in Annals of Neurology. MeCP2 (Methyl-CpG binding protein 2) gene was first identified in mice by Adrian Bird. Huda Zoghbi, MD discovered that mutations in MECP2 cause Rett Syndrome. KO mouse models were developed by Adrian Bird, and Rudolf Jaenisch. Human induced pluripotent stem cell (iPSC) model of Rett was developed. 1966 1983 1992 1999 2001 2009 Danielle Feldman Sally Kwok Mriganka Sur MIT

Claire Medicine offers only supportive measures Feeding tubes, orthopedic braces, surgeries, seizure medications Need a more high-throughput method for drug testing = BETTER MODEL! Danielle Feldman Mriganka Sur MIT Clairescrusade.org

MeCP2 and Rett Syndrome Mutations are almost always de novo 8 most common mutations account for 70% of mutations Region of mutation can determine severity XCI enhances phenotypic variability Danielle Feldman Mriganka Sur MIT Williamson and Christodoulou 2006

X-inactivation Danielle Feldman Mriganka Sur MIT Sciencebuddies.org

Mecp2 Transcriptional modulator MeCP2 Target Genes Danielle Feldman Mriganka Sur MIT Chahrour et al. 2008 Chahrour and Zoghbi, 2007

Good news: Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice. Luikenhuis S, Giacometti E, Beard CF, Jaenisch R. Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6033-8. Epub 2004 Apr 6. Reversal of neurological defects in a mouse model of Rett syndrome. Guy J, Gan J, Selfridge J, Cobb S, Bird A. Science. 2007 Feb 23;315(5815):1143-7. Epub 2007 Feb 8. Partial reversal of Rett Syndrome-like symptoms in MeCP2 mutant mice (by an active peptide fragment of Insulin-like Growth Factor 1 (IGF-1)) Tropea D, Giacometti E, Wilson NR, Beard C, McCurry C, Fu DD, Flannery R, Jaenisch R, Sur M. Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):2029-34. Danielle Feldman Mriganka Sur MIT Tropea et al. 2009

Ideas of treating Rett Modifier gene Increase MeCP2 level Correct the pathway downstream of MeCP2 Small molecules that can help MeCP2 fold properly Danielle Feldman Sally Kwok Mriganka Sur MIT

Overview Stem cells Rett Syndrome Embryonic stem cells Somatic cell nuclear transfer Induced pluripotent stem cells Rett Syndrome Rett iPSC modeling Danielle Feldman Mriganka Sur MIT

iPSC models of RTT Danielle Feldman Mriganka Sur MIT Walsh and Hochedlinger, 2010

Controls Issues with controls in iPS models Donor specific differences in genome Differences introduced during the processes of reprogramming and derivation We utilize XCI and expand single iPS clones into large clonal populations expressing only one allele TALEN technology: specifically target MeCP2 in hES and hiPS cells Knockout gene Insert disease relevant mutations in MeCP2 Correction of point mutation in MeCP2 mutant line Danielle Feldman Mriganka Sur MIT

Generating isogenic iPSC-derived lines Schematics of TALEN and the active TALEN/DNA complex for double stranded DNA cleavage. ( a ) TALEN protein is comprised of a TAL effector (TALE) and the FokI DNA cleavage domain (FokI). The architecture shown here is of a full-length TAL effector (e.g., AvrXa7) with the number of amino acids of each domain indicated above. The C-terminus of the TALE contains nuclear localization signals (NLS) directing the TALEN into the nuclei of target cells. AD is the activation domain. ( b ) Paired TALENs (homo- or heterodimeric). Danielle Feldman Mriganka Sur MIT Li and Yang, 2013

iPS NPC Neuron Danielle Feldman Mriganka Sur Goulburn et al. 2011 MIT UPDATE FROM LAST TIME Danielle Feldman Mriganka Sur MIT Goulburn et al. 2011 Shi et al. 2012

Patch Clamping Copyright 2013 Discovery Medicine. Danielle Feldman Mriganka Sur MIT Copyright 2013 Discovery Medicine.

MeCP2 in non-neuronal cells Rett syndrome microglia damage dendrites and synapses by the elevated release of glutamate. Maezawa I, Jin LW. J Neurosci. 2010 Apr 14;30(15):5346-56. Rett syndrome astrocytes are abnormal and spread MeCP2 deficiency through gap junctions. Maezawa I, Swanberg S, Harvey D, LaSalle JM, Jin LW. J Neurosci. 2009 Apr 22;29(16):5051-61. Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Ballas N, Lioy DT, Grunseich C, Mandel G. Nat Neurosci. 2009 Mar;12(3):311-7. Epub 2009 Feb 22. A role for glia in the progression of Rett's syndrome. Lioy DT, Garg SK, Monaghan CE, Raber J, Foust KD, Kaspar BK, Hirrlinger PG, Kirchhoff F, Bissonnette JM, Ballas N, Mandel G. Nature. 2011 Jun 29;475(7357):497-500. doi: 10.1038/nature10214. Danielle Feldman Sally Kwok Mriganka Sur MIT

Transplantation into mice Danielle Feldman Mriganka Sur MIT Espuny-Camacho et al. 2013

Thank You! Danielle Feldman Mriganka Sur MIT