Human Stem Cells & Applications
Danielle Feldman
July 17, 2013
Danielle Feldman
Mriganka Sur
MIT
Overview
• Stem cells
– 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
– 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
– 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
– 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
– 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
•
Danielle Feldman
Mriganka Sur
MIT
The “ideal”
• Non-invasive method of
obtaining/generation
• No rejection from immune
system upon
transplantation
• No controversial issues
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
– 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 Xlinked 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
1966
1983
1992
1999
2001
2009
• 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.
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!
Clairescrusade.org
Danielle Feldman
Mriganka Sur
MIT
MeCP2 and Rett Syndrome
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•
•
•
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
Chahrour et al. 2008
Chahrour and Zoghbi, 2007
Danielle Feldman
Mriganka Sur
MIT
MeCP2 Target Genes
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.
Tropea et al. 2009
Danielle Feldman
Mriganka Sur
MIT
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
– 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
Danielle Feldman
Mriganka Sur
MIT
Li and Yang, 2013
iPS
Danielle Feldman
Mriganka Sur
MIT
NPC
Neuron
Goulburn et al. 2011
Shi et al. 2012
7/16/13
Patch Clamping
www.discoverymedicine.com/Jeffrey-J-Clare/files/2010/03/clare_no46_figure_2a.jpg.jhtml?id=2|attachment_7
Figure 2A. Diagram of the whole cell patch clamp technique. See text for explanation.
Danielle Feldman
Mriganka Sur
MIT
Copyright 2013 Discovery Medicine.
Copyright 2013
Print Discovery
This Page 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