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

2. 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

3. 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

4. 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

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

6. 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

7. 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

8. Somatic Cell Nuclear Transfer
Danielle Feldman
Mriganka Sur
MIT

9. Somatic Cell Nuclear Transfer
Danielle Feldman
Mriganka Sur
MIT

10. 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

11. 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

12. Nobel Prize 2012
Danielle Feldman
Mriganka Sur
MIT

13. 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

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

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

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

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

18. 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

19. 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

20. 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

21. 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

22. 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

23. X-inactivation
Danielle Feldman
Mriganka Sur
MIT
Sciencebuddies.org

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

25. 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 Apr 20;101(16): 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 Feb 23;315(5815): 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 Feb 10;106(6):
Danielle Feldman
Mriganka Sur
MIT
Tropea et al. 2009

26. 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

27. 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

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

29. 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

30. 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

31. 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

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

33. MeCP2 in non-neuronal cells
Rett syndrome microglia damage dendrites and synapses by the elevated release of glutamate.
Maezawa I, Jin LW.
J Neurosci Apr 14;30(15):
Rett syndrome astrocytes are abnormal and spread MeCP2 deficiency through gap junctions.
Maezawa I, Swanberg S, Harvey D, LaSalle JM, Jin LW.
J Neurosci Apr 22;29(16):
Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.
Ballas N, Lioy DT, Grunseich C, Mandel G.
Nat Neurosci Mar;12(3): 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 Jun 29;475(7357): doi: /nature10214.
Danielle Feldman
Sally Kwok
Mriganka Sur
MIT

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

35. Thank You!
Danielle Feldman
Mriganka Sur
MIT