1. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation
Jessica Ruth Schein
December 5, 2011
Cheung A., Horvath L.M., Grafodaskaya D., Pasceri P., Weksberg R., Hotta A., Carrel L., Ellis J. (2011) Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X- chromosome inactivation. Human Molecular Genetics, 20,

2. Introduction to Rett Syndrome
Objectives
Experimental Design and Results
Conclusion
Future Work
Personal Critique
Questions

3. Rett Syndrome (RTT) Neurodevelopmental autism spectrum disorder
Affects 1 in 10,000 live female births
Developmental arrest
Microcephaly
Hand wringing
Autistic features
Retardation
Loss of language

4. Rett Syndrome
95% of classic RTT paitents have loss-of-function mutation in an X-linked gene
Encodes methyl-CpG binding protein 2 (MECP2)
MECP2 functions as a transcriptional activator and repressor
Binding to methylated CpG dinucleotides of target genes
Binds to methyl-CpG binding domain (MBD)
Recruits chromatin remodeling proteins via transcriptional repression domain (TRD)

5. MECP2 X-linked disorder
Subject to effect of X-chromosome inactivation (XCI) in female cells
Usually, 50% maternal inactivated and 50% paternal inactivated
XCI can occasionally be nonrandom
Leading to phenotypic variability in RTT patients
It has been observed that human induced pluripotent stem (hiPS) cells can retain an inactive X-chromosome in a nonrandom pattern

6. MECP2
Most knowledge of RTT has come from the study of Mecp2 mouse models
Access to human neurons is limited
Mouse models do not accurately reflect the early onset symptoms of RTT
Mouse onset of RTT is in adulthood

7. Objectives Introduction to Rett Syndrome
Experimental Design and Results
Conclusion
Future Work
Personal Critique
Questions

8. Objectives
Isolate MECP2-null human induced pluripotent stem cells and create an isogenic control through a nonrandom pattern of X-chromosome inactivation that can be used in future research of the role of MECP2 in Rett Syndrome.

9. Translation Found a RTT patient with deletion in MECP2
Characterized the deletion
Created human induced pluripotent stem cells
Showed that they display X-inactivation
Determined pattern of X-inactivation was nonrandom
Exploited this pattern to produce an isogenic mutant/control to be used in future research

10. Introduction to Rett Syndrome
Objectives
Experimental Design and Results
Conclusion
Future Work
Personal Critique
Questions

11. Experimental Procedure and Results
Characterize the MECP2 mutation

12. Experimental Procedure and Results
1. Characterize the MECP2 mutation
Multiple ligation dependent probe amplification indicated deletion of exons 3 and 4 of MECP2
Map the Δ3-4 MECP2 mutation
Sequencing determined two deletions remove important domains

13. Experimental Procedure and Results
Large deletion: g.61340_67032delinsACTTGTGCCAC
Associated with an 11bp insert
Removal of entirety of exon 3 and 5’ end of exon 4 including MBD and TRD
Detected a AluSx element spanning the 5’ end which could trigger Alu-recombination-mediated deletions
Small deletion: g.67072_67200del.
3bp microhomology was seen flanking this region
Microhomology-mediated process

14. Experimental Procedure and Results
qPCR to determine copy number variants along MECP2 locus

15. Experimental Procedure and Results
PCR amplification using Δ3-4 MECP2 spanning primers to amplify to mutant MECP2 allele

16. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells

17. Experimental Procedure and Results
2. Generation and characterization of RTT-hiPS cells
Δ3-4 fibroblasts transduced with OCT4, SOX2, KLF4, and c-MYC retroviral vectors and EOS lentiviral vector that reports pluripotency
Expression of pluripotency-associated markers REX1, ABCG2, DNMT3B, and TRA1-60 indicated full reprogramming and pluripotency
3 cell lines propagated extremely successfully, these were Δ3-4 hiPS #6, #20, and #37 cell lines
There ability to differentiate was shown in vitro and in vivo
DNA fingerprinting indicated all RTT-hiPS cells came from their fibroblast of origin

18. Δ3-4 hiPS #37 express pluripotency markers by immunocytochemistry an qRT-PCR
Δ3-4 hiPS cell lines #6, #20, and #37 express pluripotent-associated markers indicating full reprogramming and pluripotency

19. Δ3-4 hiPS #37 differentiate into the three germ layers in vitro via embryoid body formation
Not only did the three lines of Δ3-4 hiPS cells regain pluripotency but they are able to differentiate into neuronal derivatives in vitro
Ectoderm
Mesoderm
Endoderm

20. Δ3-4 hiPS #37 differentiate into the three germ layers in vivo via teratoma formation by injection into immunodeficient mice
Not only did the three lines of Δ3-4 hiPS cells regain pluripotency but they are able to differentiate into neuronal derivatives in vivo
Ectoderm
Mesoderm
Endoderm

21. Δ3-4 hiPS cells carry identical short tandem repeat profiles as their parental fibroblasts of origin and are distinct from other cells

22. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells
Investigate RTT-hiPS cell’s XCI status

23. Experimental Procedure and Results
3. Investigate RTT-hiPS cell’s XCI status
Probed for expression of XIST RNA by RNA-FISH
Performed immunocytochemistry for histone H3 lysine 27 trimethylation (H3K27me3)
Accumulates on the inactive X-chromosome

24. Experimental Procedure and Results
Probed for expression of XIST RNA by RNA-FISH
67-100% of colonies showed positive signal
>90%
>0% 0%

25. Experimental Procedure and Results
Immunocytochemistry for histone H3 lysine 27 trimethylation (H3K27me3)
Similar results to RNA-FISH: % showed positive signal

26. Experimental Procedure and Results
Data suggest Δ3-4 hiPS cells retain an inactive X-chromosome
The lack of XIST RNA and H3K27me3 in some cells can be interpreted in three ways:
Loss of an X-chromosome
Reactivation of the inactive X-chromosome
Inactive X-chromosome that has lost its XIST RNA and H3K27me3 but remains inactive

27. Experimental Procedure and Results
Data suggest Δ3-4 hiPS cells retain an inactive X-chromosome
The lack of XIST RNA and H3K27me3 can be interpreted in three ways:
Loss of an X-chromosome
Reactivation of the inactive X-chromosome (data shown later)
Inactive X-chromosome that has lost its XIST RNA and H3K27me3 during in vitro cultures but remains inactive

28. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells
Investigate Δ3-4 hiPS cell’s XCI status
Investigate pattern of XCI in Δ3-4 hiPS cells

29. Experimental Procedure and Results
4. Investigate pattern of XCI in Δ3-4 hiPS cells
Androgen receptor (AR) assay
Detect heterozygous trinucleotide repeat polymorphisms in the first exon of the X-linked AR gene by PCR
distinguish between paternal and maternal X-chromosome
First digested with methylation-sensitive enzymes prior to PCR

30. Experimental Procedure and Results
Androgen receptor (AR) assay of Δ3-4 hiPS cells
Shows extreme skewing pattern indicating a nonrandom pattern of XCI
Indicates inactive X-chromosome

31. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells
Investigate Δ3-4 hiPS cell’s XCI status
Investigate pattern of XCI in Δ3-4 hiPS cells
Compare MECP2 expression pattern and XCI pattern in RTT-hiPS cells and their neuronal derivatives

32. Experimental Procedure and Results
Investigate if RTT-hiPS cells retain an inactive X-chromosome despite the loss of XIST RNA and H3K27me3 in some cells
Investigate whether the extreme XCI skewing represents a nonrandom XCI pattern

33. RT-PCR and qRT-PCR using primers that would detect the presence of WT MECP2 but not the mutant
Δ3-4 hiPS cell lines #6 and #37 express WT MECP2
Δ3-4 hiPS cell line #20 expresses mutant MECP2

34. WT MECP2
Mutant MECP2
WT MECP2
These results confirm that the skewing pattern seen in the AR assay does in fact represent an expression pattern reflective of nonrandom XCI in hiPS cell lines
Not only were WT MECP2 transcripts being expressed but AR assay confirmed the same X-chromosome was still inactive

35. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells
Investigate Δ3-4 hiPS cell’s XCI status
Investigate pattern of XCI in Δ3-4 hiPS cells
Compare MECP2 expression pattern of XCI in RTT-hiPS cells and their neuronal derivatives
Determine whether the pattern of WT and mutant-specific expression of MECP2 seen in the different Δ3-4 hiPS cell lines is maintained upon differentiation

36. Experimental Procedure and Results
6. Determine whether the pattern of WT and mutant-specific expression of MECP2 seen in different Δ3-4 hiPS cell lines is maintained upon differentiation
Directed differentiation of Δ3-4 hiPS #20 and #37 cells into neuronal lineage
WT MECP2 protein can be seen using immunocytochemistry
Results confirmed with qRT-PCR
Additional AR assay was preformed to double check skewing pattern after differentiation (data not shown)

37. Directed differentiation of #20 and #37 Δ3-4 hiPS cells into neuronal lineage
WT MECP2 protein can be seen using immunocytochemistry
qRT-PCR supports results
Indicates the generation of isogenic system

38. Experimental Procedure and Results
Characterize the MECP2 mutation
Generation and characterization of RTT-hiPS cells
Investigate Δ3-4 hiPS cell’s XCI status
Investigate pattern of XCI in Δ3-4 hiPS cells
Compare MECP2 expression pattern of XCI in RTT-hiPS cells and their neuronal derivatives
Determine whether the pattern of WT and mutant-specific expression of MECP2 seen in the different Δ3-4 hiPS cell lines is maintained upon differentiation
Demonstrate the utility of isogenic control and mutant Δ3-4 hiPS cells

39. Experimental Procedure and Results
Phenotyping Δ3-4 hiPS cell-derived neurons by soma size
Mutant neurons show decreased soma size
Mutant
Wild-type/Control

40. Introduction to Rett Syndrome
Objectives
Experimental Design and Results
Conclusion
Future Work
Personal Critique
Questions

41. Conclusion
A large complex genetic mutation has been mapped, patient fibroblasts successfully reprogrammed and a disease specific phenotype observed in cell types that differentiated from experimental hiPS cell lines generated from the same patient
Confirmed recent finding that female hiPS cells retain an inactive X-chromosome in a nonrandom pattern
Showed that nonrandom pattern can be exploited to generate isogenic control and experimental hiPS cell lines from heterozygous X-linked diseases

42. Introduction to Rett Syndrome
Objectives
Experimental Design and Results
Conclusion
Personal Critique and Future Work
Questions

43. Critique
Preformed multiple overlapping experiments to thoroughly support hypothesis
Extremely thorough in excluding other factors from their conclusions
Did not mention how their phenotypic findings related to RTT nor a cause for the change soma size

44. Future Work
Application of Δ3-4 hiPS cell lines to identify phenotypes that are specific to the MECP2 mutation
Affect of mixing the mutant and the isogenic control Δ3-4 hiPS cells in different relative proportions prior to neuronal differentiation to create a mosaic mutant and WT expression pattern
Generation of Δ3-4 hiPS derived-neurons to ethically study MECP2 function in human neurons and for disease phenotyping
Drug screening for developing treatments

45. Questions?