Supplementary Materials and Methods s2

Supplementary Materials and Methods

Genotyping of Mecp2308 mutation and Southern blot analysis

PCR with oIMR3912, oIMR3913, and oIMR3914 primers on genomic DNA from mouse embryonic fibroblasts (MEFs) and resulting iPS cell lines yielded amplicons of 396 bp and 318

308 bp for wild-type Mecp2 and truncated Mecp2 alleles, respectively (Supplementary Table 1).

Genomic DNA was isolated using phenol/chloroform/isoamyl alcohol extraction. Southern transfer and hybridization were by standard procedures.

Mouse iPS cell induction and cell culture iPS cell induction from MEFs (isolated from Mecp2 mutant mice1 [B6.129S-Mecp2tm1Hzo/J strain on a C57BL/6 genetic background; Stock No. 005439, Jackson Laboratory] and wild-type littermate controls) and subsequent culture was performed as previously described.2 Preliminary experiments with individually picked three-factor reprogrammed colonies indicated poor survival, therefore multiple colonies were picked into a single well and passaged before subcloning to establish sublines for the initial WT and HET inductions. For the hemizygous

308/y +/y mutant Mecp2 (308) and wild-type Mecp2 male (WT ♂) iPS cell inductions, clonal lines were established by picking one colony into a single well for each individual cell line. Control mouse ES cells were cultured in similar conditions (lacking puromycin) as for mouse iPS cells.2

Alkaline phosphatase staining and immunocytochemistry

Alkaline phosphatase staining and immunocytochemistry assessing pluripotency marker expression and three germ layer staining was performed as previously described.2 Alkaline

1 phosphatase images were taken with a Leica DM IL microscope equipped with a Leica DC500 digital color camera and PerkinElmer OpenLab software. Immunocytochemistry images were captured with Leica DMI4000B microscope equipped with a Leica DFC340FX camera and

Leica Application Suite software, and a Zeiss Axiovert 200M microscope equipped with a

AxioCam HRm camera and Zeiss AxioVision Image Analysis Software for pluripotency and germ layer staining, respectively. For neuron immunocytochemistry, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and permeabilized with 0.25% Triton X-

100-containing blocking buffer (5% NGS and 1% BSA in 1X PBS) for 10 minutes. Cells were subsequently incubated with primary antibody in Triton X-100-containing blocking buffer overnight at 4°C, washed twice with blocking buffer, and incubated with appropriate Alexa Fluor secondary antibody (Invitrogen) in Triton X-100-containing blocking buffer for 1 hour at room temperature. After two washes, nuclei were stained with DAPI for 10 minutes. Images were taken using a Zeiss Axiovert 200M microscope equipped with a Hamamatsu C9100-13 EMCCD camera and PerkinElmer Volocity software. For MeCP2 counts in HET iPS cell-derived neurons, images were processed with ImageJ software (http://rsbweb.nih.gov/ij). All antibodies used in this study are listed in Supplementary Table 2.

RNA isolation and PCR analysis

Total RNA was isolated using TRIzol Reagent (Invitrogen) according to manufacturer’s specifications. 1 µg of total RNA was treated with DNase I and SuperScriptTM II reverse transcriptase (both Invitrogen) to yield complementary DNA for RT-PCR. Primers are listed in

Supplementary Table 1. J1 ES cells and MEFs were used as positive and negative controls for endogenous pluripotency marker expression, respectively.3 For the RT-PCR assessing exogenous

2 viral transgene expression, the EOS3F-24 published partially reprogrammed iPS cell line and J1

ES cells were included as positive and negative controls, respectively.2,4

Karyotype analysis

Standard G-banding chromosome analysis was performed with a 400–500 banding resolution at

TCAG (The Centre for Applied Genomics, Hospital for Sick Children, Canada).

Neuronal differentiation

Neuronal differentiation of iPS cell lines was performed using methods adapted with modifications from the differentiation protocol published by Bibel et al.6,7 for generating glutamatergic neurons from mouse embryonic stem cells. In brief, iPS cellular aggregates cultured in suspension in EB media for 4 days were subsequently cultured in suspension with 5

µM all-trans-retinoic acid (Sigma) for an additional four days. Resulting neuronal precursors were dissociated and plated on poly-L-ornithine (Sigma)/laminin (Roche)-coated dishes in neural medium containing DMEM, 1X N2 supplement and 4 mM penicillin/streptomycin/glutamine (all

Invitrogen). Neuronal precursors were then differentiated into neurons in differentiation medium containing DMEM, 1X B27 supplement, 4 mM Glutamax, and 4 mM penicillin/streptomycin (all

Invitrogen). For conditioned media experiments, conditioned B27 differentiation media was collected from WT neuronal cultures, centrifuged at 1000g for 5 min, filtered, and transferred immediately afterwards onto 308 neuronal cultures on Days 8, 12, and 14 as previously described.8

3 Electrophysiology

To record and study membrane properties, the external recording solution was composed of (in mM, pH 7.35): 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 15 HEPES, and 10 glucose. Recording electrodes (4–7 MΩ) were filled with a solution containing (in mM, pH 7.2): 154 K +-gluconate,

2+ 10 HEPES, 2 EGTA, 1 MgCl2, and 2 Mg -ATP. In whole-cell voltage-clamp, membrane potentials were held at –70 mV and currents were evoked by stepping the membrane potentials to a series of potentials from –80 mV to +60 mV (in 10-mV increments) for 400 ms. Input resistance refers to the average resistances obtained by hyperpolarizing or depolarizing step of 10 mV. In whole-cell current-clamp, action potentials were triggered from around –60 mV membrane potential by current injection from –50 pA to +230 pA (in 30-pA increments) for 1 s.

The first evoked action potential recorded for each cell was used for the analysis of action potential parameters. Action potential threshold was determined by calculating the derivative of data of the action potential using Origin software (OriginLab Corporation). Action potential amplitude was measured from the threshold to peak. Rise time and decay time were calculated between 10% and 90% of the spike amplitude, and action potential duration (D1/2) was measured at half of the spike amplitude.

For miniature excitatory synaptic currents (mEPSC) recordings, membrane potentials were held at –60 mV and whole-cell voltage clamp recordings were made from WT or HET cells treated with TTX, GABAA receptor blocker bicuculline, and glycine receptor blocker strychnine. The external solution was composed of (in mM, pH 7.35): 140 NaCl, 5.4 KCl, 2 CaCl2, 15 HEPES,

25 glucose, 0.0005 TTX, 0.001 glycine; 0.01 bicuculline, and 0.01 strychnine. The internal solution was composed of (in mM; pH 7.2): 105 Cs-methanesulfonate, 15 Cs2SO4, 10 HEPES, 10

2+ BAPTA, 1 MgCl2, 4 Mg -ATP. In some experiments, 2 mM MgCl2 was added to the external 4 solution. mEPSC events were detected and analyzed by Mini Analysis Program (Synaptosoft Inc,

NJ, USA). AMPA receptor-mediated current was measured from the baseline to the peak (in 5 ms) and NMDA receptor-mediated current was measured 15 ms after the peak.9 Results are shown as mean ± SEM. Statistical analysis was estimated by Student's t-test unless stated otherwise, and *P < 0.05 was considered significant.

5 Supplementary References

1. Shahbazian M, Young J, Yuva-Paylor L, Spencer C, Antalffy B, Noebels J et al. Mice

with truncated MeCP2 recapitulate many Rett syndrome features and display

hyperacetylation of histone H3. Neuron 2002; 35: 243–254.

2. Hotta A, Cheung AY, Farra N, Vijayaragavan K, Séguin CA, Draper JS et al. Isolation of

human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat Methods

2009; 6: 370–376.

3. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene

results in embryonic lethality. Cell 1992; 69: 915–926.

4. Fussner E, Djuric U, Strauss M, Hotta A, Perez-Iratxeta C, Lanner F et al. Constitutive

heterochromatin reorganization during somatic cell reprogramming. EMBO J 2011; 30:

1778–1789.

5. Hotta A, Cheung AY, Farra N, Garcha K, Chang WY, Pasceri P et al. EOS lentiviral

vector selection system for human induced pluripotent stem cells. Nat Protoc 2009; 4:

1828–1844.

6. Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M et al. Differentiation of

mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci 2004; 7:

1003–1009.

6 7. Bibel M, Richter J, Lacroix E, Barde YA. Generation of a defined and uniform

population of CNS progenitors and neurons from mouse embryonic stem cells. Nat

Protoc 2007; 2: 1034–1043.

8. Ballas N, Lioy DT, Grunseich C, Mandel G. Non-cell autonomous influence of MeCP2-

deficient glia on neuronal dendritic morphology. Nat Neurosci 2009; 12: 311–317.

9. Yu XM, Askalan R, Keil GJ 2nd, Salter MW. NMDA channel regulation by channel-

associated protein tyrosine kinase Src. Science 1997; 275: 674–678.

7 Supporting Figure Legends

Supplementary Figure 1. All wild-type and Mecp2308 iPS cells are pluripotent. (a) WT #1,2,4 and HET #2,3 iPS cell lines express alkaline phosphatase, Nanog, and SSEA-1 pluripotency markers by immunocytochemistry. (b) DAPI-colocalized H3K27me3 foci representing the inactive X-chromosome are absent in the iPS cell lines, but can be observed in female MEFs

(arrows). Scale bars: 250 µm. (c) Immunofluorescence analysis using a C-terminal-specific

MeCP2 antibody unable to detect the truncated MeCP2308 protein reveals heterozygous iPS cell lines (representative image of HET #4) express wild-type MeCP2 protein in all cells (left), indicating reactivation of the inactive X-chromosome. The MeCP2+ iPS cells express the stem cell-specific EOS-EGFP pluripotency vector (right) following reprogramming. Scale bar: 10 µm.

Supplementary Figure 2. Derivation of female WT and HET iPS cell lines. (a) WT iPS cell lines lack the Mecp2308 allele, which is present in the HET iPS cell lines, as confirmed by PCR

(top and bottom panels). WT, HET, and 308 represent genotyped DNA controls, respectively. (b)

PCR for the SRY gene reveals the WT iPS cell lines are female. ♂ and ♀ serve as male and female DNA controls, respectively, with β-actin as a loading control.

Supplementary Figure 3. All iPS cell lines are functionally pluripotent in vitro and in vivo. (a)

WT #1,2,4 and HET #2,3 differentiate into the three germ layers (ectoderm, Tuj1/βIII-tubulin; mesoderm, α-SMA; endoderm, GATA4) following EB-mediated in vitro differentiation. (b) WT

#1,2,4 and HET #2,3 generate structures corresponding to the three germ layers (ectoderm, neural structures/keratinizing epithelium; mesoderm, cartilage; endoderm, endodermal epithelium) in vivo in teratoma formation assays. Scale bars: 250 µm.

8 Supplementary Figure 4. Southern blot analysis of viral transgene integration and copy number. Genomic DNAs were digested by BamH1 restriction enzyme and integrated pMXs viral transgenes were detected by probes to the endogenous gene sequences for Oct4, Sox2, and Klf4

(left to right). Molecular weight in kb.

Supplementary Figure 5. MeCP2 protein colocalizes with DAPI foci by immunocytochemistry in HET #1 iPS-derived neurons. Scale bars: 10 µm.

Supplementary Figure 6. iPS cells can be directed to produce excitatory glutamatergic neurons.

(a) Following directed differentiation into glutamatergic neurons, iPS-derived cells express excitatory presynaptic marker VGLUT1. (b) Immunofluorescence for GAD65/67 reveals the inhibitory neuron marker is expressed in a minority of cells. Representative images of HET #4 iPS-derived neurons. Scale bars: 10 µm.

Supplementary Figure 7. Spontaneous action potential frequency is not significantly different between WT and HET iPS-derived neurons. Histogram illustrating spontaneous action potential number in neurons derived from WT (grey, n=7) and HET (black, n=11) iPS cell lines (0.7 ± 0.2 versus 1.1 ± 0.6 Hz, P = 0.55).

Supplementary Figure 8. Immunofluorescence for astrocyte marker GFAP (representative image of HET #4 iPS-derived neurons) reveals directed neuronal differentiation of the iPS cell lines produces cultures with minimal astrocytes. Scale bar: 10 µm.

9 Supplementary Figure 9. Action potentials in HET iPS-derived neurons are prolonged in duration. (a) Histogram showing threshold of action potentials in WT iPS-derived neurons (grey) and HET (black) iPS-derived neurons. The groups of bars on the left and middle show the four individual WT and HET sublines, respectively. On right are the overall averages for WT (n=48) and HET (n=42) cells (–30.0 ± 1.4 versus –27.1 ± 1.1 mV, respectively, P = 0.11). (b) Histogram of action potential duration in the WT and HET sublines and overall groups as in panel a (2.8 ±

0.3 versus 4.3 ± 0.6 ms, *P < 0.05). For action potential threshold and duration, n=2, 11, 21, 14 for WT #1,2,3,4, and n=8, 9, 12, 13 for HET #1,2,3,4, respectively.

Supplementary Figure 10. iPS-derived neurons produce voltage-gated inward and outward currents. (a) A typical trace in voltage-clamp mode shows inward and outward currents evoked by a step from a holding potential of −70 mV to –10 mV (left) and the current-voltage relationship in this neuron (right) in a WT iPS-derived neuron. K+ currents were measured at the end of the 400-ms step. (b) A typical trace of inward and outward currents evoked when the holding potential of −70 mV was stepped to –10 mV for 400 ms in a HET iPS-derived neuron

(left) and the current-voltage relationship in this neuron (right).

Supplementary Figure 11. Neither the membrane potential nor input resistance is significantly different between WT iPS-derived and HET iPS-derived non-neuronal cells. (a) Histogram illustrating membrane potentials without current injection in WT (grey) and HET (black) non- neuronal cells where action potentials were not generated spontaneously or by injecting depolarizing current (–29.1 ± 4.1 versus –30.5 ± 3.1 mV, P = 0.84). (b) Input resistance in WT

10 and HET iPS-derived non-neuronal cells (2170 ± 457.9 versus 1752 ± 468.6 mΩ, P = 0.6).

Numbers of cells were 37 and 15 for WT and HET, respectively.

Supplementary Figure 12. Synaptic activity is decreased in HET iPS-derived neurons. (a)

Typical traces of mEPSCs under untreated (left), APV (50 µM; middle), and APV + CNQX (10

µM; right) conditions in a WT iPS-derived cell. A plot (far right) illustrates averaged mEPSCs before (untreated, grey) or during (APV, black) the application of APV in this cell. (b) mEPSCs recorded from a HET iPS-derived cell at −60mV with 2 mM MgCl2 in the external recording solution in a typical trace (left). A plot (middle) of the averaged traces recorded at holding potentials of –60 and +60 mV in this cell. Current-voltage relationship (right) of normalized

AMPA receptor-mediated and NMDA receptor-mediated mEPSCs in this cell. (c) Normalized mEPSC amplitude in WT (grey) and HET (black) iPS-derived cells (1.0 ± 0.1 versus 1.2 ± 0.1, P

= 0.35, Mann-Whitney test, one-tailed). (d) Normalized frequency of mEPSCs in WT and HET iPS-derived cells (1.0 ± 0.5 versus 0.5 ± 0.1, *P < 0.05, Mann-Whitney test, one-tailed). For mEPSC amplitude and frequency, n=7 for WT and n=22 for HET.

Supplementary Figure 13. Southern blot analysis of viral transgene integration and copy number in WT ♂ and 308 iPS cell lines. Genomic DNAs were digested by BamH1 restriction enzyme and integrated pMXs viral transgenes were detected by probes to the endogenous gene sequences for Oct4, Sox2, and Klf4 (top to bottom). Molecular weight in kb.

Supplementary Figure 14. Derivation of karyotypically normal WT ♂ and 308 iPS cell lines.

(a) G-banding analysis indicates the iPS cell lines (from left to right, WT ♂ #11 and 308 #1) are

11 karyotypically normal. (b) PCR analysis for the Mecp2308 allele confirms hemizygous mutant male 308 iPS cell lines lack a WT Mecp2 allele, the only allele present in the WT ♂ iPS cell lines (top panel). HET, WT, and 308 represent genotyped DNA controls, respectively. PCR for the SRY gene (bottom panel) confirms the WT ♂ and 308 iPS cell lines are all male. ♀ serves as a female DNA control, with β-actin as a loading control.

Supplementary Figure 15. WT ♂ and 308 iPS cells are pluripotent. (a) WT ♂ #2,7,11,12,18 iPS cell lines express alkaline phosphatase (left), Nanog, and SSEA-1 (right) pluripotency markers by immunocytochemistry. (b) 308 #1,2,10,29,33,42,45 iPS cell lines are positive for stem cell markers alkaline phosphatase (left), Nanog, and SSEA-1 (right). Scale bars: 250 µm.

Supplementary Figure 16. WT ♂ iPS cell lines are functionally pluripotent in vitro and in vivo.

(a) WT ♂ #2,7,11,12,18 differentiate into the three germ layers (ectoderm, Tuj1/βIII-tubulin; mesoderm, α-SMA; endoderm, GATA4) following EB-mediated in vitro differentiation. (b) WT

♂ #2,7,11,12,18 generate structures corresponding to the three germ layers (ectoderm, neural structures/keratinizing epithelium; mesoderm, cartilage; endoderm, endodermal epithelium) in vivo in teratoma formation assays. Scale bars: 250 µm.

Supplementary Figure 17. 308 iPS cell lines are functionally pluripotent in vitro and in vivo.

(a) Following EB-mediated differentiation, 308 #1,2,10,29,33,42,45 differentiate into the three germ layers (ectoderm, Tuj1/βIII-tubulin; mesoderm, α-SMA; endoderm, GATA4) in vitro. (b)

308 #1,2,10,29,33,42,45 generate structures corresponding to the three germ layers (ectoderm,

12 neural structures/keratinizing epithelium; mesoderm, cartilage; endoderm, endodermal epithelium) in vivo in teratoma formation assays. Scale bars: 250 µm.

Supplementary Figure 18. Mecp2308/y male iPS-derived neurons reproduce neurophysiology phenotypes observed in HET iPS-derived neurons. (a) Histogram showing action potential amplitude in 5 cell lines of WT ♂ iPS-derived neurons (grey) and in 7 cell lines of 308 iPS- derived neurons (black). (b) Histogram of action potential rise time in multiple cell lines of WT

♂ and 308 iPS-derived neurons as in panel a. (c) Input resistances in WT ♂ iPS-derived neurons compared to 308 iPS-derived neurons as in panel a. For action potential amplitude and rise time, n=13,12,12,11,5 for WT ♂ #2,7,11,12,18, and n=13,13,13,14,12,12,11 for 308

#1,2,10,29,33,42,45, respectively. For input resistance, numbers of cells were 14, 12, 12, 11 and

5 for WT ♂ #2,7,11,12,18, and 13, 13, 13, 14, 12, 12 and 12 for 308 #1,2,10,29,33,42,45, respectively.

Supplementary Figure 19. Additional neurophysiology phenotypes of Mecp2308/y male iPS- derived neurons. (a) Histogram showing action potential threshold in neurons derived from multiple WT ♂ (grey) and 308 (black) iPS cell lines. The groups of bars on the left and middle show the individual WT ♂ and 308 lines, respectively. On right are the overall averages for WT

♂ (n=53) and 308 (n=88) cells (–30.8 ± 1.4 versus –28.1 ± 1.1 mV, P = 0.13). Numbers of cells were 13, 12, 12, 11 and 5 for WT ♂ #2,7,11,12,18, and 13, 13, 13, 14, 12, 12, 11 for 308

#1,2,10,29,33,42,45, respectively. (b) Histogram of decay time of action potentials in WT ♂

(n=49) and 308 (n=81) iPS-derived neurons as in panel a (3.6 ± 0.4 versus 3.7 ± 0.3 ms, P =

0.81). n=10,12,12,11,4 for WT ♂, and n=13,10,11,13,12,11,11 for the 308 lines. (c) Histogram

13 of action potential duration in WT ♂ (n=53) and 308 (n=87) iPS-derived neurons as in panel a

(3.6 ± 0.4 versus 4.5 ± 0.4 ms, P = 0.13, respectively). Numbers of cells were 13, 12, 12, 11 and

5 for WT ♂, and 13, 13, 12, 14, 12, 12 and 11 for the 308 lines. (d) Histogram of resting membrane potentials in WT ♂ iPS-derived neurons compared to 308 (n=54 and 89, respectively) as in panel a (–41.3 ± 2.1 versus –38.2 ± 1.6 mV, P = 0.24). n=14,12,12,11,5 for WT ♂

#2,7,11,12,18, and n=13,13,13,14,12,12,12 for 308 #1,2,10,29,33,42,45, respectively. (e)

Spontaneous action potential frequency is not significantly different between WT ♂ and 308 iPS- derived neurons. Histogram showing spontaneous action potential number in neurons derived from WT ♂ (n=18) and 308 (n=24) iPS cell lines (0.8 ± 0.3 versus 0.5 ± 0.1 Hz, P = 0.23).

Compared to WT ♂, 308 iPS-derived neurons exhibited more depolarized membrane potentials and increased decay times and action potential duration, consistent with recordings from the

HET lines though not statistically significant.

Supplementary Figure 20. Conditioned media from WT cultures ameliorates neurophysiology deficits in Mecp2308/y neurons (308 #29 iPS-derived neurons). (a) Histogram summary of action potential amplitude from untreated 308 (grey, n=13) and conditioned media-cultured 308 (black, n=14) iPS-derived neurons (47.5 ± 6.1 versus 61.8 ± 5.3 mV, P = 0.09, Mann-Whitney test). (b)

Histogram of action potential rise time of untreated 308 iPS-derived neurons (n=13) compared to

308 iPS-derived neurons in conditioned media (n=14) (2.8 ± 1.0 versus 1.3 ± 0.3 ms, *P < 0.05,

Mann-Whitney test). (c) A plot depicting the numbers of evoked action potentials elicited by a series of current injection from +20 pA to +230 pA (in 30-pA increments) in untreated 308 iPS- derived neurons and conditioned media-treated 308 neurons (*P < 0.05, **P < 0.01, ***P <

0.001, Mann-Whitney test).