Reprogramming, PSC culture and characterization

Fibroblasts were seeded at 150,000 cells per 12-well in 5 wells per line (resulting in 5 PSC sub-clones) and transduced with 4 CytoTune Sendai viruses (Life Technologies, A1378002) encoding Oct-4, Sox2, Klf4 and c-Myc each at an MOI of 3¹⁶. Cells were fed daily with fibroblast medium. At day 7, the cells were dissociated using trypsin-EDTA (Gibco-Life Technologies, 25300-054) and replated onto 10-cm MEF (GlobalStem, GSC-6105M)-coated plates in HES medium supplemented with 0.5 mM VPA (Tocris Bioscience, 2815), as described in Zeltner et al,¹⁸. Cells were fed daily with HES medium/VPA until day 14, then with HES medium only. Between day 20 and 40 PSC colonies were manually picked, expanded and frozen. PSCs were fed daily with HES medium and passaged manually or with trypsin-EDTA¹⁸ approximately once per week onto MEFs. PSC cultures were routinely tested for mycoplasma contamination every other week. For the characterization of PSC lines, the cells were immuno-stained with Oct-4 (mIgG2b, Santa Cruz, sc-5279), Nanog (goat, R&D, AF1997) and Sendai virus (rabbit, MBL International, PD029). IKBKAP splicing in PSCs was conducted as described for fibroblasts. The karyotypes of the PSC clones were normal, except at high passages in S3 and M2.

HES media, KSR differentiation media, N2 differentiation media, matrigel dish coating and PO/LM/FN dish coating, plating of PSC for differentiation and day 0 to day 11 dual-Smad inhibition differentiation were all done as described in Zeltner et al.¹⁸.

For the in vitro differentiation to CNS neurons, PSCs were detached using Accutase (Innovative Cell Technologies, AT104) for 20 min. at 37°C, washed twice with 1X PBS and plated onto matrigel (BD, 354234)-coated 6-wells in HES medium supplemented with 10 μM Y-27632 dihydrochloride (Tocris, 1254). When 100% confluency was reached the cells were fed daily with KSR- or N2-medium supplemented with LDN193189 (Stemgent, 04-0074) and SB431542 (Tocris, 1614)¹⁸. On day 11 they were accutased, washed twice and replated onto Poly-L Ornithine hydrobromide (Sigma, P3655)/Laminin-1 (R&D Systems, 3400-010-01)/Fibronectin (BD Biosciences, 356008)-coated plates in droplets of approximately 100–150,000 cells per 10 μl as described in Zeltner et al.¹⁸, in N2 medium supplemented with 0.02 μg/ml BDNF (R&D Systems, 248-BD), 0.2 mM Ascorbic Acid (Sigma, A4034) and 0.1 μM Purmorphamine (Stemgent, 04-0009). On day 13 the cells were fed with N2 medium/AA/BDNF/Purmorphamine, on day 15, 17 and 19 they were fed with N2 medium/AA/BDNF/ 0.01 mM DAPT (R&D, 2634/50) and on day 20 they were fixed with 4% PFA (Affymetrix, 19943) and immuno-stained with Tbr1 (rabbit, Millipore, AB10554) and Tuj1 (mIgG2a, Covance, MMS-435P-250).

PSCs were plated as described previously¹⁸. The cells were fed daily with RPMI medium containing Glutamax (Invitrogen, 61870-036) supplemented with 0.5% FBS and 100 μg/ml ActivinA (R&D, 338-AC), fixed on day 5 and immuno-stained for Sox17 (goat, R&D, af1924) and DAPI.

Cardiac differentiations were done as described previously^37,38 with the following modifications: RPMI Medium 1640 (Life Technologies, 11875-093)/B27 (Life Technologies, 17504044) media was used instead of StemPro34. Embryoid bodies (EBs) were treated with 10 ng/ml ActivinA (R&D Systems, 338-AC) and 10 ng/ml of BMP4 (R&D Systems, 314-BP) from day 1–3, followed by 10 μM of XAV939 (Stemgent, 04-0046) from day 3–5. Differentiation cultures were assessed for mesoderm formation by flow cytometry staining for KDR/VEGFR2 (Alexa Fluor 647 anti-human CD309, BioLegend, 7D4-6) and PDGFRA (PE anti-human CD140a, BioLegend, 16A-1). The differentiations were carried on until day 20 in RPMI Medium 1640 (Life Technologies, 11875-093)/B27 (Life Technologies, 17504044) media and stained for the anti-cardiac isoform of TroponinT (cTnT) (Thermo Scientific Lab Vision, clone 13–11) by flow cytometry as well as plated onto plastic and stained for cTnT via IF on day 30 of differentiation.

For the quantification of neurons, endoderm and cardiac cells, images from two wells of each of 3–5 differentiations were counted for Tbr1⁺/Tuj1⁺ cells or Sox17⁺ or TroponinT⁺, respectively and the percentage to total DAPI+ cells was reported.

rNC differentiations, FACS isolation, HNK1, Pax6, αAP2a, ASCL1 and Tuj1 staining were carried out as described in Zeltner et al.¹⁸. To measure the proliferation rate, FACS isolated rNC cells were plated at 30,000 cells/96-well¹⁸. 24 hrs. later they were fed with N2/FGF2/EGF media containing EdU using the Click-iT Plus EdU Alexa Fluor 555 Imaging kit (Life technologies, {"type":"entrez-nucleotide","attrs":{"text":"C10638","term_id":"1535709","term_text":"C10638"}}C10638). 48 hrs. later the cells were fixed and stained according to the manufacturer’s instructions and EdU⁺/DAPI⁺ cells were counted for the quantification. The IKBKAP splicing PCR was conducted as described for fibroblasts. The migration (scratch assay) was conducted as described in Zeltner et al.¹⁸. In short, FACS isolated rNC cells were plated in PO/LM/FN-coated 96-wells at 20,000–60,000 cells/well and scratched 24 hrs. later using a 200 μl pipet tip. 6–8 wells were fed with media containing 10 ng/ml Hoechst33342 trihydrochloride, trihydrate (Invitrogen, H3570) and reference pictures (0 hrs. = before migration) were taken immediately. The media in the other wells was changed to remove dead cells. The cells were allowed to migrate for 48 hrs. and stained with Hoechst33342 before pictures were taken. The number of migrated cells was quantified automatically by the Konstanz information miner (KNIME) workflow³⁹. Results are expressed as percentage of cells migrated into a ROI (Region of interest) of the total number of cells per picture. Ascl1 qRT PCR was conducted on RNA isolated from FACS sorted rNC cells on day 18 of differentiation. Taqman gene expression assay (ABI, 4304437) for ASCL1 (primers: ABI, Hs00269932_m1 (ASCL1) and 4326321E (HPRT)) was conducted on an Eppendorf Mastercycler machine. Fold-changes were calculated using the comparative Ct method (ΔΔ Ct method) and then normalized to the expression in undifferentiated HES cells. Immuno-staining of rNC cells was conducted one week post FACS isolation as described in Zeltner et al.¹⁸.

The SN differentiations were carried out as described in Chambers et al.,¹⁹ with few modifications. In short, PSC were plated on matrigel coated dishes and differentiations were induced once 100% confluency was reached, using a gradient of KSR and N2 differentiation media, supplemented with LDN193189 and SB431542¹⁸. From day 2 to 11, 1.5 μM CHIR99021 (GSK3β-inhibitor, activation of Wnt signaling; Stemgent, 04-0004), 10 μM SU5402 (FGF inhibition; BioVision, 1645-1) and 10 μM DAPT (Notch inhibition; Tocris, 2634) were added to the media. On day 12, the cells were detached using Accutase, washed twice and replated into PO/LM/FN coated 96-wells at 280,000 cells per well in N2 differentiation media supplemented with 50X B-27 supplement (Life technologies, 12587-010), 20 ng/ml BDNF (R&D Systems, 248-BD), 20 ng/ml GDNF (Peprotech 450-10), 25 ng/ml β-NGF (PeproTech, 450-01), 100 μg/ml Primocin (InvivoGen, ant-pm-2), 0.6 μg/ml Laminin-1 (R&D Systems, 3400-010-1), 0.6 μg/ml Fibronectin (Fisher Scientific, CB40008A-BD) and 10 μM Y-27632 (Tocris, 1254). For maintenance, the cells were fed with the same media without Y-27632 every 2–3 days. On day 14, 17, 20, 23 and 26 the cells were fixed in 4% PFA and stained with Brn3a (mIgG1, 1:100, Millipore, MAB1585), Tuj1 (rabbit, 1:1000, Covance, MRB-435P-100) and DAPI (1:1000, Sigma-Aldrich, D9542-5M), cleaved Caspase3 (rabbit, 1:100, Cell Signaling, 9661S), Sox10 (goat, 1:100, Santa Cruz, sc-17342), Ki67 (rabbit, 1:500, SP6, RM9106-S1), Sox2 (rabbit, 1:400, Cell Signaling, 3579P) and αSMA (mIgG2a, 1:1000, Sigma, A5228-200UL). For the quantification, Brn3a⁺/Tuj1⁺ cells in pictures taken from 2–3 wells from 3–9 independent differentiations were counted (Figure 2b) or the percentage of DAPI⁺ of Brn3a⁺ cells were quantified using the Meta Express software (Figure 5b). IKBKAP splicing was done was described for fibroblasts. For the Sox10 expression time course, cells were harvested for RNA extraction at day 4, 8, 12 and 16 without replating. The real-time RT PCR was conducted using SYBR SsoFast EvaGreen Supermix (BioRad, 172–5202) and the QuantiTect Primer Assay for Sox10 (Qiagen, QT00055405). The alpha₂adrenergic receptor PCR was previously described⁴⁰. Drug treatment of SNs for the rescue of FD phenotypes were done either from day 0 throughout the entire differentiation or from replating at day 12. Kinetin (Sigma-Aldrich, K0753-5G) and {"type":"entrez-protein","attrs":{"text":"SKF86466","term_id":"1155968155","term_text":"SKF86466"}}SKF86466 hydrochloride (Tocris, 3866) were used at 10 μM.

PSCs were plated and dual-Smad inhibition differentiation until day 11 was conducted as reported in Zeltner et al.¹⁸. For Wnt-based NC induction the culture was supplemented with 3 μM CHIR99021 from day 2 to 14 and the cells were sorted for CD49d (1:800, PE-Cy7 anti-human CD49d, BioLegend, 304314) on day 11. The cells were aggregated in Ultra Low Attachment 24-well culture plates (Fisher Scientific, 3471) as 3D spheroids (500,000 cells/well) in Neurobasal (NB, Life Technologies, 21103-049) media supplemented with N2 supplement (Stem Cell Technologies, 07156) and B27, 3 μM CHIR99021 and 10 ng FGF2 (R&D Systems, 233-FB-001MG/CF). On day 14, the spheroids were plated onto PO/LM/FN coated 24-wells in NB+N2 supplement+B27 containing GDNF and Ascorbic Acid (Sigma, A8960-5G). On day 25, cells were fixed in 4% PFA and stained for Phox2a (rabbit, 1:800, kindly gifted from Dr. J-F Brunet, France), Tuj1 and DAPI. For the AN marker expression, RNA was extracted at day 30, 40, 50 and 60 and real-time RT-PCR was conducted either using SYBR SsoFast EvaGreen Supermix (QuantiTect primer assays for ASCL1: QT0023775, Phox2a: QT00215467, ChAT: QT00029624, EDNRB: QT00014343, Six1: QT00010584, CRNA3: QT00015386, CRNB4: QT00010570, VMAT1: QT00028420, VMAT2: QT00059857) or Taqman gene expression assays (primers for Scg10: Hs00199796_m1, TH: Hs00165941_m1, DBH: Hs01089840_m1). For the stress conditions in the AN survival assay, the cells were maintained either in the above mentioned media or in NB only or in NB only supplemented with 0.1 μM Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma, C2759-100MG). For intracellular FACS analysis, the cells were dissociated using Accutase, then fixed and permeabilized using BD Cytofix/Cytoperm (BD Bioscience, 554723) according to the manufacturer’s instructions. Primary antibody staining for ASCL1 (mIgG1, 1:200, BD Pharmingen, 556604) and TH (rabbit, 1:700, Pel-Freez, P40101-0) was done at 4 °C over night and secondary antibody staining was done at RT for 30 min. using AlexaFluor 647 goat anti mouse IgG1 (Invitrogen, A21240) for ASCL1 and AlexaFluor 647 goat anti rabbit (Invitrogen, A21245) for TH. The analysis was done on a flow Cytometer and using the FlowJo software. Intracellular FACS and immunohistochemistry analysis was done as described above. ISL1 (mouse IgG2b, 1:200, DSHB, 39.4D5-c).

The 3′ homology arm spans from intron18 to intron 20 of the IKBKAP gene. The FD point mutation is located in intron 20, 5bps downstream of exon 20. The gRNA binding site is 38bps upstream of the FD mutation. The selection cassette is located in intron 20, thus upon correction of the splicing site (FD mutation) it will be removed from the protein. The 3′ homology arm spans intron 20.

The 3′ and 5′ homology arms are 697 bps and 682 bps long, respectively and were amplified from C1.2 PSC genomic DNA using primers 5′CCCAGGGAGATTGTACACAC and 5′CGACTCTAGAGGATCCCAATGCCAAGCT or 5′CGGTACCCGGGGATCAGTGGGAGAGCTAAGGCACA and 5′TATGGATCCGCTAGCTTCACAGGCCACCTAAAACC, respectively. The selection cassette consists of the human PGK promoter followed by the puromycin resistance gene. The three fragments were ligated into a linearized pUC19 vector backbone using the Gibson Assembly Master Mix (New England Biolabs, E2611S) and the plasmid insert was sequenced entirely.

The CRISPR/Cas9 technology³¹ was used to design the gRNA and induce the double strand break to enhance homologous recombination to repair the FD mutation in IKBKAP. Option A from the hCRISPR gRNA synthesis protocol (deposited www.addgene.org/41824) was used to generate the gRNA. The website http://www.genome-engineering.org/crispr/?page_id=41 was used to generate the gRNA target sequence as well as to check for potential off-target effects. The gRNA with the best score was chosen (shown in bold below), synthesized as a gBlock from IDT and cloned into a TOPO vector (Invitrogen, 450245). The target sequence is located 39 bps upstream of the FD mutation.

U6 promoter/target sequence/gRNA scaffold/termination signal TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGTTGTTCATCATCGAGCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA

10 million S2 FD PSCs were nucleofected with 10 μg IKBKAP donor plasmid, 5 μg gRNA-TOPO vector and 5 μg Cas9-GFP plasmid (Addgene, plasmid #44719) using solution I and program A23 from the Amaxa Human Stem Cell Nucleofector Kit 1 (Lonza, VPH-5012). The cells were plated on MEFs in half fresh HES media and half HES media conditioned on S2 FD PSCs supplemented with 10 μM Y-27632 dihydrochloride. 48 hours later the cells were detached with Accutase and GFP⁺ cells were isolated by FACS using low pressure/μm nozzle settings. The 122,000 GFP⁺ cells were plated on fresh puromycin-resistant MEFs in a 10 cm dish. 72 hours post sort puromycin selection was initiated (1 μg/ml Millipore, 540411-100MG) and the cells were fed daily until colonies appeared, which were manually picked, expanded and frozen.

Genomic DNA was extracted from 21 clones (DNeasy Blood & Tissue kit (Qiagen, 69506) and screened using a 3 primer PCR strategy. Primers 5′GCTAGCGGATCCATAACT and 5′TGCCATTTGATTTCAGTCTCC bind on either side of the homology cassette and primer 5′AGGGGGCTGGAAGAGCTA binds inside the selection cassette. The expected amplicon sizes are shown in Fig. 4a. To sequence the FD mutation, a 483 bp amplicon surrounding the FD mutation was PCR amplified and sequenced.

The top 5 predicted off-target sites in coding and non-coding regions each were Sanger sequenced. For each of the 10 sites, two PCR primer pairs were chosen. The PCR was performed on genomic DNA from S2-iPSCs, S2rescue1-iPSCs, S2rescue5-iPSCs and S2rescue7-iPSCs. The resulting PCR product was cut from the agarose gel, purified and sequenced with two sequencing primers each. Primers: ID#1: NZ50f-5′CACAGAGGACCTGTGCTCAA, NZ50r-5′CCTGTGAAGTTGTGGGACCT, NZ51f-5′TGCCCACTTTCTCTGTTCCT and NZ51r5′-CCTGTGAAGTTGTGGGACCT, NZ52fseq-5′CCTCCCCTTCTATTCCGAAG, NZ53fseq-5′CCTGCAACTGACGCTGTATC. ID#2: NZ54f-5′ATCCCATGCTCTTCCTCCTT, NZ54r-5′GACAATCCAGGCAACCTTGT, NZ55f-5′CCTCCTTCTCTCTGGCCTTT, NZ55r-5′TAGCATTTTGGCATGAGTGG, NZ56fseq-5′ CCCTGGGAGCTGTCTGTAAG, NZ57fseq-5′TGGTTTCCTCCCTGACACTT. ID#3: N58f-5′ TGAACTTTTCCGGACTGACC, NZ58r-5′GGAGACCTGGTCGTAGTGGA, NZ59f-5′CGGACTACGATCCTTTCCTG, NZ59r-5′TGACAGTCGATGGAGACCTG, NZ60fseq-5′ACTACAGAGCCCGGGAATG, NZ61fseq-5′GGGAAGGTGGGACTCTGTG. ID#4: NZ62f-5′GTGCTGGGGGAGCTCTAAG, NZ62r-5′CTGCCTTGTTCCCTTGATGT, NZ63f-5′CTAGAGAAGCTGGGGTGCTG, NZ63r-5′TGTCCCAGAGCTGAGACTCC, NZ64fseq-5′ CACCATGTTCCTGCACTGAC, NZ65fseq-5′GAGCTCTAAGGGGGCTGTTC. ID#5: NZ66f-5′ TGTCTTTCCCTCCCTTCCTT, NZ66r-5′TGTTTATGGAGCCCGGATAC, NZ67f-5′TGTCTTTCCCTCCCTTCCTT, NZ67r-5′ATGAGCCAGACTGCCATCTC, NZ68fseq-5′AGGCAGTTTGTGGTGA, NZ69fseq-5′TGTTTTAGGGGGAAGC. ID#6: NZ70f-5′CAGTGGAAGGAGAGGAGCAG, NZ70r-5′TGGGTGGAAGGTCAAGAAAG, NZ71f-5′ CAGTGGAAGGAGAGGAGCAG, NZ71r-5′CCAACACAAGGAGTGGGACT, NZ72fseq-5′ CTCTGAGGAAGCCAGTCCAC, NZ73fseq-5′CCCTTACCCAGGAGGTCACT. ID#7: NZ74f-5′GGCTCTCTGCCCAATTCATA, NZ74r-5′TGGGGATTTTTCCCCTTTAG, NZ75f-5′AACCCTCCATCCCACCTTAC, NZ75r-5′CAGGCACACCCCATTTTAAG, NZ76fseq-5′AGAAAGCTTGCCATCCTTGA, NZ77fseq-5′TTCTGGAAGCAGTAGCACCA. ID#8: NZ78f-5′CAGCTTTGGGTCCTATTTGC, NZ78r-5′AATGGCCCCTTGGTGAATAC, NZ79f-5′TCTGGCTGGAATGATGTTTG, NZ79r-5′TCTTGGTCCATCCAAAATGG, NZ80fseq-5′CTGCCTCATGGTGAAGAGGT, NZ81fseq-5′TCTCCTTATCCATCTGCCTCA. ID#17: NZ82f-5′GCAGCTGCAGGATAGGGTAG, NZ82r-5′GACAGCTCCTGCATCAGGTT, NZ83f-5′ATAGGGTAGCCCTCCTCCAC, NZ83r-5′GAGGACAGCTCCTGCATCA, NZ84fseq-5′TACTGCGCTTGCTCAAAAAC, NZ85fseq-5′ACATTCTCGCGAAGGGTCT. ID#22: NZ86f-5′GCCTCAGCCTGTTGTTCTTC, NZ86r-5′CGATCTCGTGGCTGATAACC, NZ87f-5′TGTTCTTCGTCCTGGCCTAC, NZ87r-5′GTCGATCTCGTGGCTGATAA, NZ88fseq-5′GCCTTCCTCTTCTCCATCG, NZ89fseq-5′TCAACAACCTCAACGGCTTC. No mutations were detected in any of the sequences. In ID#3, S2rescue1 and in ID#22, all three clones did not yield PCR products or sequences that would allow the determination of mutations.