Supplemental Information

Supplemental Experimental Procedures

Primary Antibodies

Antibody / Isotype / Source / Dilution
α-Actinin / monoclonal
mouse IgG / Sigma-Aldrich / 1:150
AFP
(α-feto-protein) / polyclonal
goat IgG / Santa Cruz / 1:100
α-Bungarotoxin / NA / Invitrogen / 1:500
Homer1 / polyclonal
rabbit IgG / SYSY / 1:500
Homer1 / polyclonal
guineapig IgG / SYSY / 1:500
HB9 / MNR2 / polyclonal
mouse IgG / DSHB / 1:50
Islet 1 / polyclonal
rabbit IgG / Abcam / 1:200
MAP2 / polyclonal
rabbit IgG / Millipore / 1:500
MHC class I
(myosin heavy chain) / polyclonal
rabbit IgG / Abcam / 1:500
phospho-IkB-α / monoclonal
mouse IgG / CellSignaling / 1:200
Piccolo / polyclonal
rabbit IgG / SYSY / 1:500
ProSAP1 / rabbit IgG / 1 / 1:600
SMI-32 (NF-H) / monoclonal
mouse IgG / Covance / 1:1000
Synaptophysin / polyclonal
rabbit IgG / Abcam / 1:500
Synaptophysin / polyclonal
guineapig IgG / SYSY / 1:500
Tuj1
(tubulin beta 3) / polyclonal
chicken IgG / Millipore / 1:1000
Tuj1
(tubulin beta 3) / polyclonal
rabbit IgG / Covance / 1:1500
VAChT
(vesicularacetylcholinetransporter) / polyclonal
guineapig IgG / Chemicon / 1:500
StemLite Pluripotency Kit / CellSignaling

Abcam, Cambridge, CB, UK,

Cell Signaling, Danvers, MA, USA,

Covance, Princton, NJ, USA,

Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA,

Invitrogen, Carlsbad, CA, USA,

Millipore, Billerica, MA, USA,

Synaptic Systems (SYSY), Goeppingen, BW, Germany,

Sigma-Aldrich, St. Louis, MO, USA,

Santa Cruz, Santa Cruz, CA, USA,

Primary myocyte culture

Neonate mice (1-7 days old) were briefly anesthetized and then decapitated. Muscle tissue was taken from M. quadriceps femoris, M. biceps femoris, M. triceps surae, M. biceps brachii and M. triceps brachii. Tissue samples were harvested, minced and centrifuged at 1000 x g for 5 min. Cells were digested with 0.2 % pronase (Roche) for 1 hour at 37 °C under constant agitation. After digestion tissue samples were washed with Hank’s buffered salt solution (HBSS, Invitrogen) and isolated by trituration with a fire-polished glass pipette to a quasi single-cell suspension. Suspension was filtered through a 70 m nylon filter and centrifuged at 1000 g for 5 min. Subsequently the pelleted cells were resuspended in 1.5 ml primary myoblast medium and layered onto a Percoll (Sigma-Aldrich) gradient containing a 35 %- and 70 % percoll fraction. The gradient was centrifuged without brake at 1250 g for 20 min. After centrifugation, cells were harvested from the interphase between 0-35 and 35-70 and seeded on PLO / laminin coated dishes. Myoblasts were kept in DMEM supplemented with 15 % heat inactivated horse serum (Invitrogen), 3% chick embryo extract (CEE, USbiologicals, Swampscotts, MA, USA, 1 % Antibiotic-Antimycotic, 1 % sodium pyruvate (Invitrogen), 10 mM HEPES (Invitrogen) and 2 mM GlutaMAX (proliferation medium). To induce differentiation, proliferation medium was changed to differentiation medium without CEE. Fusion into myotubes was observed four days after medium change and at 90 % confluence. For prolonged passaging in an undifferentiated state, cells were cultured in HAM’s F-12 medium (Biochrom, Berlin, Germany, supplemented with 20 % FBS, 1 % Antibiotic-Antimycotic and 2.5 ng/ml FGF2.

Differentiation of hiPSCs into motoneurons

After lifting hiPSCs via dispase (Stemcell Technologies) digestion, hiPSCs formed embryoid bodies (EBs) in suspension. EBs were cultured in DMEM/F12, supplemented with 20 % Knockout Serum Replacement, 2 mM GlutaMAX, 100 µM nonessential amino acids, 1 % Antibiotic-Antimycotic and 100 µM ß-mercaptoethanol for the next 4 days. Addition of ROCK-inhibitor Y-27632 (Ascent Scientific, Avonmouth, BS, UK, for the first 24 hours improved survival of hiPSC single cells2 and promoted generation of EBs as well. On day 4 medium was changed to neural differentiation medium containing DMEM/F12, N2-supplement (Invitrogen), 100 μM nonessential amino acids, 1 % Antibiotic-Antimycotic and 2 µg/ml heparin (Sigma-Aldrich, St. Louis, USA, For neural differentiation 10 mM cAMP, 20 ng/ml ascorbic acid, brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), insulin-like growth factor1 (IGF1) (all 10 ng/ml, Peprotech) were added immediately before use. After one week in suspension, clusters were attached to laminin-coated dishes (20 µg/ml, Roche, Basel, Switzerland, Primitive neuroepithelial cells were posteriorized by addition of 0.1 µM retinoid acid (RA, Sigma-Aldrich) at day 10. Neural tube-like rosettes were loosely attached to the substrate whereas the non-neural cells were much more fixated to the Petri dish. At day 14 neuroepithelial cells were rinsed off with a 5-ml serological pipette while the nonneural cells remained attached. Isolated neuroepithelial cells were cultured in the same neural media in the presence of 0.1 µm RA, 1 µm purmorphamine (PU, Calbiochem, Gibbstown, NJ, USA, and B27 supplement without vitamin A (Invitrogen) in suspension. From day 28 onward the motoneuron progenitors were seeded on poly-L-ornithine (PLO, Sigma-Aldrich) and laminin (20 µg/ml) coated dishes in neural differentiation medium with a reduced concentration of RA (0.05 µm) and PU (0.5 µm).

Lentivirus generation

Lentivirus containing a policistronic expression cassette encoding for Oct4, Sox2, Klf4 and cMyc3 was produced in 70 % confluent 10 cm dishes with Lenti-X 293T cells (Clontech, Mountain View, CA, USA, by cotransfection of the policistronic vector (8 μg) (kind gift of G. Mostoslavsky, Boston), the pMD2 vector (2 μg) and the psPAX2 (5,5 μg) vector (both Addgene, Cambridge, MA, USA, using 100 μl of the PolyFect transfection reagent (Qiagen, Hilden, Germany, Viral supernatant was collected at 48 and 96 hours after transfection, concentrated using the Lenti-X Concentrator Kit, (Clontech), resuspended in EpiLife medium and stored in aliquots at -80 °C.

Semi-quantitative real-time one-step RT-PCR

Semi-quantitative real-time one-step RT-PCR was carried out using the Rotor-Gene Q System (Qiagen) and amplification was monitored and analyzed by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. 1 μl of total RNA was reversely transcribed and subsequently amplified using the QuantiFast SYBR Green RT-PCR Kit (Qiagen) and 0.5 mM of both sense and antisense primers. Tenfold dilutions of total RNA were used as external standards. Internal standards and samples were simultaneously amplified. After amplification, melting curves of the RT-PCR products were acquired to demonstrate product specificity. Results are expressed relative to the housekeeping gene hydroxymethylbilane synthase (HMBS). All primers were purchased as validated primer pairs (Quantitect primer assay, Qiagen).

Supplemental Figure Legends

Supplemental Figure 1.(A) HiPSCs derived from human keratinocytes express the nuclear factors Oct4, Sox2 and NANOG as well as pluripotent surface markers SSEA4, TRA-1-60 and TRA-1-81 (all red). (B) Stem cell colonies from human keratinocytes were able to differentiate in all three primary germ layers in vitro. After formation of embryoid bodies hiPSCs spontaneously differentiate into ectodermal (Tuj1, red), mesodermal (a-actinin, red) and endodermal (AFP, a-fetoprotein, red) cells. Nuclei were labeled with DAPI (blue). All scale bars are 10 μm. (C) DIC imagines showing morphological changes during differentiation from hiPSCs into motor neurons according to Figure 1A.

Supplemental references

1Grabrucker AM, Knight MJ, Proepper C, et al. Concerted action of zinc and ProSAP/Shank insynaptogenesisandsynapsematuration.EmboJ. 2010;30:569-581.

2Gauthaman K, Fong CY, Bongso A. Effect of ROCK inhibitor Y527632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev. 2010;6:86595.

3Sommer CA, Stadtfeld M, Murphy GJ, et al. Induced pluripotent stem cell generation using a singlelentiviralstemcell cassette.StemCells. 2009;27:5435549.