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Supplemental Information
Supplemental Experimental Procedures
Primary Antibodies
Antibody
α-Actinin
AFP
(α-feto-protein)
α-Bungarotoxin
Homer1
Homer1
HB9 / MNR2
Islet 1
MAP2
MHC class I
(myosin heavy chain)
phospho-IkB-α
Piccolo
ProSAP1
SMI-32 (NF-H)
Synaptophysin
Synaptophysin
Tuj1
(tubulin beta 3)
Tuj1
(tubulin beta 3)
VAChT
(vesicular acetylcholine transporter)
Isotype
monoclonal
mouse IgG
polyclonal
goat IgG
NA
Source
Sigma-Aldrich
Dilution
1:150
Santa Cruz
1:100
Invitrogen
1:500
polyclonal
rabbit IgG
polyclonal
guinea pig IgG
polyclonal
mouse IgG
polyclonal
rabbit IgG
polyclonal
rabbit IgG
polyclonal
rabbit IgG
monoclonal
mouse IgG
polyclonal
rabbit IgG
rabbit IgG
SYSY
1:500
SYSY
1:500
DSHB
1:50
Abcam
1:200
Millipore
1:500
Abcam
1:500
Cell Signaling
1:200
SYSY
1:500
1
1:600
monoclonal
mouse IgG
polyclonal
rabbit IgG
polyclonal
guinea pig IgG
polyclonal
chicken IgG
polyclonal
rabbit IgG
polyclonal
guinea pig IgG
Covance
1:1000
Abcam
1:500
SYSY
1:500
Millipore
1:1000
Covance
1:1500
Chemicon
1:500
StemLite Pluripotency Kit
Cell Signaling
Abcam, Cambridge, CB, UK, www.abcam.com
Cell Signaling, Danvers, MA, USA, www.cellsignal.com
Covance, Princton, NJ, USA, www.covance.com
Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA, www.dshb.biology.uiowa.edu
Invitrogen, Carlsbad, CA, USA, www.invitrogen.com
Millipore, Billerica, MA, USA, www.millipore.com
Synaptic Systems (SYSY), Goeppingen, BW, Germany, www.sysy.com
Sigma-Aldrich, St. Louis, MO, USA, www.sigmaaldrich.com
Santa Cruz, Santa Cruz, CA, USA, www.scbt.com
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, www.usbio.net), 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, www.biochrom.de) 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, www.ascentscientific.com)
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,
www.sigmaaldrich.com). 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,
www.roche.com).
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, www.emdchemicals.com) 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, www.clontech.com) 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, www.addgene.org) using 100 μl of the PolyFect
transfection reagent (Qiagen, Hilden, Germany, www.qiagen.com). 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
1
Grabrucker AM, Knight MJ, Proepper C, et al. Concerted action of zinc and
ProSAP/Shank in synaptogenesis and synapse maturation. Embo J. 2010;30:569-581.
2
Gauthaman 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.
3
Sommer CA, Stadtfeld M, Murphy GJ, et al. Induced pluripotent stem cell generation
using a single lentiviral stem cell cassette. Stem Cells. 2009;27:5435549.