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BME395F 2018 BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES E...
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BME395F 2018 BIOMEDICALSYSTEMSENGINEERINGIICELLSAN...
BME395F_2018_BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES_E.pdf-UNIVERSITY OF TORONTO FACULTY OF APPLIED
BME395F 2018 BIOMEDICALSYSTEMSENGIN...
BME395F_2018_BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES_E.pdf-UNIVERSITY OF TORONTO FACULTY OF APPLIED
Page 7
LETTERS
relevant neuron subtypes after short differentiation periods
(-
19 d), compared with 30-50 d when using stromal feeder—
mediated induction protocols 3,22.
Recent publications have reported the reprogramming of human
somatic cells into hiPS cells
1,21,21.
We wanted to next determine
whether dual-SMAD inhibition could be used to reliably generate a
broad repertoire of hiPS cell—derived neural cell types. Given the
expected intrinsic variability among hiPS cell clones, reproducible
differentiation results would confirm the robustness of our differen-
tiation protocol. Two hiPS cell clones (iPS-14, iPS-27;
Fig.
3a)
were
generated using lentiviral transduction of human fetal lung fibroblasts
with cMYC, KLF4, OCT4 and SOX2. Both clones express the pluri-
potency markers including Nanog, Tra-1-60 and SSEA-3 at the
undifferentiated state and are capable of differentiating into derivatives
of the three germ layers (data not shown). Upon neural induction by
means of the Noggin/SB43 1542 protocol, both clones yielded nearly
homogeneous populations of PAW cells by day 11 of differentiation
(Fig.
3b).
Using the strategies described above to manipulate cell
density, passage and patterning factors, both hiPS cell clones could
be readily biased toward generating
HNKle
putative neural crest
progeny (Fig. 3c), hiPS cell—derived R-NS cells
(Fig.
3d) and specific
hiPS cell—derived neuron subtypes, including somatic motoneurons
(Fig.
3e) and dopamine neurons (Fig. 3f). These data demonstrate
robustness and extension of the dual-SMAD-inhibition strategy
beyond hES-cell differentiation. The protocol offers an efficient,
defined and robust platform for the rapid generation of hiPS cell—
derived neural cell types.
In this report, we describe a method of neural differentiation that
combines the inhibitors Noggin and SB43 1542 to block SMAD
signaling.
Noggin and SB431542 act on pluripotent cells at multiple
stages of differentiation and provide access to an early intermediate
progenitor capable of giving rise to known populations of R-NS and
NCS cells
(Fig. 4).
Whereas for most of the studies presented here an
11-d treatment period was used, preliminary studies indicate that
similar levels of neural induction can be achieved when the treatment is
shortened to the first 5 d of differentiation (Supplementary.
Fig. 6
online; and data not shown). This should further reduce complexity
and cost, particularly of recombinant Noggin. Noggin/SB43 1542 treat-
ment greatly improves
on
current methods of generating neural tissue
by inducing rapid and uniform neural conversion of human pluripo-
tent cells under adherent culture conditions without the need for
embryoid body formation or MS5 stromal feeder co-culture. The
protocol allows for the derivation of relevant neuron subtypes
after much shorter differentiation periods
('..
19 d) compared with
30-50 d of differentiation for stromal feeder—mediated induction
protocols
3
'
22
. Given the need for defined protocols that induce rapid
and complete neural conversion, this technique may become the
standard strategy for driving differentiation of human pluripotent cells.
METHODS
Cells and culture
conditions.
hES cell (WA-09; passages 35-45) and iPS lines
(iPS-14, iPS-27; passages 4-10) were cultured on mouse embryonic fibroblasts
plated at 12-15,000 cells/cm
2
(MEFs, Glohalstem). A medium of DMEM/F12,
20% knockout serum replacement (Gibco), 0.1 mM b-mercaptoethanol,
6 nglml FGF-2 was changed daily. Cells were passaged using 6 U/rnl of dispase
in hES-cell media, washed and replated at a dilution of 1:5 to 1:10.
IPS cell
generation.
The cDNAs encoding h0ct4, hSox2, hKlf4 and c-myc
(Open Biosystems) were suhcloned into self-inactivating lentiviral vectors
driven by the human phosphoglycerate kinase (PGK) promoter. Lentiviral
vector supernatants were produced by triple co-transfection of the plasmid
DNA encoding the vector, pCMVAR8.91 and pUCMD.G into 293T cells.
NATURE BIOTECHNOLOGY
VOLUME 27 NUMBER 3 MARCH 2009
Human fetal lung fibroblasts (MRC-5) purchased from ATCC (CCL-171) were
seeded at 1.5 x 104 cells/cm
2
in Eagle's Minimum
Essential Medium supple-
ntcnted with 10% FBS (PBS). The following day the fibroblasts were transduced
with equal amounts of supernatants of the four lentiviral vectors in the
presence of 4 Ltg/ml polybrene for
16 h. Six days after transduction,
fibroblasts were harvested by trypsinization and plated at 2 x 10 cells per
60 mni dish on a feeder layer of mytomycin C-treated mouse embryonic
fibroblasts (CF-1). The next day, the medium was switched to hES-cell
medium. The iPS lines were confirmed positive for Tra-1-81, Tra-1-60,
SSEA-4 and Nanog by immunofluorescence and flow cytometry. In both hiPS
cell clones, all four vector-encoded transgenea were found to be silenced.
Neural induction.
hES-cell cultures were disaggregated using aceutase for
20 mm, washed using hES-cell media and pre-plated on gelatin for 1 h at
37 C
in
the presence of ROCK inhibitor to remove MEFs. The non-
adherent hES cells were washed and plated on Matrigel at a density of
10,000-25,000 cells/cm
2
on
Matrigel (BD)-coated dishes in MEP conditioned
hES
-
cell medium spiked with 10 ng/ml
of FGF
-
2 and ROCK
-
inhibitor. Ideal cell
density was found tube 18,000 cells/em
2
. The ROCK inhibitor was withdrawn,
and
hES cells were allowed
to expand
in cell medium for 3 d or until they were
nearly confluent. The initial differentiation media conditions included knockout
serum replacement media with 10 tM TGF-h inhibitor (Tocris) and 500 rig/ml
of Noggin (R&D). Upon day 5 of differentiation, the TGF-b inhibitor was
withdrawn and increasing amounts of N2 media (25%, 50%, 75%) was added
to the knockout serum replacement medium media every 2 d while maintaining
500 ng/ml of Noggin. For MS5 induction, established methods previously
reported were used 22.
Quantitative real-time PCR.
Total RNA was extracted using an RNeasy kit
(Qiagen). For each sample, 1 jig
of total
RNA was treated for DNA contamina-
tion and reverse transcribed using the Quantitect RT kit (Qiagen). Amplified
material was detected using Quantitect SYBR green probes and PCR kit
(Qiagen) on a Mastercycler RealPlex2 (Eppendorf). All results were normalized
to a HPR'I' control and are from 44) technical replicates of 2-3 independent
biological samples at each data point.
Neuronal
patterning and differentiation.
Dopaminergic patterning was
initiated using BDNF, ascorbic acid, sonic hedgehog and FGF8 in N2 media
as
previously reported
22
, and maturation was performed in the presence of
BDNF, ascorbic acid, GDNF, TGtb-1 and cyclic-AMP. Motoneuron patterning
was performed using BDNF, ascorbic acid, sonic hedgehog and retinoic acid in
N2 media as previously reported".
Microscopy, antibodies and flow cytometry.
Tissue was fixed using 4%
paraformaldehyde for 20 mm, washed with PBS, permeablized using 0.3%
Triton X in PBS and blocked using
1%
BSA in PBS. Primary antibodies used
for microscopy included PAX6 (Covanee), Oct4 (Biovision), AP2 (Novus
Biologicals), GBX2 (Sigma), HNK1 (Sigma), HOXB4 (Developmental Studies
Hybridonsa Bank (DSHB)), Nestin (R&D), NKX6.1 (DSHB), OTX2 (gift), p75
(Advanced Target Systems), PAX7 (DSHB), PLZF (Calbiochem),
TUJ1
(Coy-
altec), ZOl (Zynsed), BF1 (FOXGI, gift Esseng Lai), TH (Sigma), HB9 (DSHB)
and ISLl (DSHB). CD105-PE (eBioscience) was used for excluding MSS
stromal cells for flow cytometry on a FACScan (BD).
Note: Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS
We are grateful to F. Vacearino for providing 0tx2 antibody and E. Lai for BF1
antibody. This work was supported in part by the Starr Foundation, NINDS
grant 1R01NS052671, the Starr Stem Scholar fellowship (S.M.C.) and the a
New York Stem Cell Foundation fellowship (C.A.F.).
AUTHOR CONTRIBUTIONS
S.M.C. and L
.
S. designed the study. E.t'.P., MT., L.S. and M.S. designed and
generated the hiPS clones. S
.
M
.
C. and L.S. analyzed the data and wrote the
manuscript. S
.
M
.
C. and C.A.F. performed the experiments.
279
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