Download Motor Neuron Differentiation from Pluripotent Stem Cells and Other

Stem Cell Rev and Rep
DOI 10.1007/s12015-014-9541-0
Motor Neuron Differentiation from Pluripotent Stem
Cells and Other Intermediate Proliferative Precursors
that can be Discriminated by Lineage Specific Reporters
Balendu Shekhar Jha & Mahendra Rao & Nasir Malik
# Springer Science+Business Media New York (outside the USA) 2014
Abstract We have used a four stage protocol to generate
spinal motor neurons (MNs) from human embryonic stem
cells (ESCs) and human induced pluripotent stem cells
(iPSCs). These stages include the pluripotent stem cell
(PSC) stage, neural stem cell (NSC) stage, OLIG2 expressing
motor neuron precursor (MNP) stage, and HB9 expressing
mature-MN stage. To optimize the differentiation protocol
reporter lines marking the NSC and MNP stages were used.
The NSC stage is a pro-proliferative precursor stage at which
cells can be directed to differentiate to other neural types like
cortical neurons also, in addition to MNs; thus, NSCs can be
expanded and stored for future differentiation to different
neural types thereby, shortening the differentiation interval
as compared to the complete process of differentiation from
ESCs or iPSCs. Additionally, we find that OLIG2 positive
cells at the MNP stage can be cryopreserved and then recovered to continue the process of MN differentiation, thereby
providing a highly stable and reproducible technique for bulk
differentiation. MNPs were differentiated to MNs expressing
the marker HB9 demonstrating that mature-MNs can be generated with this protocol.
Keywords Motor neuron . Motoneuron . Neural Stem Cell .
Motor neuron progenitor . Stem cell differentiation . Neural
differentiation . Neural induction . Motor neuron
differentiation
Abbreviations
PSC
Pluripotent stem cell
iPSC
Induced pluripotent stem cell
hESC
Human embryonic stem cell
B. S. Jha : M. Rao : N. Malik (*)
Laboratory of Stem Cell Biology, National Institutes of Health
(NIH), Bethesda, MD, USA
e-mail: [email protected]
NSC
MNP
MN
CNS
O-L plate
bFGF
BMP
RA
SHH
BDNF
GDNF
HD
Neural stem cell
Motor neuron precursor
Motor neuron
Central nervous system
Ornithine-laminin coated plate
Basic fibroblast growth factor
Bone morphogenetic protein
Retinoic acid
Sonic hedgehog
Brain derived neurotrophic factor
Glial cell line derived neurotrophic factor
Homeodomain
Introduction
Pluripotent stem cells (PSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem
cells (iPSCs) are being increasingly utilized in regenerative
medicine and the biomedical industry. PSC use in applications
like disease modeling, drug screening, and cellular replacement therapies, often requires that the pluripotent stem cells
(PSCs) be differentiated to the cell type of interest. A variety of
protocols are being developed and optimized to differentiate
PSCs to enriched populations of differentiated functional cells
including neural cells (1–5), cardiac cells (6), skeletal muscle
cells (7), osteoblasts (8), and hematopoietic cells (9). This
paper describes a highly optimized four stage protocol for
spinal motor neuron (MN) differentiation from the PSCs.
The protocol is based on the well-established factors that
regulate the native pathways critical for the development of
motor neurons from the notochord (10, 11).
The spinal motor neurons occupy a well-defined region in
the neuroanatomy of the central nervous system (CNS). They
form a part of the hindbrain and are located caudal to the rest
Stem Cell Rev and Rep
of the CNS. In the spinal cord, the motor neurons are
concentrated in the ventral region. This precise level of
regionalization of the motor neurons is achieved by the
signaling pathways of several defined morphogens (11, 12).
Initially, the ectodermal germline cells attain a rostral character forming neuroectoderm under the effect of FGF, BMP, Wnt
and Activin/Nodal signaling pathways (13–16). The caudal
spinal positional identity is primarily regulated by retinoic
acid (RA) provided by the paraxial mesoderm in the native
environment (17–19). After regionalizing caudally, the differentiating precursor cells acquire the motor neuron precursor
(MNP) identity under the ventralizing influence of the Sonic
hedgehog (SHH) released in a concentration gradient initially
by the the notochord and later by floor plate cells (10, 12).
This gradient effect of SHH is triggered by the patterned
expression of PAX6 and NKX6.1 homeodomain (HD) and
OLIG2 basic helix-loop-helix (bHLH) transcription factors
(12, 18). Continued action of these factors drive the MNPs
out of their cell cycle resulting in the expression of downstream HD protein MNX2, also known as HB9, which is
expressed during the final division cycle of the MNPs and
acts as a dedicated determinant of MN identity (10, 18, 20).
Differentiated mature and functional MNs present great
opportunities to study MN diseases like spinal muscular
atrophy (21) and amyotrophic lateral sclerosis (22), to perform
drug screenings and for cell therapeutics. Many protocols
have been developed to differentiate MNs from PSCs from
both healthy and diseased cell populations. These include cocultures with MS5 stromal feeders supplemented with Noggin
to inhibit BMP pathway (23), or developing embryoid bodies
in suspension (24, 25) for neural induction, followed by
patterning of the cells using RA and SHH to MNs. Differentiation of PSCs to MNs by early exposure to RA to caudalize
them has been shown to yield a higher efficiency of differentiated MNs (26, 27). Furthermore, virus mediated gene delivery systems with MN inducing transcription factors have been
developed that allows the differentiation process to be completed in relatively shorter duration (28, 29). While all these
MN differentiation protocols are promising, each has its limitations. Drawbacks include the amount of time required for
differentiation, need for animal feeder cells or repeated viral
infections and genetic manipulations that limit their use to
non-clinical applications. Additionally all published MN differentiation protocols go directly from a PSC to a terminally
differentiated MN with no intermediate NSC stage. We have
developed a four-stage MN differentiation strategy that overcomes some of the difficulties associated with other protocols
(Fig. 1). By utilizing iPSC reporter lines that express the
neuronal stem cell marker NESTIN and the MNP marker
OLIG2, we have been able to optimize the protocol and track
neural stem cell (NSC) generation and subsequent differentiation of these NSCs to MNPs.
At the first two stages, the PSC stage and the NSC stage,
the cells are very stable and can be indefinitely expanded,
propagated and cryopreserved (30, 31). The third stage is the
MNP stage where cells are marked by OLIG2 expression.
Although the cells cannot be expanded at this stage as they
are exiting the cell cycle, they can be cryopreserved and
Fig. 1 Motor neuron differentiation schematic. Passage the PSCs cultured in E8 medium to Geltrex coated plates and then culture in NIM for
12 days. When cells differentiate and become Nestin positive, change the
medium to StemPro hESC SFM supplemented with RA, SHH, bFGF and
Activin. After 2 days, passage these cells on ornithine-laminin coated
plates in StemPro hESC SFM supplemented with RA, SHH and bFGF.
After approximately 16 days, majority of cells should be OLIG2 positive
and become MNPs. Change the medium to StemPro hESC SFM supplemented with BDNF and GDNF, and continue the culture for additional
20–25 days. Majority of cells should differentiate to become mature-MNs
marked by positive HB9 expression
Stem Cell Rev and Rep
Culture and Maintenance of PSCs
recovered for further differentiation to the mature MN stage.
This allows culturing cells in bulk until they are in the MNP
stage, and then cryopreserving them in large batches for
decreased variability and high reproducibility, making it
possible for the large scale MN production required for
clinical applications. Additionally the availability of an
expandable and cryopreservable NSC state allows for the
generation of many different central nervous system cell
types. The NSCs we have derived are positionally unspecified
and can be differentiated to the mature neural cell types like
forebrain neuron, midbrain neurons, spinal neurons and to glial
cells like astrocytes and oligodendrocytes (4, 31). It is also
possible to differentiate OLIG2 positive neural progenitors to
oligodendrocytes instead of mature-MNs (32).
The four staged protocol we describe is highly optimized
and the time points for each stage were confirmed with the
help of the reporter lines used in this protocol. A NESTINGFP reporter line was generated to determine the exact time
point when the PSCs were maximally differentiated to NSCs.
This allowed us to switch the neuroinduction medium to MN
differentiation medium at its optimal time. The use of OLIG2
reporter cell line(33) allowed us to identify the time-point
when NSCs differentiated to MNP cells. Identifying this
time-point is critical in MN differentiation protocol as SHH
needs to be withdrawn at the specific interval when the cells
start expressing OLIG2; early withdrawal can direct the progenitors to differentiate to interneurons (34), and continued
exposure to SHH can direct them to differentiate to oligodendrocytes (32). We believe that this differentiation protocol will
provide a highly effective means of generating motor neurons
that can be used for disease modeling and that the protocol is
easily modifiable for the production of cells that can be used
for cellular replacement therapies.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Materials
Maturation of Motor Neuron Progenitors
General Supplies
1. StemPro hESC SFM (Life Technologies, A1000701)
2. Brain derived neurotrophic factor (BDNF) (R&D Systems, # 248-BD)
3. Glial cell derived neurotrophic factor (GDNF) (R&D
Systems, # 212-GD)
1.
2.
3.
4.
5.
6.
6-well cell culture plates (Corning Costar, # 3506)
15 ml conical tubes (Fisher Scientific, # 14-959-49D)
T-75 flasks (BD Biosciences, # 13-680-65)
Cryotube vials (Thermo Scientific, # 375418)
Coverslips for cell culture (NeuVitro, # GG-25-1.5-pre)
Permanox Lab-Tek™ Chamber Slides™ (Fisher, # 12565-21)
Cell Lines Used
1. CY2-NESTIN-Puro cell line
2. R-OLIG2 reporter cell line (33)
3. H9 cell line
1.
2.
3.
4.
5.
6.
7.
Matrigel (BD, # 354230)
Essential 8 medium (Life technologies, # A1517001)
EDTA, 0.5 M (Cellgro, # 46-034-CI)
Sodium Chloride (J.T. Baker, # 3624–01)
DPBS (Life Technologies, # 14190)
Y-27632 dihydrochloride (Tocris, # 1254)
Dimethyl sulphoxide (DMSO) (Sigma, # D2650)
Differentiation of iPSCs/ESCs to NSCs
1. Geltrex (Life Technologies, # A1413202)
2. PSC Neural Induction Medium (Life Technologies, #
A1647801)
3. StemPro Accutase (Life Technologies, # A11105-01)
4. Y-27632 dihydrochloride (Tocris, # 1254)
5. Dimethyl sulphoxide (DMSO) (Sigma, # D2650)
Caudalization and Ventralization of NSCs to Motor Neuron
Progenitors
Poly-ornithine (Sigma, # P4957)
Laminin (Life Technologies, # 23017015)
StemPro hESC SFM (Life Technologies, A1000701)
Retinoic Acid (Sigma, # R2625)
Sonic Hedgehog (R&D Systems, # 1845-SH-100)
bFGF (Peprotech, # 100-18B)
Activin A (Peprotech, # 120-14E)
StemPro Accutase (Life Technologies, # A11105-01)
Dimethyl sulphoxide (DMSO) (Sigma, # D2650)
Immunostaining
1. Phosphate buffered saline (PBS) (Life Technologies, #
70011–044)
2. Paraformaldehyde (Electron Microscopy Sciences, #
15710)
3. Triton X-100 (American Analytical, # AB02025-00100)
4. Tween 20 (Affymetrix, T1003)
5. BSA (Life Technologies, # A10008-01)
Stem Cell Rev and Rep
6. Hoechst 33342 (Life Technologies, # H3570)
7. Primary and secondary antibodies:
Primary Antibody
Nanog
Tra-1-60
Nestin
Host
Rabbit
Mouse
Mouse
Dilution
1:1000
1:500
1:250
Sox1
Goat
1:100
Tuj1
Olig2
HB9
MAP2
GFAP
Mouse
Rabbit
Mouse
Rabbit
Rabbit
1:250
1:200
1:100
1:500
1:1000
Secondary
Antibody
Alexa-Fluor 488
Alexa-Fluor 594
Alexa-Fluor 488
Alexa-Fluor 594
Alexa-Fluor 594
Host α Reactivity Dilution Company, Cat. No.
Goat α Mouse
Goat α Mouse
Goat α Rabbit
Goat α Rabbit
Donkey α Goat
1:500
1:500
1:500
1:500
1:500
Company, Cat. No.
Peprotech, # 500-P236
Millipore, # MAB4360
BD Biosciences,
# 611658
R&D Systems,
# AF3369
Millipore, # MAB1637
IBL America, # 18953
DSHB, # 81.5C10
Millipore, # AB5622
Dako, # Z0334
Invitrogen, # A11001
Invitrogen, # A11005
Invitrogen, # A11034
Invitrogen, # A11012
Invitrogen, # A11058
3. Fast SYBR Green Master Mix (Applied Biosystems,
# 4385612)
4. Primers:
Gene
Forward primer
OLIG2
5′-CCTGAGGCTTTT
CGGAGC-3′
HB9
5′- CTTTTTGCTGCG
TTTCCATT-3′
ISL1
5′- CATGCTTTGTTA
GGGATGGG-3′
PERIPHERIN 5′- AGACCATTGAGA
CCCGGAAT-3′
CHAT
5′- AACGAGGACG
AGCGTTTG-3′
NKX6.1
5′- ATTCGTTGGGGA
TGACAGAG-3′
SCL18A3
5′- GATAAGTACCCG
GAGGAGCC-3′
HOXB4
5′- GTCGTCTACCCC
TGGATGC-3′
Reverse primer
bp
5′-CTGGCGTCCGAG
TCCAT-3′
5′- GCACCAGTTCAA
GCTCAACA-3′
5′- ACGCATCACGAA
GTCGTTC-3′
5′- GGCCTAGGGCAG
AGTCAAG-3′
5′- TCAATCATGTCC
AGCGAGTC-3′
5′- CCGAGTCCTGCT
TCTTCTTG-3′
5′- GCGAACTCATAG
AGGATGCC-3′
5′- TTCCTTCTCCAG
CTCCAAGA-3′
120
133
113
128
122
114
113
123
Methods
Reagent Setup
Timing (Fig. 1)
1. EDTA: Add 500 μL of 0.5 M EDTA and 0.9 g Sodium
Chloride to 500 mL DPBS (Calcium/Magnesium free).
Filter the final solution.
2. Y-27632: Dissolve 10 mg of Y27632 in 3.12 mL DMSO to
give 10 mM stock solution. Working concentration: 10 μM.
3. Retinoic Acid (RA): Dissolve 50 mg RA in 1.65 mL
DMSO to give 100 mM stock solution. Working concentration: 50 μM.
4. Sonic Hedgehog (SHH): Dissolve 100 μg of SHH in
1 mL of 0.1 % BSA in PBS to give 100 μg/mL stock
concentration. Working concentration: 200 ng/mL.
5. bFGF: Dissolve 100 μg of bFGF in 1 mL of 0.1 % BSA in
PBS to give 100 μg/mL stock concentration. Working
concentration: 8 ng/mL.
6. Activin: Dissolve 50 μg of Activin in 1 mL of 0.1 % BSA
in PBS to give 50 μg/mL stock concentration. Working
concentration: 10 ng/mL.
7. BDNF and GDNF: Dissolve 100 μg of each growth factor
in 1 mL of 0.1 % BSA in PBS to give 100 μg/mL stock
concentration. Working concentration: 10 ng/mL.
i. Step 1, Confluent PSCs: 2–3 days
ii. Steps 2–8, Generation of NSCs: 12 days
iii. Steps 9–12, Generation of motor neuron progenitors: 15–
17 days
iv. Steps, 13–15, Maturation of progenitors: 22–25 days
qPCR
1. RNeasy Mini Kit (Qiagen, # 74104)
2. SuperScript III First-Strand Synthesis SuperMix (Life
Technologies, # 18080–400)
Culture and Maintenance of PSCs
i. Culture PSCs (iPSCs/ESCs) in monolayer on Matrigel
coated plates in Essential 8 (E8) medium (Life Technologies). The medium needs to be changed every day. On
confluency, the cells can be passaged at a ratio of 1:6 using
sterilized 0.5 mM EDTA as the cell detaching agent.
Passaged cells should be seeded in the E8 medium supplemented with 10 μM Rock inhibitor – Y27632 (Tocris).
Matrigel coating: Thaw Matrigel at 4 °C. Dilute
Matrigel 1:60 in the DMEM medium. Coat each well
of 6-well plate with 1.5 mL of Matrigel solution.
Incubate for 1 h at 37 °C or overnight at 4 °C. Warm
the plates from 4 °C for 20–30 min at 37 °C before
using them. Aspirate Matrigel immediately before
seeding the cells; no washing is required. Plates
incubated overnight at 4 °C can be store for 2 weeks
at 4 °C. CRITICAL: Matrigel forms gel at room
Stem Cell Rev and Rep
temperature. Do not let it to come to room temperature before coating the plates.
Passaging cells: Aspirate the E8 medium and wash
twice in DPBS (Gibco). Add 1 mL/well of 6-well
plate 0.5 mM EDTA and incubate at 37ºC for approximately 5–8 min. During the wait time, prepare
15 mL centrifuge tubes with 5 mL of fresh E8 medium. When the cells start rounding up, aspirate the
EDTA and detach the cells from the plates using
DPBS and 5 mL pipette. Transfer the cells suspension in DPBS to the 15 mL centrifuge tube with E8
medium. Centrifuge the tubes at x300g for 5 min.
After confirming the pellet formation, aspirate the
suspension. Re-suspend the pellet in fresh E8 medium and seed onto the prepared plate.
Differentiation of iPSCs/ESCs to NSCs
ii. Day −1: Passage 1 well of 6-well plate of confluent PSC
culture to 3 wells of Geltrex coated 6-well plate in the
ratio of 1:2, 1:3, and 1:6 respectively. Use E8 medium
supplemented with 10 μM Y27632 to seed the cells on
Geltrex. Follow the passaging protocol of PSCs.
Geltrex coating: Thaw Geltrex at 4 °C. Dilute
Geltrex 1:200 in the DMEM medium. Coat each well
of 6-well plate with 1.5 mL of Geltrex solution.
Incubate for 1 h at 37 °C or overnight at 4 °C. Warm
the plates from 4 °C for 20–30 min at 37 °C before
using them. Aspirate Geltrex immediately before
seeding the cells; no washing is required. Plates
incubated overnight at 4 °C can be store for 2 weeks
at 4 °C. CRITICAL: Geltrex forms gel at room
temperature. Don’t let it to come to room temperature
before coating the plates.
iii. Day 0: Aspirate E8+Y27632 medium from all the 3
wells and wash the wells once with PSC Neural
Induction Medium (NIM) (Life Technologies) to get
rid of Y27632 completely. Add 2 mL of NIM/well of
6-well plate.
iv. Day 1–6: Change the medium with fresh NIM as
required. By day 4, 4 mL of NIM per well may be
required for high cell count. Observe the cells daily,
and continue with the well with optimal cell density
(4). Wells with very high or very low cell count on
day 3 can be discarded.
v. Day 7: Passage the well 1:2 to 2 wells of Geltrex coated 6well plate using Accutase (Life Technologies) as the dissociation agent. Seed the cells in NIM supplemented with
Y27632 in 1 well; 1 well in NIM without Y27632.
Passaging cells:Aspirate the NIM from the culture
wells. Add 1 mL of Accutase per well of the 6-well
plate. Incubate at 37ºC for 5 min. When the cells
appear to detach from the plate, add 1 mL of NIM per
well of the plate and pipette up and down 2–3 times
to get the cells in suspension. Transfer the cell suspension to 15 mL centrifuge tubes and centrifuge
them at x300g for 5 min. . After confirming the pellet
formation, aspirate the suspension. Re-suspend the
pellet in fresh NIM medium and seed the cells on the
fresh prepared plate.
vi. Day 8: If good cell survival is seen in the well without
Y27632, then discard the other well. Otherwise retain the
well in which NIM was supplemented with Y27632.
Aspirate the medium and add 2 mL of fresh NIM per
well of 6-well plate (no Y27632).
vii. Day 8–14: Continue culture by passaging the cells 1:2
when they reach maximal confluence (usually by day 3
after 1:2 passaging). In each passage of the well where
Y27632 was used in its previous passage, in its next
passage withdraw Y27632 in one of the seeded wells
and continue culturing this well if good cell survival is
seen. Once cell survival after passaging without Y27632
is established, discontinue use of this Rock inhibitor in
future passages.
If NESTIN reporter line is used, GFP expression will
mark the differentiation of PSCs to NSCs. For nonreporter cell lines, at each passage, seed some cells on
glass slides and stain for NESTIN expression using the
anti-Nestin antibody (1:250: BD, # 611658).
viii. When >90 % cells are NESTIN positive, they can be
expanded in the ratio of 1:4 and stored using 10 % DMSO
in NIM for cryopreservation in liquid Nitrogen. They can
also be differentiated to distinct regionalized neural subtypes like cortical neurons, midbrain neurons, MNs, astrocytes and oligodendrocytes.
Caudalization and Ventralization of NSCs to Motor Neuron
Progenitors
ix. Day 12: When cells are NESTIN positive in NIM, at 80 %
confluency the medium is changed to StemPro hESC SFM
supplemented with SHH (200 ng/mL), RA (50 μM), bFGF
(8 ng/mL) and Activin (10 ng/mL).
x. Day 14: At 100 % confluency, passage cells to ornithinelaminin (O-L) coated plates using Accutase with StemPro
hESC SFM using the protocol for passaging mentioned in
Step 5. Seed cells at a density of 100,000 cells per cm2 of
the O-L coated plates. For immunostaining, passage and
culture cells on O-L coated Permanox Lab-Tek™ Chamber Slides™ and/or on O-L coated culture grade coverslips
Stem Cell Rev and Rep
inside culture plates. Medium should be supplemented
with SHH (200 ng/mL) and bFGF (8 ng/mL). This medium needs to be changed every alternate day. 50 μM RA
(1 μL of 100 mM to 2 mL medium) needs to be added to
2 mL of StemPro hESC SFM+SHH medium every day.
CRITICAL: As RA is very unstable it is important to add it
to the medium every day.
Ornithine-laminin coating: Dilute poly-ornithine
1:5 in sterile water. Thaw laminin overnight at
4 °C. Coat each well of 6-well plate with 1.5 mL of
poly-ornithine solution. Incubate the plates for 2 h at
37 °C or overnight at 4 °C. Rinse the plates 2x with
sterile water. Coat the plates with 20 μg/mL laminin
solution in sterile water. Incubate the plates for 2 h at
37 °C or overnight at 4 °C. Rinse the plate 1x with
DPBS before use. CRITICAL: Laminin absorbs
plastic and forms aggregates at room temperature.
Avoid storing laminin in plastic vials and always
thaw it at 4 °C.
xi. Day 24: By this time, the regionalization phase for most
of the cells should be midway and some cells start expressing OLIG2. If the OLIG2 reporter cell line is used,
this would be marked by GFP expression in some cells.
Between day 24-day 27, the cells can be dissociated
using Accutase for cryopreservation in the culture medium+10 % DMSO. However, this interval varies with
different cell lines; cells cryopreserved at an earlier time
point show better recovery.
xii. Day 28–30: The regionalization phase should last till day
30. During this time (day 12 – day 30) it might be
necessary to passage cells once around day 24. By day
30, differentiating OLIG2 reporter cells should express
GFP. For other cell lines, they should be analyzed for the
expression of the OLIG2 marker using anti-Olig2 antibody (1:200, IBL, # 18953) to confirm differentiation of
NSCs to MNPs.
Maturation of Motor Neuron Progenitors
xiii. Day 30: Prepare fresh StemPro hESC SFM medium
without SHH. Supplement the medium with 10 ng/mL
of BDNF and GDNF. Aspirate old medium from MNPs
cell- or OLIG2 expressing cell- cultures and feed the
cultures with fresh medium containing BDNF and
GDNF. RA is also withdrawn from the culture medium.
xiv. Day 31–52: The maturation phase takes ~3 weeks and
during this time the medium should be changed every
2–3 days.
xv. After maturing for 3–4 weeks, analyze the differentiated
cells for the expression of mature motor neuron marker -
HB9 by immunostaining using anti-HB9 antibody
(1:100, DSHB, # 81.5C10).
Immunostaining
i.
Prepare the blocking buffer (BB) comprising of 1 % BSA
in PBS plus 0.1 % Tween 20.
ii. Fix the cells in ice cold 4 % paraformaldehyde in PBS
pH 7.4 for 20 min at room temperature. CRITICAL: Paraformaldehyde is toxic and should be used in a fume hood.
iii. Wash the samples three times with ice cold PBS (5 min
each wash).
iv. Incubate the samples with 0.25 % Triton X-100 in BB for
15 min.
v. Wash samples once with BB.
vi. Incubate the samples in BB for 1 h at room temperature
or overnight at 4 °C.
vii. Aspirate the BB and wash once with fresh BB.
viii. Add primary antibodies with appropriate dilutions in
fresh BB. Incubate samples for 1 h at room temperature
or overnight at 4 °C.
ix. Wash the samples three times with BB (5 min each
wash).
x. Incubate samples with secondary antibodies with appropriate dilutions in fresh BB for 1 h at room temperature in
dark.
xi. Aspirate the secondary antibody solution and add 0.5 μg/
mL of Hoechst stain in BB. Incubate for 5–10 min are
room temperature in dark.
xii. Wash the samples three times with BB (5 min each
wash).
qPCR Assay
i.
Extract RNA from the cell pellets following the manufacturer’s protocol for RNeasy Mini Kit (Qiagen,
#74104)
ii. Obtain cDNA from the extracted RNA following the
manufacturer’s protocol for SuperScript III First-Strand
Synthesis SuperMix (Life Technologies, # 18080–400).
iii. Run the qPCR assay following the manufacturer’s protocol for Fast SYBR Green Master Mix (Applied
Biosystems, # 4385612) using appropriate primers.
Cell Identification
The cells at different stages have distinct cellular and/or cellular aggregate morphology and gene expression which can
Stem Cell Rev and Rep
A
B
Phase
Phase
C
Phase
I
D
E
H
Nanog
Tra-1-60
Hoechst / GFP
Hoechst / Nestin
F
G
J
K
Hoechst / Sox1
Hoechst / GFP
Hoechst / Sox1
Hoechst /
Nanog / Tra-1-60
Fig. 2 iPSC to NSC differentiation. a–c Phase image of non-confluent
iPSCs (a), confluent iPSCs when they are ready to be passaged for NSC
differentiation protocol (b), rosettes visible (red circles) when iPSCs
differentiate to NSCs. d–g Immunostained CY2-Nestin-Puro reporter cell
line at iPSC stage positive for Nanog (green) (d) and Tra-1-60 (red) (e)
and negative for NSC marker – Sox1 (red) (f) and the GFP expression (g).
h–k Immunostained CY2-NESTIN-Puro reporter cell line at NSC stage
positive for GFP expression (h), Nestin (green) (i) and Sox1 (red) (j) and
negative for iPSC pluripotency markers – Nanog (green) and Tra-1-60
(red) (k). Scale bar in (a) for (a–c)=400 μm, in (d) for (d–h, j–k)=25 μm,
and in (i)=50 μm
used to determine when the culture is ready to proceed ahead
to the next stage.
4.1 Seeded PSCs appear a bit round in morphology with
prominent euchromatin. The cells tend to double in
Fig. 3 NSC to mature-MN
differentiation. a–d CY2-NestinPuro reporter cell line at NSC
stage in phase (a) is positive for
GFP (b), Sox1 (red) (c), and
negative for Olig2 (green) and
TuJ1 (red) (d). e–h Differentiated
OLIG2 reporter cell line at MNP
stage show neurite processes in
phase (e), are positive for Olig2
(red) and GFP (green) (f). g, h
Differentiated H9 cell line at the
MNP stage are also positive for
Olig2 (green) and TuJ1 (red). i–l
Differentiated H9 cells at matureMN stage appear arranged in
clusters interconnected with long
processes in phase (g), are
positive for HB9 (red) (j–l) and
MAP2 (green) (k and l). Cells still
in MNP stage are positive for
Olig2 (green) (j). Scale bar in (a)
for (a, e–f, i–j)=400 μm, in (b, c,
g, and k)=25 μm, in (d) for (d, h,
and l)=50 μm
A
E
I
Phase
Phase
Phase
B
F
J
Hoechst / Nestin_GFP
Hoechst / Olig2_GFP/ Olig2
Hoechst / Olig2/ HB9
C
G
K
Hoechst / Sox1
Hoechst /
Olig2 / TuJ1
Hoechst /
HB9 / MAP2
D
H
L
Hoechst / Olig2 / TuJ1
Hoechst / Olig2 / TuJ1
Hoechst / HB9 / MAP2
NSCs
MNPs
Mature MNs
Stem Cell Rev and Rep
Fig. 4 qPCR results showing
relative expression of specific
markers at NSC, MNP and
mature-MN stages of motor
neuron differentiation protocol
number in approximately every 16 h. They tend to grow in
high densities in colonies with clearly defined edges
(Fig. 2a). The PSCs should be positive for the nuclear
marker Nanog and the Tra-1-60 cell surface marker
(Fig. 2d–g).
4.2 The PSCs are ready to passage when the culture is
confluent as determined by the very high density of cells
which are packed together to form sheet-like structures
(Fig. 2b).
4.3 When the PSCs are successfully differentiated to NSCs, the
cells cluster themselves together forming rosettes (Fig. 2c).
There is no expression of PSCs markers, instead NSCs
markers are expressed. In this study, the NSC markers used
were NESTIN and SOX1 (Fig. 2h–k, Fig. 3a–e).
4.4 The differentiation of NSCs to MNPs is marked by the
exit of the cells from their cell cycle. The cells start to
aggregate in clusters and they also start sending out
processes form connections with other cells. There is
expression of MNP marker- OLIG2 in this study
(Fig. 3e–h).
Table 1 Additional markers at
different stages of iPSC to MN
differentiation protocol
4.5 When MNPs mature to MNs, they have completely
exited the cell cycle and are in post-mitotic stage. They
aggregate themselves into clusters with established connections between the different cell clusters from by the
cell processes. Mature MN markers are expressed at
this stage- HB9 expression was tested in this study
(Fig. 3i–l).
4.6 In this study, the cells pellets were collected at
each stage of differentiation from 1 well of a 6well plate. RNA was extracted from these pellets
and qPCR was run using SYBR Green PCR Master Mix using SYBR Green-Based Gene Expression
Analysis protocol by Applied Biosystems by Life
Technologies. The results of the qPCR analysis
confirm the expression of distinct markers at the
different stages of the MN. With the expression of
established mature-MN markers in its final stage,
this analysis also confirms that this protocol can be
used to differentiate mature and functional MNs
(Fig. 4).
PSC stage markers
NSC stage markers
MNP stage markers
Mature MN markers
• NANOG
• NESTIN
• OLIG2
• HB9
• TRA-1-60
• SOX 1/2
• ISLET 1/2
• OCT4
• PAX6
• HOXB4
• ZFP42
• ZBTB16
• CHAT
• MSXI1
• TAU (axonal)
• SSCA 1/4
• MAP2 (dendritic)
• E-CADHERIN
• SYNAPTOPHYSIN (synaptic)
Stem Cell Rev and Rep
TuJ1 / GFAP
Fig. 5 Co-culture of differentiating MNs with fetal astrocytes. Immunostained image at day 40 of iPSC to MN differentiation using the same
medium in MN differentiation protocol showing highly stable co-culture
model with neurons expressing TuJ1 (red) and astrocytes expressing
GFAP (green). Scale bar=50 μm
Notes
5.1 Troubleshooting:
i. Step 7: Problem – Non-neural, non-rosette forming
cells in the cultures.
Potential cause – Cell-line specific incomplete differentiation.
Solution – Identify and manually scrape the nonneural cells.
ii. Step 11: Problem - Poor recovery or survival of
MNPs after seeding/passaging.
Potential cause – Cell damage during enzymatic
dissociation
Solution – Plate cells at a higher density.
iii. Step 12: Problem – Only few or no OLIG2 positive
cells.
Potential Cause – Bad stock of retinoic acid.
Solution – Ensure proper handling of retinoic acid
in dark conditions.
iv. Step 15: Problem – Only few or no HB9 positive
cells.
Potential Cause – NSCs not caudalized or additional culture time required by the specific cell line.
Solution – If cells were not caudalized (checked
by OLIG2) expression by day 30, refer to retinoic
acid troubleshooting. Alternatively culture for additional 5–6 days and then stain for HB9 expression.
v. Contamination – Due to the long duration of this
protocol, negligence of sterile techniques can result
in contaminated cultures. Sometimes repeated washing with Hank’s Balanced Salt Solution (HBSS)
followed by addition of antibiotic for 1 week can
restore the culture. However, if the contamination
persists, the cultures must be destroyed using Sodium
Hydroxide or discarded completely if possible, and
the incubator should be decontaminated.
5.2 Retinoic acid is a highly unstable compound. Its
stock should be replaced every 3 months for optimal effects and it should be always protected from
light.
5.3 There are many other distinct markers for the stages of
MN differentiation as listed in Table 1. For more elaborate study, testing a couple of additional markers for each
stage is recommended.
5.4 A variation of the protocol to differentiate PSCs to NSCs
by Sterneckert et al. has been tested in our laboratory to
be highly efficient in yielding pure populations of NSCs
(35). However, it is more labor intensive protocol, and
the efficiency can be variable as it requires embryoid
body formation and rosette selection.
5.5 Puromorphamine, a SHH agonist, can replace the SHH
in this protocol as a more economical alternative. However it is important to note that because of its direct
downstream effects its dose window is very narrow
and OLIG2 expressing MNPs appear sooner (32).
5.6 Our laboratory also tested that astrocytes and differentiating MN can be co-cultured in the same MN media
(Fig. 5). It has already been established that the coculture setup provides a more native environment to
the differentiating MNs and the survival rate of the
differentiating cells is much higher because of the
molecules secreted from the astrocytes (36). Furthermore, the problem of the detachment of MN processes
during the process of their differentiation is highly minimized when they are co-cultured with astrocytes.
5.7 In conjunction with the growth factors and morphogens,
the extracellular matrix environment has been shown to
play an important role in the patterning of PSCs and in
inducing MN fate (37). Three-dimensional tissue
engineered scaffolds have been utilized in cell cultures
and their effects of the characteristics and architecture of
the scaffolds on cell proliferation, migration, their
phenotype, and protein expressions has been confirmed
(38, 39). In a recent study, it was demonstrated that the
mechanical properties of the extracellular matrix complements the effect of the morphogens in differentiating
and regionalizing the pluripotent stem cells, and that
softer substrates improve the purity and yield of
functional MNs differentiated from PSCs (40). These
findings show the potential of functionalized threedimensional tissue engineered scaffolds with varying
mechanical properties and adhesion molecules to further
optimize the MN differentiation protocol.
Conflict of Interest The work in this manuscript was funded by the
NIH Common Fund, National Institutes of Health, Bethesda, USA. The
authors declare no potential conflicts of interest.
Stem Cell Rev and Rep
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