Motor Neuron Differentiation from Pluripotent StemCells and Other Intermediate Proliferative Precursorsthat 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 generatespinal motor neurons (MNs) from human embryonic stemcells (ESCs) and human induced pluripotent stem cells(iPSCs). These stages include the pluripotent stem cell(PSC) stage, neural stem cell (NSC) stage, OLIG2 expressingmotor neuron precursor (MNP) stage, and HB9 expressingmature-MN stage. To optimize the differentiation protocolreporter lines marking the NSC and MNP stages were used.The NSC stage is a pro-proliferative precursor stage at whichcells can be directed to differentiate to other neural types likecortical neurons also, in addition to MNs; thus, NSCs can beexpanded and stored for future differentiation to differentneural types thereby, shortening the differentiation intervalas compared to the complete process of differentiation fromESCs or iPSCs. Additionally, we find that OLIG2 positivecells at the MNP stage can be cryopreserved and then recov-ered to continue the process of MN differentiation, therebyproviding a highly stable and reproducible technique for bulkdifferentiation. MNPs were differentiated to MNs expressingthe marker HB9 demonstrating that mature-MNs can be gen-erated with this protocol.

Keywords Motor neuron .Motoneuron . Neural StemCell .

Motor neuron progenitor . Stem cell differentiation . Neuraldifferentiation . Neural induction .Motor neurondifferentiation

AbbreviationsPSC Pluripotent stem celliPSC Induced pluripotent stem cellhESC Human embryonic stem cell

NSC Neural stem cellMNP Motor neuron precursorMN Motor neuronCNS Central nervous systemO-L plate Ornithine-laminin coated platebFGF Basic fibroblast growth factorBMP Bone morphogenetic proteinRA Retinoic acidSHH Sonic hedgehogBDNF Brain derived neurotrophic factorGDNF Glial cell line derived neurotrophic factorHD Homeodomain

Introduction

Pluripotent stem cells (PSCs), which include human embry-onic stem cells (hESCs) and human induced pluripotent stemcells (iPSCs) are being increasingly utilized in regenerativemedicine and the biomedical industry. PSC use in applicationslike disease modeling, drug screening, and cellular replace-ment therapies, often requires that the pluripotent stem cells(PSCs) be differentiated to the cell type of interest. Avariety ofprotocols are being developed and optimized to differentiatePSCs to enriched populations of differentiated functional cellsincluding neural cells (1–5), cardiac cells (6), skeletal musclecells (7), osteoblasts (8), and hematopoietic cells (9). Thispaper describes a highly optimized four stage protocol forspinal motor neuron (MN) differentiation from the PSCs.The protocol is based on the well-established factors thatregulate the native pathways critical for the development ofmotor neurons from the notochord (10, 11).

The spinal motor neurons occupy a well-defined region inthe neuroanatomy of the central nervous system (CNS). Theyform a part of the hindbrain and are located caudal to the rest

B. S. Jha :M. Rao :N. Malik (*)Laboratory of Stem Cell Biology, National Institutes of Health(NIH), Bethesda, MD, USAe-mail: malikn@mail.nih.gov

Stem Cell Rev and RepDOI 10.1007/s12015-014-9541-0

of the CNS. In the spinal cord, the motor neurons areconcentrated in the ventral region. This precise level ofregionalization of the motor neurons is achieved by thesignaling pathways of several defined morphogens (11, 12).Initially, the ectodermal germline cells attain a rostral charac-ter forming neuroectoderm under the effect of FGF, BMP,Wntand Activin/Nodal signaling pathways (13–16). The caudalspinal positional identity is primarily regulated by retinoicacid (RA) provided by the paraxial mesoderm in the nativeenvironment (17–19). After regionalizing caudally, the differ-entiating precursor cells acquire the motor neuron precursor(MNP) identity under the ventralizing influence of the Sonichedgehog (SHH) released in a concentration gradient initiallyby the the notochord and later by floor plate cells (10, 12).This gradient effect of SHH is triggered by the patternedexpression of PAX6 and NKX6.1 homeodomain (HD) andOLIG2 basic helix-loop-helix (bHLH) transcription factors(12, 18). Continued action of these factors drive the MNPsout of their cell cycle resulting in the expression of down-stream HD protein MNX2, also known as HB9, which isexpressed during the final division cycle of the MNPs andacts as a dedicated determinant of MN identity (10, 18, 20).

Differentiated mature and functional MNs present greatopportunities to study MN diseases like spinal muscularatrophy (21) and amyotrophic lateral sclerosis (22), to performdrug screenings and for cell therapeutics. Many protocolshave been developed to differentiate MNs from PSCs fromboth healthy and diseased cell populations. These include co-cultures withMS5 stromal feeders supplemented with Noggin

to inhibit BMP pathway (23), or developing embryoid bodiesin suspension (24, 25) for neural induction, followed bypatterning of the cells using RA and SHH to MNs. Differen-tiation of PSCs to MNs by early exposure to RA to caudalizethem has been shown to yield a higher efficiency of differen-tiated MNs (26, 27). Furthermore, virus mediated gene deliv-ery systems with MN inducing transcription factors have beendeveloped that allows the differentiation process to be com-pleted in relatively shorter duration (28, 29). While all theseMN differentiation protocols are promising, each has its lim-itations. Drawbacks include the amount of time required fordifferentiation, need for animal feeder cells or repeated viralinfections and genetic manipulations that limit their use tonon-clinical applications. Additionally all published MN dif-ferentiation protocols go directly from a PSC to a terminallydifferentiated MN with no intermediate NSC stage. We havedeveloped a four-stage MN differentiation strategy that over-comes some of the difficulties associated with other protocols(Fig. 1). By utilizing iPSC reporter lines that express theneuronal stem cell marker NESTIN and the MNP markerOLIG2, we have been able to optimize the protocol and trackneural stem cell (NSC) generation and subsequent differenti-ation 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 theMNP stage where cells are marked by OLIG2 expression.Although the cells cannot be expanded at this stage as theyare exiting the cell cycle, they can be cryopreserved and

Fig. 1 Motor neuron differentiation schematic. Passage the PSCs cul-tured in E8 medium to Geltrex coated plates and then culture in NIM for12 days. When cells differentiate and become Nestin positive, change themedium to StemPro hESC SFM supplemented with RA, SHH, bFGF andActivin. After 2 days, passage these cells on ornithine-laminin coatedplates in StemPro hESC SFM supplemented with RA, SHH and bFGF.

After approximately 16 days, majority of cells should be OLIG2 positiveand become MNPs. Change the medium to StemPro hESC SFM supple-mented with BDNF and GDNF, and continue the culture for additional20–25 days. Majority of cells should differentiate to becomemature-MNsmarked by positive HB9 expression

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recovered for further differentiation to the mature MN stage.This allows culturing cells in bulk until they are in the MNPstage, and then cryopreserving them in large batches fordecreased variability and high reproducibility, making itpossible for the large scale MN production required forclinical applications. Additionally the availability of anexpandable and cryopreservable NSC state allows for thegeneration of many different central nervous system celltypes. The NSCs we have derived are positionally unspecifiedand can be differentiated to the mature neural cell types likeforebrain neuron, midbrain neurons, spinal neurons and to glialcells like astrocytes and oligodendrocytes (4, 31). It is alsopossible to differentiate OLIG2 positive neural progenitors tooligodendrocytes instead of mature-MNs (32).

The four staged protocol we describe is highly optimizedand the time points for each stage were confirmed with thehelp of the reporter lines used in this protocol. A NESTIN-GFP reporter line was generated to determine the exact timepoint when the PSCs were maximally differentiated to NSCs.This allowed us to switch the neuroinduction medium to MNdifferentiation medium at its optimal time. The use of OLIG2reporter cell line(33) allowed us to identify the time-pointwhen NSCs differentiated to MNP cells. Identifying thistime-point is critical in MN differentiation protocol as SHHneeds to be withdrawn at the specific interval when the cellsstart expressing OLIG2; early withdrawal can direct the pro-genitors to differentiate to interneurons (34), and continuedexposure to SHH can direct them to differentiate to oligoden-drocytes (32). We believe that this differentiation protocol willprovide a highly effective means of generating motor neuronsthat can be used for disease modeling and that the protocol iseasily modifiable for the production of cells that can be usedfor cellular replacement therapies.

Materials

General Supplies

1. 6-well cell culture plates (Corning Costar, # 3506)2. 15 ml conical tubes (Fisher Scientific, # 14-959-49D)3. T-75 flasks (BD Biosciences, # 13-680-65)4. Cryotube vials (Thermo Scientific, # 375418)5. Coverslips for cell culture (NeuVitro, # GG-25-1.5-pre)6. Permanox Lab-Tek™ Chamber Slides™ (Fisher, # 12-

565-21)

Cell Lines Used

1. CY2-NESTIN-Puro cell line2. R-OLIG2 reporter cell line (33)3. H9 cell line

Culture and Maintenance of PSCs

1. Matrigel (BD, # 354230)2. Essential 8 medium (Life technologies, # A1517001)3. EDTA, 0.5 M (Cellgro, # 46-034-CI)4. Sodium Chloride (J.T. Baker, # 3624–01)5. DPBS (Life Technologies, # 14190)6. Y-27632 dihydrochloride (Tocris, # 1254)7. 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 NeuronProgenitors

1. Poly-ornithine (Sigma, # P4957)2. Laminin (Life Technologies, # 23017015)3. StemPro hESC SFM (Life Technologies, A1000701)4. Retinoic Acid (Sigma, # R2625)5. Sonic Hedgehog (R&D Systems, # 1845-SH-100)6. bFGF (Peprotech, # 100-18B)7. Activin A (Peprotech, # 120-14E)8. StemPro Accutase (Life Technologies, # A11105-01)9. Dimethyl sulphoxide (DMSO) (Sigma, # D2650)

Maturation of Motor Neuron Progenitors

1. StemPro hESC SFM (Life Technologies, A1000701)2. Brain derived neurotrophic factor (BDNF) (R&D Sys-

tems, # 248-BD)3. Glial cell derived neurotrophic factor (GDNF) (R&D

Systems, # 212-GD)

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)

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6. Hoechst 33342 (Life Technologies, # H3570)7. Primary and secondary antibodies:

Primary Antibody Host Dilution Company, Cat. No.

Nanog Rabbit 1:1000 Peprotech, # 500-P236

Tra-1-60 Mouse 1:500 Millipore, # MAB4360

Nestin Mouse 1:250 BD Biosciences,# 611658

Sox1 Goat 1:100 R&D Systems,# AF3369

Tuj1 Mouse 1:250 Millipore, # MAB1637

Olig2 Rabbit 1:200 IBL America, # 18953

HB9 Mouse 1:100 DSHB, # 81.5C10

MAP2 Rabbit 1:500 Millipore, # AB5622

GFAP Rabbit 1:1000 Dako, # Z0334

SecondaryAntibody

Host α Reactivity Dilution Company, Cat. No.

Alexa-Fluor 488 Goat α Mouse 1:500 Invitrogen, # A11001

Alexa-Fluor 594 Goat α Mouse 1:500 Invitrogen, # A11005

Alexa-Fluor 488 Goat α Rabbit 1:500 Invitrogen, # A11034

Alexa-Fluor 594 Goat α Rabbit 1:500 Invitrogen, # A11012

Alexa-Fluor 594 Donkey α Goat 1:500 Invitrogen, # A11058

Reagent Setup

1. EDTA: Add 500 μL of 0.5 M EDTA and 0.9 g SodiumChloride to 500 mL DPBS (Calcium/Magnesium free).Filter the final solution.

2. Y-27632: Dissolve 10 mg of Y27632 in 3.12 mL DMSO togive 10 mM stock solution. Working concentration: 10 μM.

3. Retinoic Acid (RA): Dissolve 50 mg RA in 1.65 mLDMSO to give 100 mM stock solution. Working concen-tration: 50 μM.

4. Sonic Hedgehog (SHH): Dissolve 100 μg of SHH in1 mL of 0.1 % BSA in PBS to give 100 μg/mL stockconcentration. Working concentration: 200 ng/mL.

5. bFGF: Dissolve 100 μg of bFGF in 1 mL of 0.1 %BSA inPBS to give 100 μg/mL stock concentration. Workingconcentration: 8 ng/mL.

6. Activin: Dissolve 50 μg of Activin in 1 mL of 0.1 % BSAin PBS to give 50 μg/mL stock concentration. Workingconcentration: 10 ng/mL.

7. BDNF and GDNF: Dissolve 100μg of each growth factorin 1 mL of 0.1 % BSA in PBS to give 100 μg/mL stockconcentration. Working concentration: 10 ng/mL.

qPCR

1. RNeasy Mini Kit (Qiagen, # 74104)2. SuperScript III First-Strand Synthesis SuperMix (Life

Technologies, # 18080–400)

3. Fast SYBR Green Master Mix (Applied Biosystems,# 4385612)

4. Primers:

Gene Forward primer Reverse primer bp

OLIG2 5′-CCTGAGGCTTTTCGGAGC-3′

5′-CTGGCGTCCGAGTCCAT-3′

120

HB9 5′- CTTTTTGCTGCGTTTCCATT-3′

5′- GCACCAGTTCAAGCTCAACA-3′

133

ISL1 5′- CATGCTTTGTTAGGGATGGG-3′

5′- ACGCATCACGAAGTCGTTC-3′

113

PERIPHERIN 5′- AGACCATTGAGACCCGGAAT-3′

5′- GGCCTAGGGCAGAGTCAAG-3′

128

CHAT 5′- AACGAGGACGAGCGTTTG-3′

5′- TCAATCATGTCCAGCGAGTC-3′

122

NKX6.1 5′- ATTCGTTGGGGATGACAGAG-3′

5′- CCGAGTCCTGCTTCTTCTTG-3′

114

SCL18A3 5′- GATAAGTACCCGGAGGAGCC-3′

5′- GCGAACTCATAGAGGATGCC-3′

113

HOXB4 5′- GTCGTCTACCCCTGGATGC-3′

5′- TTCCTTCTCCAGCTCCAAGA-3′

123

Methods

Timing (Fig. 1)

i. Step 1, Confluent PSCs: 2–3 daysii. Steps 2–8, Generation of NSCs: 12 daysiii. Steps 9–12, Generation of motor neuron progenitors: 15–

17 daysiv. Steps, 13–15, Maturation of progenitors: 22–25 days

Culture and Maintenance of PSCs

i. Culture PSCs (iPSCs/ESCs) in monolayer on Matrigelcoated plates in Essential 8 (E8) medium (Life Technolo-gies). The medium needs to be changed every day. Onconfluency, the cells can be passaged at a ratio of 1:6 usingsterilized 0.5 mM EDTA as the cell detaching agent.Passaged cells should be seeded in the E8 medium sup-plemented with 10 μM Rock inhibitor – Y27632 (Tocris).

Matrigel coating: Thaw Matrigel at 4 °C. DiluteMatrigel 1:60 in the DMEMmedium. Coat each wellof 6-well plate with 1.5 mL of Matrigel solution.Incubate for 1 h at 37 °C or overnight at 4 °C. Warmthe plates from 4 °C for 20–30 min at 37 °C beforeusing them. Aspirate Matrigel immediately beforeseeding the cells; no washing is required. Platesincubated overnight at 4 °C can be store for 2 weeksat 4 °C. CRITICAL: Matrigel forms gel at room

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temperature. Do not let it to come to room tempera-ture before coating the plates.Passaging cells: Aspirate the E8 medium and washtwice in DPBS (Gibco). Add 1 mL/well of 6-wellplate 0.5 mM EDTA and incubate at 37ºC for ap-proximately 5–8 min. During the wait time, prepare15 mL centrifuge tubes with 5 mL of fresh E8 medi-um. When the cells start rounding up, aspirate theEDTA and detach the cells from the plates usingDPBS and 5 mL pipette. Transfer the cells suspen-sion in DPBS to the 15 mL centrifuge tube with E8medium. Centrifuge the tubes at x300g for 5 min.After confirming the pellet formation, aspirate thesuspension. Re-suspend the pellet in fresh E8 medi-um and seed onto the prepared plate.

Differentiation of iPSCs/ESCs to NSCs

ii. Day −1: Passage 1 well of 6-well plate of confluent PSCculture to 3 wells of Geltrex coated 6-well plate in theratio of 1:2, 1:3, and 1:6 respectively. Use E8 mediumsupplemented with 10 μM Y27632 to seed the cells onGeltrex. Follow the passaging protocol of PSCs.

Geltrex coating: Thaw Geltrex at 4 °C. DiluteGeltrex 1:200 in the DMEMmedium. Coat each wellof 6-well plate with 1.5 mL of Geltrex solution.Incubate for 1 h at 37 °C or overnight at 4 °C. Warmthe plates from 4 °C for 20–30 min at 37 °C beforeusing them. Aspirate Geltrex immediately beforeseeding the cells; no washing is required. Platesincubated overnight at 4 °C can be store for 2 weeksat 4 °C. CRITICAL: Geltrex forms gel at roomtemperature. Don’t let it to come to room temperaturebefore coating the plates.

iii. Day 0: Aspirate E8+Y27632 medium from all the 3wells and wash the wells once with PSC NeuralInduction Medium (NIM) (Life Technologies) to getrid of Y27632 completely. Add 2 mL of NIM/well of6-well plate.

iv. Day 1–6: Change the medium with fresh NIM asrequired. By day 4, 4 mL of NIM per well may berequired 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 onday 3 can be discarded.

v. Day 7: Passage the well 1:2 to 2 wells of Geltrex coated 6-well plate using Accutase (Life Technologies) as the dis-sociation agent. Seed the cells in NIM supplemented withY27632 in 1 well; 1 well in NIM without Y27632.

Passaging cells:Aspirate the NIM from the culturewells. Add 1 mL of Accutase per well of the 6-wellplate. Incubate at 37ºC for 5 min. When the cellsappear to detach from the plate, add 1 mL of NIM perwell of the plate and pipette up and down 2–3 timesto get the cells in suspension. Transfer the cell sus-pension to 15 mL centrifuge tubes and centrifugethem at x300g for 5 min. . After confirming the pelletformation, aspirate the suspension. Re-suspend thepellet in fresh NIMmedium and seed the cells on thefresh prepared plate.

vi. Day 8: If good cell survival is seen in the well withoutY27632, then discard the other well. Otherwise retain thewell in which NIM was supplemented with Y27632.Aspirate the medium and add 2 mL of fresh NIM perwell of 6-well plate (no Y27632).

vii. Day 8–14: Continue culture by passaging the cells 1:2when they reach maximal confluence (usually by day 3after 1:2 passaging). In each passage of the well whereY27632 was used in its previous passage, in its nextpassage withdraw Y27632 in one of the seeded wellsand continue culturing this well if good cell survival isseen. Once cell survival after passaging without Y27632is established, discontinue use of this Rock inhibitor infuture passages.

If NESTIN reporter line is used, GFP expression willmark the differentiation of PSCs to NSCs. For non-reporter cell lines, at each passage, seed some cells onglass slides and stain for NESTIN expression using theanti-Nestin antibody (1:250: BD, # 611658).

viii. When >90 % cells are NESTIN positive, they can beexpanded in the ratio of 1:4 and stored using 10%DMSOin NIM for cryopreservation in liquid Nitrogen. They canalso be differentiated to distinct regionalized neural sub-types like cortical neurons, midbrain neurons, MNs, as-trocytes and oligodendrocytes.

Caudalization and Ventralization of NSCs to Motor NeuronProgenitors

ix. Day 12: When cells are NESTIN positive in NIM, at 80 %confluency the medium is changed to StemPro hESC SFMsupplemented 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 ornithine-laminin (O-L) coated plates using Accutase with StemProhESC SFM using the protocol for passaging mentioned inStep 5. Seed cells at a density of 100,000 cells per cm2 ofthe O-L coated plates. For immunostaining, passage andculture cells on O-L coated Permanox Lab-Tek™ Cham-ber Slides™ and/or on O-L coated culture grade coverslips

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inside culture plates. Medium should be supplementedwith SHH (200 ng/mL) and bFGF (8 ng/mL). This medi-um needs to be changed every alternate day. 50 μM RA(1 μL of 100 mM to 2 mL medium) needs to be added to2 mL of StemPro hESC SFM+SHH medium every day.CRITICAL: As RA is very unstable it is important to add itto the medium every day.

Ornithine-laminin coating: Dilute poly-ornithine1:5 in sterile water. Thaw laminin overnight at4 °C. Coat each well of 6-well plate with 1.5 mL ofpoly-ornithine solution. Incubate the plates for 2 h at37 °C or overnight at 4 °C. Rinse the plates 2x withsterile water. Coat the plates with 20 μg/mL lamininsolution in sterile water. Incubate the plates for 2 h at37 °C or overnight at 4 °C. Rinse the plate 1x withDPBS before use. CRITICAL: Laminin absorbsplastic and forms aggregates at room temperature.Avoid storing laminin in plastic vials and alwaysthaw it at 4 °C.

xi. Day 24: By this time, the regionalization phase for mostof the cells should be midway and some cells start ex-pressing 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 dissociatedusing Accutase for cryopreservation in the culture medi-um+10 % DMSO. However, this interval varies withdifferent cell lines; cells cryopreserved at an earlier timepoint show better recovery.

xii. Day 28–30: The regionalization phase should last till day30. During this time (day 12 – day 30) it might benecessary to passage cells once around day 24. By day30, differentiating OLIG2 reporter cells should expressGFP. For other cell lines, they should be analyzed for theexpression of the OLIG2 marker using anti-Olig2 anti-body (1:200, IBL, # 18953) to confirm differentiation ofNSCs to MNPs.

Maturation of Motor Neuron Progenitors

xiii. Day 30: Prepare fresh StemPro hESC SFM mediumwithout SHH. Supplement the medium with 10 ng/mLof BDNF and GDNF. Aspirate old medium fromMNPscell- or OLIG2 expressing cell- cultures and feed thecultures with fresh medium containing BDNF andGDNF. RA is also withdrawn from the culture medium.

xiv. Day 31–52: The maturation phase takes ~3 weeks andduring this time the medium should be changed every2–3 days.

xv. After maturing for 3–4 weeks, analyze the differentiatedcells 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%BSAin PBS plus 0.1 % Tween 20.

ii. Fix the cells in ice cold 4 % paraformaldehyde in PBSpH 7.4 for 20 min at room temperature. CRITICAL: Para-formaldehyde is toxic and should be used in a fume hood.

iii. Wash the samples three times with ice cold PBS (5 mineach wash).

iv. Incubate the samples with 0.25% Triton X-100 in BB for15 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 temperatureor overnight at 4 °C.

ix. Wash the samples three times with BB (5 min eachwash).

x. Incubate samples with secondary antibodies with appro-priate dilutions in fresh BB for 1 h at room temperature indark.

xi. Aspirate the secondary antibody solution and add 0.5 μg/mL of Hoechst stain in BB. Incubate for 5–10 min areroom temperature in dark.

xii. Wash the samples three times with BB (5 min eachwash).

qPCR Assay

i. Extract RNA from the cell pellets following the manu-facturer’s protocol for RNeasy Mini Kit (Qiagen,#74104)

ii. Obtain cDNA from the extracted RNA following themanufacturer’s protocol for SuperScript III First-StrandSynthesis SuperMix (Life Technologies, # 18080–400).

iii. Run the qPCR assay following the manufacturer’s proto-col for Fast SYBR Green Master Mix (AppliedBiosystems, # 4385612) using appropriate primers.

Cell Identification

The cells at different stages have distinct cellular and/or cel-lular aggregate morphology and gene expression which can

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used to determine when the culture is ready to proceed aheadto the next stage.

4.1 Seeded PSCs appear a bit round in morphology withprominent euchromatin. The cells tend to double in

A B C

D E

F G

H I

J K

Nanog

Phase Phase Phase

Tra-1-60

Hoechst / Sox1 Hoechst / GFP

Hoechst / GFP

Hoechst / Sox1

Hoechst / Nestin

Hoechst / Nanog / Tra-1-60

Fig. 2 iPSC to NSC differentiation. a–c Phase image of non-confluentiPSCs (a), confluent iPSCs when they are ready to be passaged for NSCdifferentiation protocol (b), rosettes visible (red circles) when iPSCsdifferentiate to NSCs. d–g Immunostained CY2-Nestin-Puro reporter cellline at iPSC stage positive for Nanog (green) (d) and Tra-1-60 (red) (e)and negative for NSCmarker – Sox1 (red) (f) and the GFP expression (g).

h–k Immunostained CY2-NESTIN-Puro reporter cell line at NSC stagepositive for GFP expression (h), Nestin (green) (i) and Sox1 (red) (j) andnegative for iPSC pluripotency markers – Nanog (green) and Tra-1-60(red) (k). Scale bar in (a) for (a–c)=400μm, in (d) for (d–h, j–k)=25μm,and in (i)=50 μm

Hoechst / Sox1

Phase Phase Phase

Hoechst / Nestin_GFP

Hoechst / Olig2 / TuJ1 Hoechst / Olig2 / TuJ1 Hoechst / HB9 / MAP2

Hoechst /HB9 / MAP2

Hoechst /Olig2 / TuJ1

Hoechst / Olig2_GFP/ Olig2 Hoechst / Olig2/ HB9

A

B

C

D

E

F

G

H L

K

J

I

NSCs MNPs Mature MNs

Fig. 3 NSC to mature-MNdifferentiation. a–d CY2-Nestin-Puro reporter cell line at NSCstage in phase (a) is positive forGFP (b), Sox1 (red) (c), andnegative for Olig2 (green) andTuJ1 (red) (d). e–h DifferentiatedOLIG2 reporter cell line at MNPstage show neurite processes inphase (e), are positive for Olig2(red) and GFP (green) (f). g, hDifferentiated H9 cell line at theMNP stage are also positive forOlig2 (green) and TuJ1 (red). i–lDifferentiated H9 cells at mature-MN stage appear arranged inclusters interconnected with longprocesses in phase (g), arepositive for HB9 (red) (j–l) andMAP2 (green) (k and l). Cells stillin MNP stage are positive forOlig2 (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

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number in approximately every 16 h. They tend to grow inhigh densities in colonies with clearly defined edges(Fig. 2a). The PSCs should be positive for the nuclearmarker Nanog and the Tra-1-60 cell surface marker(Fig. 2d–g).

4.2 The PSCs are ready to passage when the culture isconfluent as determined by the very high density of cellswhich are packed together to form sheet-like structures(Fig. 2b).

4.3 When the PSCs are successfully differentiated toNSCs, thecells cluster themselves together forming rosettes (Fig. 2c).There is no expression of PSCs markers, instead NSCsmarkers are expressed. In this study, the NSCmarkers usedwere NESTIN and SOX1 (Fig. 2h–k, Fig. 3a–e).

4.4 The differentiation of NSCs to MNPs is marked by theexit of the cells from their cell cycle. The cells start toaggregate in clusters and they also start sending outprocesses form connections with other cells. There isexpression of MNP marker- OLIG2 in this study(Fig. 3e–h).

4.5 When MNPs mature to MNs, they have completelyexited the cell cycle and are in post-mitotic stage. Theyaggregate themselves into clusters with established con-nections between the different cell clusters from by thecell processes. Mature MN markers are expressed atthis stage- HB9 expression was tested in this study(Fig. 3i–l).

4.6 In this study, the cells pellets were collected ateach stage of differentiation from 1 well of a 6-well plate. RNA was extracted from these pelletsand qPCR was run using SYBR Green PCR Mas-ter Mix using SYBR Green-Based Gene ExpressionAnalysis protocol by Applied Biosystems by LifeTechnologies. The results of the qPCR analysisconfirm the expression of distinct markers at thedifferent stages of the MN. With the expression ofestablished mature-MN markers in its final stage,this analysis also confirms that this protocol can beused to differentiate mature and functional MNs(Fig. 4).

Fig. 4 qPCR results showingrelative expression of specificmarkers at NSC, MNP andmature-MN stages of motorneuron differentiation protocol

Table 1 Additional markers atdifferent stages of iPSC to MNdifferentiation protocol

PSC stage markers NSC stage markers MNP stage markers Mature MN markers

• NANOG

• TRA-1-60

• OCT4

• ZFP42

• NESTIN

• SOX 1/2

• PAX6

• ZBTB16

• MSXI1

• SSCA 1/4

• E-CADHERIN

• OLIG2 • HB9

• ISLET 1/2

• HOXB4

• CHAT

• TAU (axonal)

• MAP2 (dendritic)

• SYNAPTOPHYSIN (synaptic)

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Notes

5.1 Troubleshooting:

i. Step 7: Problem – Non-neural, non-rosette formingcells in the cultures.

Potential cause – Cell-line specific incomplete dif-ferentiation.

Solution – Identify and manually scrape the non-neural cells.

ii. Step 11: Problem - Poor recovery or survival ofMNPs after seeding/passaging.

Potential cause – Cell damage during enzymaticdissociation

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 addi-

tional culture time required by the specific cell line.Solution – If cells were not caudalized (checked

by OLIG2) expression by day 30, refer to retinoicacid troubleshooting. Alternatively culture for addi-tional 5–6 days and then stain for HB9 expression.

v. Contamination – Due to the long duration of thisprotocol, negligence of sterile techniques can resultin contaminated cultures. Sometimes repeated wash-ing with Hank’s Balanced Salt Solution (HBSS)followed by addition of antibiotic for 1 week canrestore the culture. However, if the contaminationpersists, the cultures must be destroyed using SodiumHydroxide or discarded completely if possible, andthe incubator should be decontaminated.

5.2 Retinoic acid is a highly unstable compound. Itsstock should be replaced every 3 months for opti-mal effects and it should be always protected fromlight.

5.3 There are many other distinct markers for the stages ofMN differentiation as listed in Table 1. For more elabo-rate study, testing a couple of additional markers for eachstage is recommended.

5.4 Avariation of the protocol to differentiate PSCs to NSCsby Sterneckert et al. has been tested in our laboratory tobe highly efficient in yielding pure populations of NSCs(35). However, it is more labor intensive protocol, andthe efficiency can be variable as it requires embryoidbody formation and rosette selection.

5.5 Puromorphamine, a SHH agonist, can replace the SHHin this protocol as a more economical alternative. How-ever it is important to note that because of its directdownstream effects its dose window is very narrowand OLIG2 expressing MNPs appear sooner (32).

5.6 Our laboratory also tested that astrocytes and differenti-ating MN can be co-cultured in the same MN media(Fig. 5). It has already been established that the co-culture setup provides a more native environment tothe differentiating MNs and the survival rate of thedifferentiating cells is much higher because of themolecules secreted from the astrocytes (36). Further-more, the problem of the detachment of MN processesduring the process of their differentiation is highly min-imized when they are co-cultured with astrocytes.

5.7 In conjunction with the growth factors and morphogens,the extracellular matrix environment has been shown toplay an important role in the patterning of PSCs and ininducing MN fate (37). Three-dimensional tissueengineered scaffolds have been utilized in cell culturesand their effects of the characteristics and architecture ofthe scaffolds on cell proliferation, migration, theirphenotype, and protein expressions has been confirmed(38, 39). In a recent study, it was demonstrated that themechanical properties of the extracellular matrix com-plements the effect of the morphogens in differentiatingand regionalizing the pluripotent stem cells, and thatsofter substrates improve the purity and yield offunctional MNs differentiated from PSCs (40). Thesefindings show the potential of functionalized three-dimensional tissue engineered scaffolds with varyingmechanical properties and adhesion molecules to furtheroptimize the MN differentiation protocol.

Conflict of Interest The work in this manuscript was funded by theNIH Common Fund, National Institutes of Health, Bethesda, USA. Theauthors declare no potential conflicts of interest.

TuJ1 / GFAPFig. 5 Co-culture of differentiating MNs with fetal astrocytes. Immuno-stained image at day 40 of iPSC to MN differentiation using the samemedium in MN differentiation protocol showing highly stable co-culturemodel with neurons expressing TuJ1 (red) and astrocytes expressingGFAP (green). Scale bar=50 μm

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