TITLE: Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use
AUTHORS: Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson’s disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson’s disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.</div></div>

[STEPS]
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
Dissociate hPSCs into single cells using Accutase (Cell Technologies, #AT104), and plate at 400K cells/cm2 onto Geltrex (Life Technologies, #A1413201) coated dishes with Neurobasal (Life Technologies)/N2 (Stem Cell Technologies)/B27(Life Technologies) media containing  L-glutamine,  SHH C25II (R&D systems #464-SH),  LDN (Stemgent # 04-0074-02),  SB431542 (R&D systems #1614),  CHIR99021 (R&D systems #4432), and  Rock inhibitor (Y-27632, R&D systems #1254), which represents day 0 of differentiation, and culture until day 3 without Rock inhibitor from day 1.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 4, expose cells to different concentration of CHIR , , , and  until day 10.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 7, withdraw LDN, SB, and SHH.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 10, change media to Neurobasal/B27/L-Glu supplemented with BDNF (brain-derived neurotrophic factor, ; R&D #248-BD), ascorbic acid (, Sigma #4034), GDNF (glial cell line-derived neurotrophic factor, ; Peprotech # 450-10), TGFb3 (transforming growth factor type b3, ; R&D #243-B3), dibutyryl cAMP (; Sigma #4043), and CHIR .
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 11, dissociate cells using Accutase and replate under high cell density (800K cells/cm2) on polyornithine (PO; )/ laminin ()/ fibronectin () coated dishes in mDA differentiation media [(NB/B27/L-Glu, BDNF, ascorbic acid, GDNF, dbcAMP, and TGFb3 until day 16 with adding DAPT ( R&D #2634)] from day 12.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 16, dissociate cells and plate as same procedure of day 11 and culture until day 25 using mDA differentiation media.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
On day 25, dissociate cells using Accutase and replate under low cell density (200K∼300K cells/cm2) in mDA differentiation media until the desired experiments.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
For the cryopreservation of mDA precursor neurons, treat day16 mDA differentiated cells with Accutase for -, washing, detached, single cells, and pelleting.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
Resuspend cell pellets at a cell density of 8 million cells/mL of STEMCELLBANKER.
?. [Directed differentiation into midbrain dopamine neurons (mDA)]
Use controlled rate freezer (ThermoFisher) to cryopreserve cell product.
?. [Immunohistochemistry]
Fix cells in 4% paraformaldehyde (PFA) (Affymetrix #MFCD00133991) in DPBS for  at .
0 Room temperature
?. [Immunohistochemistry]
Wash cells subsequently with DPBS.
?. [Immunohistochemistry]
Permeabilize the samples with 0.5% Triton X-100 and block with 2% BSA in DPBS.
?. [Immunohistochemistry]
Incubate the samples with primary antibody  at .
4 °C
?. [Immunohistochemistry]
The next day, after washing with DPBS, incubate the samples with secondary antibody conjugated with Alexa Fluor 488- 555-, or 647- (Thermo Fisher) diluted at 1:400 in 2% BSA (DPBS) for  at .
4 Room temperature
?. [Immunohistochemistry]
Wash the samples with DPBS and count-stain with 40, 6-diamidino-2-phenylindole (DAPI) (Sigma, #D9542).
?. [Immunohistochemistry]
Visualize the images using an Olympus and Zeiss inverted fluorescence microscope.
?. [Immunohistochemistry]
Use donkey anti- mouse, goat, rabbit or chicken secondary antibodies conjugated with Alexa Fluor-488, Alexa Fluor-555 or Alexa Fluor-647 fluorophore (1:400, Life technologies). Counterstain nuclei by DAPI.
?. [Immunohistochemistry]
Use Mouse and chicken anti-MAP2 (1:1500, Sigma and 1:2000, Abcam), rabbit and mouse anti-TH (1:500, PelFreez and 1:1000, Immunostar), goat anti-FOXA2 (1:200, R&D), Rabbit anti-LMX1A (1:1500, Abcam), Goat anti-OTX2 (1:1000, Neuromics), rabbit and mouse anti-PAX6 (1:500, Covance and 1:200, BD-Biosciences), mouse and rabbit anti-EN1 (1:50, DSHB and 1:200 Invitrogen), goat anti-ALDH1A1 (1:250, Santa Cruz), rabbit anti-GIRK2 (1:400, Almonte), rabbit anti-CALB1 (1:2000 Swant), and mouse anti-NURR1 (1:1500, Perseus Proteomics) for immuno-fluorescent staining.
?. [Western blotting]
Collect and lyse the cultured cells and with 2X Laemmli Sample Buffer (Bio-Rad, #161-737).
?. [Western blotting]
After protein quantification using BCA protein assay kit (Pierce, #23228), load and separate the same amount of proteins from samples by NuPAGE 4%–12% Bis-Tris Protein Gel (Invitrogen, #NP0322BOX) using NuPAGE MES SDS Running Buffer (Invitrogen, #NP0060).
?. [Western blotting]
Transfer proteins electrophoretically to a nitrocellulose membrane using NuPAGE Transfer Buffer (Invitrogen, #NP0006) with 20% Methanol.
?. [Western blotting]
Block the membranes in 5% skim milk (TBS-T) for  at  and incubate primary antibodies  at .
4 Room temperature
4 °C
?. [Western blotting]
After washing with TBS-T, incubate secondary mouse or rabbit antibodies conjugated to horseradish peroxidase for  at .
4 Room temperature
?. [Western blotting]
After three times washing, perform developing the signals by using an enhanced chemiluminescence (ECL) detection kit (PerkinElmer, #NEL104001WA).
?. [RNA extraction and Real-time qRT-PCR]
Isolate the total RNAs from samples with TRIzol (QIAGEN) using the Direct-zol RNA MiniPrep kit (Zymo Research, #R2052).
?. [RNA extraction and Real-time qRT-PCR]
Use  of RNA to generate cDNA using the iScript Reverse Transcription Supermix (BioRad, #170-8841).
1 µg
?. [RNA extraction and Real-time qRT-PCR]
Perform real-time qRT-PCR using the SSoFAST EvaGreen Mix (BioRad) in a BioRad CFX96 Thermal Cycler.
?. [RNA extraction and Real-time qRT-PCR]
Perform all reactions according to the manufactured protocol.
?. [RNA extraction and Real-time qRT-PCR]
Obtain some primers from QIAGEN (Quantitect Primer assays). Normalize results to GAPDH.
Primer sequences are listed in Table S1.
?. [RNA-sequencing]
Perform RNA-seq library preparation at the MSKCC Integrated Genomics Operation Core Facility.
?. [RNA-sequencing]
Sequence libraries on an Illumina HiSeq 2500 platform with 50bp paired end reads.
?. [RNA-sequencing]
Filter the sequencing data for quality and remove adaptor sequences using Flexbar (v.2.2) (Dodt et al., 2012) and align to hg19 using STAR aligner (v.2.4.2a) (Dobin et al., 2013).
On average, we obtain ∼50M reads per sample with > 97% mapped reads.
?. [RNA-sequencing]
Generate gene read coverage using FeatureCounts (v.1.4.2) (Liao et al., 2014) using GENCODE annotation (v19) (Harrow et al., 2012).
?. [RNA-sequencing]
Perform differential gene expression using DESeq2 (v. 1.12.4) (Love et al., 2014) and annotate using biomaRt package (v. 2.28) (Durinck et al., 2009).
?. [ChIP-sequencing]
Perform chromatin immune-precipitation (ChIP) for H3K27me3 (Millipore, #07-449) and H3K4me3 (Abcam, #ab8580) from each sample using SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell signaling Tech, #9005) according to the instructions of the manufacturer.
?. [ChIP-sequencing]
Generate ChIP-sequencing library at the MSKCC Integrated Genomics Operation Core Facility.
?. [ChIP-sequencing]
Sequence the libraries on an Illumina HiSeq 2500 platform with 50bp paired end reads.
?. [ChIP-sequencing]
Process the generated each FASTQ files to remove any adaptor sequences at the end of the reads using cutadapt (v1.6).
?. [ChIP-sequencing]
Map the files using the BWA mapper (bwa mem v0.7.12).
?. [ChIP-sequencing]
Sort the SAM files after mapping and add read group tags using the PICARD tools.
?. [ChIP-sequencing]
After sorting in coordinate order, process the BAM’s with PICARD MarkDuplicates.
?. [ChIP-sequencing]
Do peak calling using the MACS program (Version 2).
?. [Electrophysiological recordings]
Perform patch-clamp electrophysiological recording on hPSC-derived mDA neurons plated on a monolayer of rat cortical  astrocytes, as described previously (Rayport et al., 1992).
?. [Electrophysiological recordings]
Conduct recording at day 40, 60, and 75 on randomly selected neurons at  in a Tyrode’s solution containing (in mM):  NaCl,  KCl,  glucose,  CaCl2,  MgCl2-6 H2O,  HEPES, and  HEPES-Na+ salt (, 270 mOsm).
4 Room temperature
?. [Electrophysiological recordings]
For whole-cell patch-clamp studies, pull borosilicate glass pipettes (G150F-4, Warner Instruments) with a tip resistance of 3-4 MΩ on a P-97 Flaming-Brown micropipette puller (Sutter Instruments) and fill with (in mM):  K-gluconate,  KCl,  HEPES,  MgCl2,  ATP-Mg,  ATP-Na2 and  GTP-Na, (, ∼280 mOsm).
?. [Electrophysiological recordings]
Visualize neurons under a 40x water immersion objective using Olympus BX51W1 microscope (Olympus), and perform recording with an Axopatch 700B amplifier (Molecular Devices) and digitize at 10 kHz with ITC-18 (HEKA Instruments Inc).
?. [Electrophysiological recordings]
Acquire data using WinWCP software (John Dempster, University of Strathclyde, UK).
?. [Electrophysiological recordings]
In each cell, monitor input resistance (measured by -100 pA, 1 s hyperpolarizing pulse), resting membrane potential and spontaneous firing frequency throughout the recording.
?. [Electrophysiological recordings]
Measure current-voltage relationship and evoked action potentials by injecting a 1 s long somatic current from -30 to +20 pA in +10 pA increments and from 0 to +250 pA in +10 pA increments, respectively.
?. [Electrophysiological recordings]
To measure HCN currents, hold cells at -50 mV in voltage-clamp mode and apply hyperpolarizing voltage steps from -70 to -160 mV.
?. [Electrophysiological recordings]
Measure KCNQ currents at -30 mV holding potential with -30 to -70 mV hyperpolarizing voltage range.
?. [Electrophysiological recordings]
Induce sodium and slow potassium currents by a depolarizing voltage step from 0 to +110 mV.
?. [Electrophysiological recordings]
Perform data analysis and statistics using Clampfit (Molecular Devices) and GraphPad Prism (GraphPad software).
Data are presented as mean ± SEM.
?. [HPLC]
For DA measurement experiments, plate mDA neurons onto PO/laminin/fibronectin coated 24-well plates in 5 × 105 cells on day 25 and use between day 60 and day 75.
HPLC with electrochemical detection (HPLC-EC) was done as previously described (Pothos et al., 1996).
?. [HPLC]
Briefly, prior to supernatant collection, incubate cells in fresh DMEM: F12 + N2 media for .
?. [HPLC]
After exposure to either Tyrode’s saline alone or supplemented with high KCl (, Sigma) for  at , collect the supernatant and mix immediately with perchloric acid ( final concentration) to deproteinize the sample and prevent dopamine auto-oxidation.
4 Room temperature
?. [HPLC]
Sonicate the samples at  for , centrifuge at  for ), store at  and analyze within the following two weeks by reverse phase HPLC-EC.
4 Room temperature
Centrifuge: 10000 34
-80 °C
?. [HPLC]
Collect the cells in each sample to normalize for protein content.
?. [HPLC]
Normalize DA concentrations in each group of samples to the levels in the corresponding control group.
Data are shown as averaged normalized values from 2 independent experiments.