TITLE: Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells
AUTHORS: Tomonori Takazawa, Gist F. Croft, Mackenzie W. Amoroso, Lorenz Studer, Hynek Wichterle, Amy B. MacDermott

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.</div></div>

[STEPS]
?. [ESC Culture]
Grow a human embryonic stem cell line with a motor neuron reporter (BAC-Hb9::GFP) under standard pluripotency maintenance conditions: on irradiated CF-1 mouse embryonic fibroblast feeder cells (0.015 M cells/cm2, GlobalStem) seeded on gelatinized (Millipore) tissue culture plastic.
?. [ESC Culture]
Feed cells daily with ES Medium comprised of Dulbecco’s Modified Eagle Medium: nutrient mixture F-12 (DMEM:F12, Invitrogen) with 20% Knockout Serum Replacer (Invitrogen),  betamercaptoethanol (BME; Sigma), L-Glutamine and Non Essential Amino Acids (NEAA; Invitrogen), and  basic fibroblast growth factor (bFGF; Invitrogen) (ESC medium), and passage weekly using  dispase for  (Invitrogen) followed by manual trituration.
?. [ESC Culture]
Karyotype parallel passages of ESCs at subsequent passages
?. [Motor Neuron Differentiation]
After normal passage, incubate washed ESC colonies for  in ESC medium with  Rho associated kinase (ROCK) inhibitor (Y-27632, Ascent Scientific).
?. [Motor Neuron Differentiation]
Trypsinize to single cells and seed in suspension at  cells/ml in ES medium with  ROCK inhibitor, and  recombinant mouse Noggin (R&D).
0.4
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Add fresh ROCK inhibitor, FGF, and Noggin daily for the first .
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Pellet embryoid bodies (EBs) at 100 G on day 4 and resuspend in DMEM F:12 plus N2 supplement (Invitrogen), NEAA, L-Glutamine, 2 mg/ml Heparin (Sigma), bFGF, Noggin, and ROCK inhibitor.
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Pellet EBs and feed with fresh medium every other day until day 31. Add ROCK inhibitor last at day 5.
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Discontinue Noggin and bFGF at day 10.
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Add 1:10 dilution of Wnt3a-L-cell conditioned medium (ATCC), all-trans retinoic acid (RA, , Sigma), ascorbic acid ( , Sigma), db-cAMP (, Sigma), and recombinant mouse Sonic hedgehog (SHH) protein (  SHH-C25II, R&D) from day 10 onward.
?. [Motor Neuron Differentiation]
Discontinue Wnt3a-conditioned medium on day 18.
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Increase SHH to  and add recombinant human brain-derived neurotrophic factor (BDNF;  , R&D).
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At day 25, switch base medium to Neurobasal with N2 and B27 (Invitrogen), LGlutamine, NEAA, ascorbic acid, db-cAMP, (Neural Differentiation Medium (NDM)), with each recombinant human BDNF, glial cell-derived neurotrophic factor (GDNF), insulin-like growth faction 1 (IGF-1), and ciliary neurotropic factor (R&D),  SHH, and  RA.
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Dissociate EBs were using trypsin on day 31 and cryopreserve using EmbryoMax 2x freezing medium (Millipore) for future use.
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Thaw separate vials for electrophysiology and morphology time series, seed on poly-ornithine/laminin coated glass coverslips, in complete day 25 NDM including all supplements.
?. [Motor Neuron Differentiation]
However, reduce RA to  and reduce SHH to  and add the following supplements:  mouse laminin (Invitrogen), BME (, Sigma), glutamate (, Sigma), forskolin (, Sigma), and IBMX (, Fisher) at 250 K cells per coverslip or 46 K cells per  coverslip.
35
15
?. [Motor Neuron Differentiation]
Change half the medium every 4 days.
?. [Immunocytochemistry]
Fix cultures for  in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at
4 °C
?. [Immunocytochemistry]
Wash 3 times for  in PBS, quench and permeabilize in wash PBS plus 0.1% Triton X-100 (Wash buffer) plus  glycine for .
?. [Immunocytochemistry]
Block samples with Wash buffer plus 10% normal donkey serum for .
?. [Immunocytochemistry]
Incubate with primary antibody in blocking buffer (chicken anti-GFP 1:1000, Invitrogen A10262; mouse antiHB9,1:50, MNR2/815C10-s, Developmental Studies Hybridoma Bank) overnight.
?. [Immunocytochemistry]
Wash cells, incubate with DyLight coupled donkey anti primary-species IgG antibodies (Jackson Immunoresearch, 1:1000).
?. [Immunocytochemistry]
Finally, wash cells and counterstain with DAPI (Invitrogen).
?. [Imaging and Image Analysis]
Mount coverslips in Fluoromount G and image on an inverted Zeiss AxioObserver Z1 using a 206 Plan-APOCHROMAT 0.8 NA objective and a 14-bit, gray scale Photometrics HQ2 CCD camera.
?. [Imaging and Image Analysis]
Export images as 16 bit gray scale images for analysis.
?. [Imaging and Image Analysis]
Score cells as HB9+ or GFP+ based on having >10 K mean gray levels of HB9- or >40 K mean gray levels of GFP-channel fluorescence intensity over local background, respectively, using the Metamorph Multiwavelength Cell Scoring module.
?. [Imaging and Image Analysis]
Trace neurites using the Neurite Outgrowth module (Metamorph) based on GFP fluorescence intensity of >3,000 g.l. over local background.
?. [Imaging and Image Analysis]
Manually adjuste a 20x field of view to capture the maximum neurite outgrowth for each cell.
This field size was sufficient to capture all neurites for almost all cells. At day 36 for example, the day at which the peak of total median outgrowth per cell occurs, less than 9% of GFP+ cells had neurites extending beyond the field of view.
?. [Imaging and Image Analysis]
Group morphological measurements by days in culture and use SigmaPlot11 for statistical analysis.
?. [Electrophysiology]
Make whole-cell patch recordings from Hb9::GFP+ motor neurons.
?. [Electrophysiology]
Transfer coverslips to the stage of a TE2000-E microscope (Nikon) and continuously perfuse at a low flow rate of  with bath recording solution containing (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, 2 MgCl2 and 5.5 glucose, pH adjusted to  with NaOH, osmolality
1
325
?. [Electrophysiology]
Pull patch pipettes with a resistance of 3-5 MΩ from borosilicate glass capillaries (0.86 mm ID, 1.5 mm OD) using a P-97 pipette puller (Sutter Instrument Co).
Intracellular solution had the following composition (in mM): 120 potassium methanesulfonate, 10 NaCl, 10 EGTA, 1 CaCl2, 10 HEPES, 0.5 NaGTP, 5 MgATP, 0.1% biocytin, pH adjusted to 7.2 with KOH,osmolality 280 mOsmol kg-1.
?. [Electrophysiology]
Fill some of the electrophysiologically recorded cells with biocytin during recording, then fix, stain and image as above. Correct junction potential before recording.
?. [Electrophysiology]
Acquire data using an Axopatch 200 B amplifier and pClamp 10 software (Molecular Devices, Sunnyvale, CA, USA).
?. [Electrophysiology]
Filter data at 2 kHz and digitized at 20 kHz.
?. [Electrophysiology]
Evoke action potentials by injecting depolarizing currents of 1 s duration and analyze using AxoGraph X software (AxoGraph Scientific, Sydney, Australia).
?. [Electrophysiology]
Measure action potential characteristics in hESMNs from resting membrane potential.
The criterion for identification of a first action potential is when a voltage response to depolarizing current injection has obvious threshold, visible as a rapidly rising membrane potential that is positive to 0 mV. Rheobase is defined as the minimum current step amplitude required to evoke an action potential. Voltage threshold for action potentials is measured as in [44].
?. [Electrophysiology]
Measure action potential half-width at rheobase.
?. [Electrophysiology]
In a given cell, calculate ISIs from recordings in which the maximum number of action potentials are evoked.
?. [Electrophysiology]
Calculate spike frequency adaptation (SFA) ratio as: ISIlast/ISI1st.
?. [Electrophysiology]
Perform the calculation only on data from neurons in which more than 5 action potentials could be evoked by a 1 s depolarizing current step.
?. [Electrophysiology]
Analyze Sag as the response following a hyperpolarizing current step that fit the following criteria:
?. [Electrophysiology]
The ratio between the size of voltage change at the steady state and the peak during a 1 sec current injection is greater than 1.1.
?. [Electrophysiology]
The voltage change peaked during the first half of the current injection period.