Application Note
Visualizing Neurite Outgrowth by Lentiviral Transduction of FluorescentProteins into Human iPSC-derived Excitatory Neurons
INTRODUCTION
Elixirgen Scientific’s proprietary transcription factor-based
technology allows rapid and reproducible differentiation ofhuman induced pluripotent stem (iPS) or embryonic stem(ES) cells into neurons without sacrificing the purity of
the cells1. Our excitatory neurons exhibit typical neuronalmorphology with outgrowing neurites and express markers
characteristic of a variety of neuronal subtypes, including
the pan-neuronal marker tubulin beta 3 class III (TUBB3),
the glutamatergic neuron marker vesicular glutamate
transporter 1 (vGLUT1), and the cholinergic neuron marker
choline acetyltransferase (ChAT).
Lentiviral delivery of a fluorescent protein reporter isan easy, non-invasive and robust method to visualize
the morphology of living cells. In this technical note, we
describe how our human iPSC-derived excitatory neurons
can be efficiently transduced by green fluorescent protein(GFP) reporter lentivirus. Nearly 100% of neurons can belabeled at an optimal multiplicity of infection (MOI) andthen analyzed by live-cell imaging-based fluorescenceintensity measurement to evaluate neurite outgrowth, test
the neurotoxicity of chemical compounds, and to perform
various functional assays.
RESULTS
Determining Optimal MOI
Human iPSC-derived excitatory neurons were transduced
4 days post-thaw using a commercial ready-to-use
lentiviral vector that harbors GFP under the humansynapsin1 (SYN1) promoter. The SYN1 promoter isneuron-specific and therefore is useful to distinguishhealthy developing neurons from other cell-types anddead cell debris (Figure 1). Neurites could be visualized
6-7 days post-thaw, and GFP expression was detected inmost of the cells on day 8 post-thaw, indicating that thesecells were efficiently committed to the neuronal lineage.
More intense fluorescence was observed when a higher
MOI was used for lentiviral transduction (Figure 2A). GFP
intensity continued to increase over several days (Figure2B), which could be due to both the increasing activity of
the SYN1 promoter with neuronal maturation as well as the
rapid outgrowth of labeled neurites. Overall, in our culture
conditions, a MOI from 2.5 to 10 appeared enough to allow
clear visualization of thin neurites. Although we did not

notice any significant toxicity at higher MOIs, damaged
neurites, namely neurite beading, were apparent uponclose observation at MOI 20 (Figure 2C). Due to the risk ofcell damage and the cost of lentiviruses, the MOI should be
kept as low as possible.

Phase

Hoechst

GFP

Merge

Figure 1: Representative phase contrast and fluorescence microscopy
images of Quick-Neuron™ Excitatory - Human iPSC-derived neurons at
day 21 post-thaw (17 days after transduction with a GFP reporter lentivirus
at MOI 2.5). Hoechst 33342 staining marks nuclei of both live and deadcells whereas green fluorescence shows only live and fully differentiatedneurons since GFP is expressed under the SYN1 promoter (scale bars =200 μm). The differential staining pattern is apparent in the merged image.

Neurotoxicity Studies
Live-cell imaging of neurite outgrowth can be used forevaluating neurotoxicity. For example, treatment withglutamate is known to induce neuronal damage by overstimulating N-methyl-D-aspartate (NMDA) receptors,
a process referred to as excitotoxicity. Using our GFPlabeled excitatory neurons, severe neurite degeneration
could be observed with a glutamate concentration of
10 μM (Figure 3). Total fluorescence intensity did notdecrease significantly, since most of the cell bodies
appeared still intact and retained their membrane integrity
at such a low concentration of the compound. Neurite
beading is reported as one of the early events of neuronaldegeneration prior to induction of cell death, and therefore
could potentially be a sensitive assay for early toxicity
prediction using high-content screening.
Functional Analysis
Lentiviral transduction is also useful for expressing modified
proteins for functional analysis of neurons. For example,

Last revised: August 10, 2021

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(A)

MOI 0

MOI 1.25

MOI 2.5

MOI 5

MOI 10(B)

MOI 20(C)

Relative Fluorescence Intensity

5,000
MOI 204,000

MOI 10

3,000

MOI 5
2,000

MOI 2.5

1,000
0MOI 1.25

5

7

9

11

13

15

17

19

MOI 0

Days

Figure 2: (A) Fluorescence microscopy images of excitatory neurons on day 19 post-thaw (15 days after transduction with a GFP reporter lentivirus) show
intensifying fluorescence at higher MOIs (scale bars = 200 μm). (B) The line chart indicates the fluorescence intensity measured in the time-course relative
to the intensity measured at day 5 post-thaw as a reference, showing that the signal increases as a function of the MOI and the time after transduction. (C)Magnified view of transduced neurons showing possible neurite damage (beading) at a high MOI of 20 (scale bars = 50 μm).

GCaMP is a genetically encoded calcium indicator basedon a fusion of calmodulin, M13 myosin light chain kinase
peptide, and GFP, which emits fluorescence when binding
to Ca2+. Expressing GCaMP6f (an improved variant of
GCaMP)2 in human iPSC-derived excitatory neurons
allows neuronal activity to be measured by detecting theintracellular Ca2+ influx evoked by action potentials (Figure4). Spontaneous calcium oscillations were observed fromday 21 post-thaw, which indicates that network maturationinduced the synchronization of neuronal firing. The moststable signals, with a regular rate of about 1 peak per minute,were obtained at day 29 post-thaw using a high MOI of 30.
The oscillation amplitude was lower than the amplitude
observed in standard calcium binding dye assays, possibly
because the promoter (CAG) used in this construct was
suboptimal for this cell-type. Nevertheless, the lentiviraltransduction of GCaMP6f offers the advantage of allowing
long-term monitoring of calcium oscillations without
significantly affecting cell viability. Changes in neuronal
activity can thus be evaluated prior to a more sensitive endpoint assay.

Lentiviral transduction of a fluorescent protein is aconvenient method to label cells and has been used
extensively to monitor neuronal cells in culture as well as invivo3. The utilization of neuron-specific promoters, such as
the one for SYN1, allows efficient tracking of differentiatedneurons4. Our results demonstrate that the majority of
viable cells in our excitatory neuron cultures are labeled(A) Day 21(B) Day 29
MOI 1

MOI 3

MOI 5

MOI 10

MOI 30Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)10 μM glutamate

Figure 3: Representative fluorescence microscopy images of excitatoryneuron cultures used for the glutamate neurotoxicity assay. Neurons weretransduced with a GFP reporter lentivirus on day 4 post-thaw and thentreated with 10 μM glutamate for 24 hours on day 21 post-thaw (right).
Neurite degeneration or neurite beading induced by excitotoxicity isevident compared to a mock-treated control (left) (scale bars = 200 μm).

DISCUSSION: ADVANTAGES AND CAVEATS

MOI 0

MOI 1

MOI 3

MOI 5

MOI 10

MOI 30Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)Time (min)FI ratio (FIt / FIt0)

FI ratio (FIt / FIt0)

FI ratio (FIt / FIt0)

FI ratio (FIt / FIt0)

MOI 0

Mock-treatedFigure 4: Waveforms of Ca2+ oscillation monitored by GCaMP6f on days 21 (A) and 29 (B) post-thaw in duplicates are shown. Neurons weretransduced with lentivirus at the indicated MOIs on day 4 post-thaw. The x-axes indicate the duration of recording (up to 20 min) and the y-axes indicate
the intensity of fluorescence detected. The waveforms for MOI 30 in (B) show the most stable signals, with a regular rate of about 1 peak per minute.

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by GFP expressed under the SYN1 promoter even at low
MOIs. This technique allows clearer visualization of neurite
outgrowth than common cell membrane dyes such as
Calcein AM and DiI, which tend to stain dead-cell debris
and/or to be released from stained cells into the medium
a few days after staining. In addition, lentiviral vectors
permanently integrate into the genome, which assures very
stable transgene expression. No decrease of fluorescencehas been observed after over 1 month of culture, thereby
making lentiviral transduction of GFP an ideal tool for longterm studies.
Some caveats of this method should be noted. MOI
and infection conditions require optimization for eachexperimental design, since they can be affected by thecell type, virus lot, timing of infection and culture volume.
Selection of the promoter should also be carefully
considered depending on the aim of the assay. For
example, the human SYN1 promoter is suited to express
a fluorescent reporter in a mature neuron-specific manner.
On the other hand, the elongation factor-1α (EF1α)
promoter is active in a wide variety of cell types, and has
been successfully used to label immature neurons. Activityof the CAG promoter in this reporter-neuron combination
was not as strong as that of the SYN1 promoter, and asignificantly high MOI had to be used to obtain detectable
fluorescence (Figure 4). Similar results were obtained with
the CMV promoter. We also made minor changes to media
formulation because the presence of antibiotics (penicillinstreptomycin) induced cell damage during infection.
The use of polybrene and other transduction enhancer
reagents is not recommended as they can have detrimental
effects on neuronal cell viability5. We found that most of ourcells died when some of these reagents were used. Finally,
the use of recombinant lentiviruses can raise biosafetyconcerns, although the most recent self-inactivating (SIN)
third generation viruses have been considerably improvedon this matter6. Relevant regulations and institutionalguidelines should be followed when lentiviruses arehandled, which generally requires biosafety level 2 (BSL2) containment. Despite these practical considerations,this Application Note clearly demonstrates that lentiviralMOI 0.1

mediated fluorescence labeling of our Quick-Neuron™
cultures is a powerful and useful method for monitoring
cultures, evaluating neurotoxicity, and performing
functional assays.
METHODS
Cell Culture
Elixirgen Scientific’s Quick-Neuron™ Excitatory - Human
iPSC-derived Neurons (Catalog number: EX-SeVCW50065) were thawed and plated at a density of 5 ×
104 cells/cm2 following the instructions in the user guide.
Minor modifications were made in media formulation for
optimal transduction and culture maintenance. On day
1 post-thaw, the medium was changed to Neurobasal
Plus (Thermo Fisher) including B-27 Plus, CultureOne
supplements, and antibiotics (penicillin-streptomycin) as
recommended by the manufacturer. Lentiviral transduction
was performed on day 4 post-thaw in Neurobasal Plus
Medium without antibiotics to avoid cell damage. Cells
were then maintained, with optional addition of antibiotics
after day 8 post-thaw, with a half medium change every 3-4
days.
Lentiviral transduction
Commercially available pre-packaged lentivirus particles
harboring GFP under the SYN1 promoter (SignaGen
Laboratories) were thawed on ice and diluted in Neurobasal
Plus Medium without antibiotics at 2 × 104 transduction
units/μL. The appropriate volume of viral suspension
was added to each culture to obtain desired MOIs (forexample, for MOI 10, 8 μl were added to each well of a
96-well plate which had been seeded with 1.6 × 104 cells/
well on D0). Cells were incubated in a 5% CO2 incubator
at 37°C for 24 hours and the medium was then changed
to fresh Neurobasal Plus Medium without antibiotics.
Lentiviruses harboring GFP under the CMV promoter orthe EF1α promoter were made using a Lentiviral High Titer
Packaging Mix with pLVSIN (Takara) and used to infectcells in a similar manner.
MOI 1.0

CMV

EF1αFigure 5: Fluorescence images of Quick-Neuron™ Excitatory - Human iPSC-derived cells
transduced with lentiviral reporter constructs are shown. In these reporters, the expression of
GFP is driven by either the CMV (top) or the EF1α (bottom) promoter. The cells were infected
at two MOIs indicated above on day 7 post-thaw, and the images were acquired on day 13
post-thaw.

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Workflow
Thawing andPlating Cells
Day 0

Lentivirustransduction

MediumChangeDay 1

Medium iN (G2P)

...

Day 4

Neurobasal Plus (P)

Day 5

...

Neurobasal Plus w/o antibiotics

Day 8

...
Neurobasal Plus

Figure 6: Schematic of the workflow of the protocol for lentiviral transduction of fluorescent proteins into Quick-Neuron™ Excitatory - Human iPSCderived Neurons.

Cellular imaging and fluorescence measurement
Fluorescence and phase contrast images were captured
using a BZ-X800 microscope (Keyence). For nuclear
staining, Hoechst 33342 solution (Therno Fisher) was
added at a final concentration of 5 μg/mL and incubatedat room temperature for 30 minutes before analysis.
Fluorescence intensity was measured with a Cytation 5
plate reader (BioTek).
Excitotoxicity assay
A stock solution of 1 mM L-Glutamic acid monosodium
salt hydrate (Fujifilm Wako) was prepared in distilled waterand filter-sterilized. The glutamate solution was diluted to
20 μM in pre-warmed medium and added to the cells witha half medium change for a final concentration of 10 μM.
Treated cells were observed 24 hours later.
Calcium oscillation measurements
Elixirgen Scientific’s Quick-Neuron™ Excitatory - Human
iPSC-derived Neurons were cultured with human primary
astrocytes (Thermo Fisher Scientific) and transduced on
day 4 post-thaw using a commercially available ready-touse lentiviral vector that harbors GCaMP6f under the CAG
promoter (SignaGen Laboratories). Calcium oscillation
was monitored with the FDSS/μCell system (Hamamatsu
Photonics K.K.) for 20 minutes on days 21 and 29.

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References
1. Goparaju, S. K. et al. Rapid differentiation of humanpluripotent stem cells into functional neurons by
mRNAs encoding transcription factors. Sci. Rep. 7,
1–12 (2017).
2. Chen, T. W. et al. Ultrasensitive fluorescent proteinsfor imaging neuronal activity. Nature 499, 295–300(2013).
3. Parr-Brownlie, L. C. et al. Lentiviral vectors as tools
to understand central nervous system biology inmammalian model organisms. Front. Mol. Neurosci.
8, 1–12 (2015).
4. Dittgen, T. et al. Lentivirus-based genetic
manipulations of cortical neurons and their optical andelectrophysiological monitoring in vivo. Proc. Natl.
Acad. Sci. U. S. A. 101, 18206–18211 (2004).
5. Bao, F. et al. Polybrene induces neural degeneration
by bidirectional Ca2+ influx-dependent mitochondrialand ER–mitochondrial dynamics. Cell Death Dis. 9,966, (2018).
6. Pauwels, K. et al. State-of-the-Art Lentiviral Vectors
for Research Use: Risk Assessment and Biosafety
Recommendations. Curr. Gene Ther. 9, 459–474(2009).

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