Stem Cell Reports, Volume 6

Supplemental Information

Xeno-Free and Defined Human Embryonic Stem Cell-Derived

Retinal Pigment Epithelial Cells Functionally

Integrate in a Large-Eyed Preclinical Model

Alvaro Plaza Reyes, Sandra Petrus-Reurer, Liselotte Antonsson, SonyaStenfelt, Hammurabi Bartuma, Sarita Panula, Theresa Mader, Iyadh Douagi, HelderAndré, Outi Hovatta, Fredrik Lanner, and Anders Kvanta

SUPPLEMENTAL FIGURES AND LEGENDS

Figure S1. Morphology of hESC-RPE on gelatin and quantification of phagocytic

activity, related to Figure 1.

(A) hESC-RPE growing on gelatin fail to cover the whole surface even after long-term

cultures. hESC-RPE on gelatin after 7 days (A1), 35 days (A2) and 77 days (A3) in culture.

(B) Transcriptional analysis for melanocytic markers PAX3 (C1) and EDNRB (C2) of hESC-

RPE differentiated on the different substrates. Values are normalized to RPLPO and GAPDH

and displayed as relative to undifferentiated hESC. Normal human epidermal melanocytes

(NHEM) were used as a positive control. Bars represent mean±SEM, from three independent

experiments.

(C) Flow cytometry analysis of BEST1 expression on hESC-RPE cells grown on the different

substrates for 47 days.

(D-E) Phagocytosis of FITC-labeled POS by hESC-RPE growing on rhLN-521, after

overnight incubation at 4°C (D) and 37°C (E). The former used as negative control, since

active-phagocytosis is temperature-dependent. (D1 and E1) Maximum intensity projection

image depicting internalized FITC-labeled POS in green and phalloidin staining of hESC-

RPE in red. (D2 and E2) Output image of phagocyted FITC-labeled POS generated by the

CellProfiler software. (D3 and E3) Z-stack confocal projections of the area delimited by

arrows in D1 and E1.

(F-G) Phagocytosis of FITC-labeled POS by hESC growing on rhLN-521, after overnight

incubation at 4°C and 37°C. The former used as negative control, since active-phagocytosis

is temperature-dependent. The images correspond to maximum intensity projections

depicting internalized FITC-labeled POS in green (not present) and hESC bounderies shown

in red by wheat germ agglutinin staining.

Scale bars: (A1) = 200μm (A2, A3) = 500μm; (D1, D2, E1, E2, F, G) = 100μm; (D3, E3) =

20μm.

Figure S2. In vivo imaging of the normal albino rabbit posterior segment and long-

term integration of hESC-RPE, related to Figure 3.

(A) En-face IR-cSLO reflectance shows the choroidal vasculature (A1). Green arrow = SD-

OCT scan plane. The corresponding cross-sectional SD-OCT b-scan of the neurosensory

retina and choroid is shown (A2).

(B) On higher magnification the individual layers of the neurosensory retina, RPE/BM, and

underlying choroid are distinguished (B1). The corresponding histologic structures are shown

(HE staining) (B2). GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear

layer), OPL (outer plexiform layer), ONL (outer nuclear layer), OLM (outer limiting

membrane), EZ (ellipsoid zone), OS (outer segments), RPE (retinal pigment epithelium), BM

(Bruch’s membrane), CC (choriocapillaris).

(C) Pigmentation is absent in the subretinal bleb area (dashed circle) one week after

transplantation (C1). Green arrow = SD-OCT scan plane. Simultaneous SD-OCT scan

shows a thickened hyperreflective RPE/BM (between closed arrowheads). Magnification of

the boxed area shows an irregular layer representing putative rhLN-521-hESC-RPE (open

arrows) beneath the hyporeflective outer segments. Twelve weeks (C2), and 34 weeks (C3)

after transplantation of rhLN-521-hESC-RPE multicolor-cSLO and SD-OCT scans

demonstrate stable pigmented donor cell integration (between arrowheads) and a well-

preserved overlying photoreceptor layer. Green arrow = SD-OCT scan plane. *Indicates the

corresponding region of the multicolor-cSLO images in C2 and C3.

(D) HE staining of the corresponding histologic section confirms presence of a preserved

neurosensory retina (D1) and pigmented hESC-RPE monolayer (D2).

(E) 8 weeks after transplantation, uninjected areas do not present pigmented cells (E1) and

show negative staining for both NuMA and BEST1 (E2). (BF = bright field).

Scale bars: Scale bars: (A1, A2, C) = 200µm; (B1) = 100µm; (B2, D) = 50µm; (E1, E2) =

10µm.

Figure S3. Undifferentiated hESC or fibroblasts do not rescue photoreceptors, related

to Figure 4.

(A-B) Transplanted undifferentiated hESC (A) or human fibroblasts (B) form transient

hyperreflective subretinal aggregates as demonstrated by SD-OCT. ORT (arrows) overlying

the transplanted bleb area is reduced as depicted by comparing the respective magnified

(boxed) areas 1 week and 4 weeks after transplantation. Note that cell aggregates have

disappeared after 4 weeks. The bleb margin is indicated (arrowheads).

Scale bars = 200µm.

SUPPLEMENTAL MOVIES

Movie S1, related to Figure 1.

Time-lapse phase contrast imaging of the hESC-RPE growing on gelatin, rhLN-111, -332, -

511 or -521. Videos are the result of time-lapse stack images taken every hour during the

first 21 days after cell seeding.

SUPPLEMENTAL TABLES

Supplemental Table S1, related to Figure 1.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Cell Culture

Human embryonic stem cell lines HS980, HS975 and HS983a were derived and cultured

under xeno-free and defined conditions according to the previously described method. [Rodin

2014b]. The cells were maintained by clonal propagation on LN-521 (Biolamina) in NutriStem

hESC XF medium (Biological Industries), in a 5% CO2/5% O2 incubator and passaged

enzymatically at 1:10 ratio every 5-6 days. For passaging, confluent cultures were washed

twice with PBS without Ca2+ and Mg2+ and incubated for 5 min at 37°C, 5% CO2/5% O2 with

TrypLE Select (GIBCO, Invitrogen). The enzyme was then carefully removed and the cells

were collected in fresh pre-warmed NutriStem hESC XF medium by gentle pipetting to obtain

a single cell suspension. The cells were centrifuged at 1100 rpm for 4 min, the pellet

resuspended in fresh prewarmed NutriStem hESC XF medium and cells plated on a freshly

rhLN-521 coated dish. Two days after passage the medium was replaced with fresh

prewarmed NutriStem hESC XF medium and changed daily subsequently.

GFP-labeled H9 human embryonic stem cell (hESC) line cells were maintained in mTESR

media (StemCell Technologies) on Matrigel (BD Biosciences) coated plates (1:60) until

confluence.

Normal human dermal fibroblasts (NHDF) from adult origin (Lonza) were maintained in

DMEM media (LifeTechnologies) with 10% FBS (LifeTechnologies) until confluence.

In vitro differentiation

Pluripotent stem cells were cultured to confluence on rhLN-521 and manually scraped to

produce embryoid bodies (EBs) using a 1000 µl pipette tip. The EBs were then cultured in

suspension in low attachment plates (Corning) at a density of 5-7x104 cells/cm2.

Differentiation was performed in custom-made NutriStem hESC XF medium in which bFGF

and TGFβ have been eliminated with media change twice a week. 10 μM Rho-kinase

inhibitor (Y-27632, Millipore) was added to the suspension cultures only during the first 24h.

Following five weeks differentiation, pigmented areas were mechanically cut out of the EBs

using a scalpel. Cells were then dissociated using TrypLE Select, followed by flushing

through a 20G needle and syringe. Cells were seeded through a cell strainer (ø 40 μm, BD

Bioscience) on LN-coated dishes at a cell density of 0.6-1.2x104 cells/cm2 and fed twice a

week with the same differentiation medium referred above.

Time-lapse microscopy

The behavior of hESC-RPE on the different substrates was monitored using the Cell-IQ live

imaging system (Chip-Man Technologies Ltd.) equipped with a 10x phase contrast objective,

an automated stage and an integrated incubator (37°C, 5% CO2).

After OVs dissociation, hESC-RPE were seeded in triplicates on the different substrates:

Gelatin (0.1%, Sigma), rhLN-111, -332, -511, and -521 (all 20μg/mL, BioLamina). On day 1

after seeding, the plates were transferred to the live cell imaging equipment. Images were

acquired for both the center (2x2 image grids) and the periphery (single images) of every

well. Every region of interest was monitored every hour for 21 days.

Cell migration was assessed for every time-lapse image stacks using NIS-Elements v.4.0

(Nikon). For each stack 10 cells were randomly chosen and manually tracked during the first

7 days of imaging. The length and trajectory of the tracks followed by the different cells was

used to compare the migration capabilities of hESC-RPE among the different substrates.

Results are presented as mean ±SD (standard deviation).

Quantitative real-time PCR

Total RNA was isolated using the RNeasy Plus Mini Kit and treated with RNase-free DNase

(both from Qiagen). cDNA was synthesized using 1 µg of total RNA in 20 µl reaction mixture,

containing random hexamers and Superscript III reverse transcriptase (GIBCO Invitrogen),

according to the manufacturer’s instructions.

Taq-polymerase together with Taqman probes (Life Technologies) for RPLPO (cat. no.

4333761F), GAPDH (cat. no. 4333764F), NANOG (cat. no. Hs02387400_g1),

POU5F1/OCT4 (cat. no. Hs03005111_g1), SOX9 (cat. no. Hs01001343_g1), PAX6 (cat. no.

Hs01088112_m1), BEST1 (cat. no. Hs00188249_m1), RPE65 (cat. no. Hs01071462_m1)

PMEL (cat. no. Hs00173854_m1), PAX3 (cat no. Hs00240950_m1) and EDNRB (cat no.

Hs00240747_m1) were used. Samples were subjected to real-time PCR amplification

protocol on StepOne™ real-time PCR System (Applied Biosystems). Biological triplicates

were performed for every condition and technical duplicates were carried for each reaction.

Results are presented as mean ±SEM (standard error of the mean).

Flow Cytometry

hESC-RPE growing on the tested substrates were dissociated into single cells using TrypLE

Select after 1 month in culture. Cells were stained with violet LIVE/DEAD fixable stain kit

(Invitrogen) following the manufacturer instructions. Samples were then fixed for 15 min in

4% methanol free formaldehyde (Polysciences) and permeabilized with 0.1% Triton X-100

(Sigma) for 15 min. Mouse anti-MITF (Abcam ab3201, clone [D5]) and mouse anti-BEST1

(Millipore, MAB5466) primary antibodies were used at a concentration of 10 μg/mL, diluted in

2% FBS, 0.1% Tween-20 (Sigma). Cell were incubated with the primary antibodies on ice for

30 min. Indirect immunostaining was completed using Alexa Fluor 488 Donkey anti-mouse

IgG secondary antibody at a concentration of 2 μg/mL (Life Technologies A21202) on ice for

another 30 min. Fluorescence minus one (FMO) controls were included for each condition to

identify and gate negative and positive cells. Stained cells were analyzed using a BD

LSRFortessa flow cytometer equipped with 488 nm, 561 nm, 405 nm and 640 nm lasers (BD

Biosciences). Analysis of the data was carried out using FlowJo v.10 software (Tree Star).

Enzyme-Linked Immunosorbent Assay (ELISA)

hESC-RPE were cultured on Transwell membranes (0.33 cm2, Millipore) coated with different

substrates. Supernatants from both the hESC-RPE apical and basal sides (meaning upper

and lower compartments of the Transwell, respectively) were collected 60 hours after the

medium was changed. VEGF and PEDF secretion levels were measured in triplicates for

each condition with commercially available human VEGF and PEDF ELISA Kits (VEGF:

Cat#DVE00, R&D Systems; PEDF: Cat#RD191114200R, BioVendor), in accordance with the

instructions of the manufacturers, after 4.5 and 7 weeks of culture, respectively. The optical

density readings were measured using SpectraMax 250 Microplate Reader

(MolecularDevices). Results are presented as mean ±SEM (standard error of the mean).

Phagocytosis assay

In order to assess the phagocytic activity of our hESC-RPE, an in vitro assay using FITC-

labelled photoreceptor outer segments (POS) was performed. FITC-labelled bovine POS

were isolated and kindly given by Dr. E.F. Nandrot from Institut de la Vision, Paris (Parinot et

al., 2014). For that purpose, hESC-RPE were cultured on Transwell membrane (0.33 cm2,

Corning) coated with different substrates for one month after seeding. Cells were incubated

at 37°C or 4°C for 16 hours with 2.42x106 thawed POS/Transwell diluted in DMEM or CO2

independent media (both from LifeTechnologies), respectively. After incubation, cells were

quenched with Trypan Blue Solution 0.2% (GIBCO, Invitrogen) for 10 minutes at room

temperature, fixed with 4% methanol free formaldehyde (Polysciences) at room temperature

for 10 min and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. Rhodamine

phalloidin staining (1:1000, 20 min at room temperature, Life Technologies) was used to

visualize the cell boundaries. Nuclei were stained with Hoechst 33342 (1:1000, 20 min at

room temperature, Invitrogen). Undifferentiated hESC were used as a negative control.

hESC were cultured under the same conditions and incubated also at 37°C and 4°C with

2.42x106 thawed POS/Transwell diluted in DMEM or CO2 independent media. Wheat Germ

Agglutinin-Alexa Fluor® 594 Conjugate (1:200, 20 min at room temperature, Invitrogen) was

used in to visualize the cell boundaries of hESC.

Images were acquired with Zeiss LSM710-NLO point scanning confocal microscope. Post-

acquisition analysis of the pictures was performed using IMARIS (Bitplane). Total number of

engulfed FITC-POS per condition was quantified with the custom-made pipeline developed

within cell image analysis software CellProfiler (Broad Institute). Results are presented as

mean ±SD (standard deviation).

Transepithelial resistance measurements

Transepithelial electrical resistance (TER) RPE cells plated on Transwells (0.33 cm2,

Millipore) was measured using the Millicell Electrical Resistance System volt-ohm meter

(Millicell ERS-2, Millipore), according to the manufacturer’s instructions. Cultures were

equilibrated outside the incubator at room temperature for 15-20 min before the experiment.

Measurements were performed in unchanged culture media in triplicates for each condition,

at three different positions of each well. Averages were used for further analysis. The

background resistance was determined from a blank culture insert in the same media coated

with the corresponding substrate but without cells, and subtracted from the respective

experiment condition. Measurements are reported as resistance in ohms times the area in

square centimeter (Ω*cm2). Results are presented as mean ±SEM (standard error of the

mean).

Immunofluorescence

Protein expression of mature hESC-RPE monolayers was assessed with

immunofluorescence. Cells were fixed with 4% methanol free formaldehyde at room

temperature for 20 min, followed by permeabilization with 0.3% Triton X-100 in Dulbecco’s

phosphate-buffered saline (DPBS) for 10 min and blocking with 4% fetal bovine serum (FBS)

and 0.1% Tween-20 in DPBS for 1 hour. Primary antibodies were diluted to the specified

concentrations in 4% FBS, 0.1% Tween-20, DPBS solution: Bestrophin 1 (BEST1) (1:100,

Millipore MAB5466), Zonula occludens-1 (ZO-1) (1:100, Invitrogen 40-2200), cellular

retinaldehyde-binding protein (CRALBP) (1:250, Abcam ab15051, clone [B2]) and alpha 1

sodium/potassium ATPase (Na/K-ATPase) (1:200, Abcam ab7671, clone [464.6[). The

primary antibodies were incubated overnight at 4°C followed by 2 hours incubation at room

temperature with secondary antibodies: Alexa Fluor 647 donkey anti-rabbit IgG and Alexa

Fluor 488 donkey anti-mouse IgG (both from Life Technologies, A31573 and A21202,

respectively) diluted 1:1000 in 4% FBS, 0.1% Tween-20, DPBS solution. Nuclei were stained

with Hoechst 33342 (1:1000, Invitrogen). Images were acquired with Zeiss LSM710-NLO

point scanning confocal microscope. Post-acquisition analysis of the pictures was performed

using IMARIS (Bitplane).

Animals

After approval by the Northern Stockholm Animal Experimental Ethics Committee 17 New

Zealand white albino rabbits (provided by Lidköpings rabbit farm, Lidköping, Sweden) aged 5

months, weighing 3.5 to 4.0 kg, where used in this study. All experiments were conducted in

accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research.

Subretinal transplantation

hESC-RPE monolayers were washed with PBS, incubated with TrypLE, and dissociated to

single cell suspension in a similar manner as here described for hESC, using a 5% CO2

incubator. Cells were then counted in a Neubauer hemocytometer chamber using 0.4%

trypan blue (GIBCO, Invitrogen). For subretinal injection, a hESC-RPE single cell suspension

was centrifuged at 2400 rpm for 4 min, the medium was carefully removed, and the cell pellet

was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The

cell suspension was then aseptically aliquoted into 100 μL units and kept on ice until surgery.

hESC were washed with PBS, incubated with Accutase (GIBCO, Invitrogen) for 2 min at

37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet

was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The

cell suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery.

NHDF cells were washed with PBS, incubated with Trypsin 1x (LifeTechnologies) for 5 min at

37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet

resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The cell

suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery.

Animals were put under general anesthesia by intramuscular administration of 35 mg/kg

ketamine (Ketaminol, 100 mg/ml, Intervet) and 5 mg/kg xylazine (Rompun vet. 20 mg/ml,

Bayer Animal Health), and the pupils were dilated with a mix of 0.75% cyclopentolate / 2.5%

phenylephrine (APL). Microsurgeries were performed on both eyes using a 3-port 25G

transvitreal pars plana technique (Alcon Accurus, Alcon Nordic). 25G trocars were inserted 1

mm from the limbus and an infusion cannula was connected to the lower temporal trocar.

The cell suspension was drawn into a 1 mL syringe connected to an extension tube and a

38G polytip cannula (MedOne Surgical Inc). Without infusion or prior vitrectomy the cannula

was inserted through the upper temporal trocar. After proper tip positioning, ascertained by a

focal whitening of the retina, 50 μL of cell suspension (equivalent to 50.000 cells) was

injected slowly subretinally approximately 6 mm below the inferior margin of the optic nerve

head, forming a uniform bleb that was clearly visible under the operating microscope. Care

was taken to maintain the tip within the bleb during the injection to minimize reflux. After

instrument removal light pressure was applied to the self-sealing suture-less sclerotomies.

No post-surgical topical steroids or antibiotics were given.

Animals were treated in three separate cohorts. In the first cohort (7 animals receiving 521-

derived hESC-RPE) 4 animals received 2 mg (100 μL) of intravitreal triamcinolone

(Triescence, Alcon Nordic) immediately after surgery, one animal received systemic

cyclosporine-A (a descending dose of 20-5 mg/kg/d sc) (Teva Sweden AB), one animal

received both triamcinolone and cyclosporine-A, and one animal was maintained without

immunosuppression. On post-operative examination, none of the eyes showed signs of

extra- or intra-ocular infection or inflammation except for one animal (one eye) that had a

uveitis one week after surgery and was sacrificed. A second animal was sacrificed 6 weeks

post-transplantation after signs of cyclosporine-A toxicity (hematuria). Analysis of cohort one

showed no beneficial effect of cyclosporine-A on donor cell integration (defined as

appearance of pigmented areas within 4 weeks post-transplantation) whereas several

animals treated with only triamcinolone had successful integration. The second cohort (6

animals receiving 521-derived hESC-RPE) was therefore treated with intravitreal

triamcinolone alone without any post-operative complications. The third cohort received

either undifferentiated hESC (2 animals) or fibroblasts (2 animals) with post-operative

triamcinolone. In animals kept for long-term evaluation, intravitreal triamcinolone was re-

administered every 3 months.

Spectral domain-optical coherence tomography (SD-OCT)

Anesthetized rabbits were placed in an adjustable mount. A commercial Spectralis HRA +

OCT device (Heidelberg Engineering) with the Heidelberg Eye Explorer Software (version

1.9.10.0) was used to obtain horizontal cross-sectional b-scans of hESC-RPE treated

animals. The Spectralis has a real-time motion tracking system that minimizes eye motion

artifacts. At least 3 cross-sectional OCT scans were obtained with simultaneous infrared-

confocal scanning laser ophthalmoscope (IR-cSLO) reflectance reference images

representing the upper, central and lower portion of the transplanted area. The best overall

image quality was obtained when the OCT setting was on high-speed acquisition with at

least 50 averaged automatic real-time images. En-face fundus images were obtained by IR-

or multicolor cSLO (a composite of three simultaneously acquired color cSLO images).

These modalities have a higher contrast level compared to conventional fundus camera

photos. The total area of the pigmented lesions was estimated manually on IR-cSLO images

using the built-in measuring tool of the Spectralis software.

Photoreceptor rescue measurements

We previously defined ORT as the SD-OCT distance between the inner nuclear layer (INL)

and RPE using the ImageJ software (http://imagej.nih.gov/ij/) (Bartuma et al., 2015). ORT of

treated regions was obtained from a section overlying at least 500 μm of continuously

integrated cells (mean of 10 random measurements). ORT of non-treated control regions

was obtained from the same scan 500 μm outside the bleb (mean of 5 random

measurements). Relative ORT was then calculated as the ratio between treated and non-

treated retina. Distances were normalized to the 200 μm scale bar of the original image. All

measurements were done one month post-transplantation, or at the nearest time-point after.

A 2-sided Student’s T-test was performed comparing the relative ORT between eyes with

integrated and with non-integrated rhLN-521-derived hESC-RPE (defined as a pigmented

area >2.5 mm2). Results are presented as mean ±SD (standard deviation).

Histology and tissue immunostaining

Immediately after sacrifice by intravenous injection of 100 mg/kg pentobarbital (Allfatal vet.

100 mg/ml, Omnidea), the eyes were enucleated and the bleb injection area marked with

green Tissue Marking Dye (TMD) (Histolab Products). An intravitreal injection of 100 μL

fixing solution (FS) consisting of 4% buffered formaldehyde (Solvenco AB) was made before

fixation in FS for 24-48 hours, and embedding in paraffin. 4 μm serial sections were made

through the TMD-labeled area and every 4 sections were stained with hematoxylin-eosin

(HE). Images were captured with an Iphone 4S mounted to a bright-field microscope (Zeiss

Axioskop 40, CarlZeiss).

For immunostaining, slides were deparaffinized in xylene, dehydrated in graded alcohols,

and rinsed with dH2O and Tris Buffered Saline (TBS, pH 7.6). Antigen retrieval was done in

10 mM citrate buffer (trisodium citrate dihydrate, Sigma-Aldrich, pH 6.0) with 1:2000 Tween-

20 (Sigma-Aldrich) at 96°C for 30 min, followed by 30 min cooling at room temperature.

Slides were washed with TBS and blocked for 30 min with 10% Normal Donkey Serum

(Abcam) diluted in TBS containing 5% (w/v) IgG and protease free bovine serum albumin

(Jackson Immunoresearch) in a humidified chamber. Primary antibodies diluted in blocking

buffer, were incubated overnight at 4°C: human nuclear mitotic apparatus protein (NuMA)

(1:200, Abcam ab84680), BEST1 (1:200, Millipore MAB5466), RPE65 (1:200, Abcam

ab78036, clone [401.8B11.3D9]) and Rhodopsin (1:2000, Millipore MAB5356). Secondary

antibodies (Alexa Fluor 555 donkey anti-rabbit IgG A31572 and Alexa Fluor 647 donkey anti-

mouse IgG A31571, both from Life Technologies) diluted 1:200 in blocking buffer, were

incubated 1 hour at room temperature. Sections were mounted with vector vectashield with

DAPI mounting medium (Vector Laboratories) in a 24x50 mm coverslip. Images were taken

with Zeiss LSM710-NLO point scanning confocal microscope. Post-acquisition analysis of

the pictures was performed using ImageJ software.

In order to enhance the signal of the NuMA, TSA-Plus-Cyanine3 System was used (Perkin

Elmer Life Sciences). An extra blocking step of 30 min with 3% H2O2 in Methanol was added

prior to serum blocking followed by incubation with NuMA. Secondary antibody chicken anti-

rabbit HRP (1:200, Santa Cruz sc-2963) diluted in the serum blocking buffer was incubated

for 30 min, followed by 8 min incubation with Tyramide-Cy3 according to manufacturer’s

instructions. Images were taken with an Olympus IX81 fluorescence microscope and post-

acquisition analysis of the pictures was performed using ImageJ software.

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