Stem Cell Research 17 (2016) 102–106

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Stem Cell Research

journal homepage: www.elsevier.com/locate/scr

Lab Resource: Stem Cell Line

Generation of urine iPS cell lines from patients with Attention Deficit
Hyperactivity Disorder (ADHD) using a non-integrative method
Jaroslaw Sochacki a, Sylvie Devalle a, Marcelo Reis a, Paulo Mattos a, Stevens Rehen a,b,⁎
a
D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro 222281, Brazil
b
Institute of Biomedical Sciences, Federal University of Rio de Janeiro (UFRJ), Avenida Carlos Chagas, 373, Rio de Janeiro 21941, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Urine samples were collected from three patients (2 males, 1 female) with clinically diagnosed Attention Deficit
Received 16 May 2016 Hyperactivity Disorder (ADHD) according to DSM-5 criteria using semi-structured interviews (K-SADS adapted
Accepted 23 May 2016 for adults) by a trained psychiatrist. Urine epithelial cell lines were established and expanded for subsequent
Available online 27 May 2016
reprogramming procedure. Induced pluripotent stem cells (iPSCs) were derived using integration-free
CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka fac-
tors Oct3/4, Sox2, Klf4 and c-Myc.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Resource table Resource details

Urine epithelial cells underwent the reprogramming procedure

using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher
Name of stem cell ADHD-4 iPSC, ADHD-5 iPSC, ADHD-10 iPSC Scientific, USA) containing reprogramming vectors with the four
construct Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc. These factors have been
Institution D'Or Institute for Research and Education repeatedly shown to be sufficient for very efficient reprogramming
Person who created Jaroslaw Sochacki, PhD
(Lieu et al. 2013; Takahashi et al. 2007). The identity of derived
resource
Contact person and Stevens Rehen, PhD, srehen@lance-ufrj.org
human iPS cell lines was confirmed by immunocytochemistry, using
email the following antibodies for pluripotency markers, TRA-1-60, Sox2,
Date archived/stock November 7th, 2015 Oct3/4 and SSEA4 (Fig. 1a). Sendai virus transgenes were undetectable
date from passage 7 (Fig. 1c). To confirm trilineage differentiation potential,
Origin Human urine epithelial cells
in vitro embryonic body (EB) formation assay was performed (Fig. 3a).
Type of resource Induced pluripotent stem cell (iPS); derived from human
urine epithelial cells Spontaneous differentiation induced the transcription of the following
Sub-type Induced pluripotent stem cell (iPS) genes: AFP (endoderm), MSX1 (mesoderm) and PAX6 (ectoderm)
Key transcription Oct3/4, Sox2, Klf4, c-Myc (Fig. 3b). Additionally, the formation of the three germ layers was con-
factors
firmed at the protein level by immunocytochemistry, which showed the
Authentication Identity and purity of cell line confirmed (Figs. 1, 2 and 3)
Link to related Not available
expression of Nestin, Tuj1, SMA and AFP (Fig. 3c). Ploidy of the 3 derived
literature iPS cell lines was analyzed by low-pass whole-genome sequencing (Fig.
Information in public Not available 2) (Wells et al. 2014).
databases
Ethics Patient informed consent obtained/Ethics Review
Materials and methods
Board-competent authority approval obtained

Derivation of human urine epithelial cell lines ADHD-4, ADHD-5 and

ADHD-10

Procedures for sample collection and iPS cell lines generation were
approved by the Institutional Ethics Committee and informed consent
⁎ Corresponding author at: D'Or Institute for Research and Education (IDOR), Rua Diniz
was obtained from the patients and/or their legal tutors. Urine cells
Cordeiro, 30, Rio de Janeiro 222281, Brazil. (UCs) were collected and cultured as described previously (Zhou et al.
E-mail address: srehen@lance-ufrj.org (S. Rehen). 2011) with small modifications. Briefly, fresh urine samples (250–

http://dx.doi.org/10.1016/j.scr.2016.05.015
1873-5061/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 J. Sochacki et al. / Stem Cell Research 17 (2016) 102–106 103

Fig. 1. Characterization of the three ADHD iPS cell lines. A) Expression of pluripotency markers: TRA-1-60, SOX2, SSEA4 and OCT3/4. Immunofluorescence staining, red/green: pluripotency
markers, Blue: DAPI, nuclei staining. Scale bars, 200 μm. B) Feeder-free ADHD iPS cell lines cultures. C) Reverse transcription PCR for the detection of Sendai transgenes after passage 7.
Transduced cell pool at passage 0 was used as positive control. C−, non-template control.

300 mL) were collected from the patients, centrifuged for 10 min at
1700 rpm and washed once with 1× PBS with penicillin/streptomycin
and then plated in 1 mL urine renal epithelial growth medium (REGM,
Lonza, USA) on a gelatin-coated 12-well plate. The cells were expanded
in standard culture conditions (37 °C, 5% CO2) and tested for Mycoplas-
ma (MycoAlert™ PLUS, Lonza, USA) before any further manipulation.
Cells at passage 3 were used for reprogramming.
After successful adaptation to feeder-free conditions of at least 3 iPS col-
onies from each patient, the cells were expanded in E8 medium using
0.5 mM EDTA passaging solution (both from Thermo Fisher Scientific,
USA) (Fig. 1b).
Immunofluorescence staining of iPS cell lines

Oct3/4, Sox2, SSEA4 and Tra-1-60 immunofluorescence staining

iPS cell generation and expansion.
Cells were reprogrammed using the integration-free CytoTune®-iPS
2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, USA), which
contains Sendai virus particles of the four Yamanaka factors
(Takahashi et al. 2007). Briefly, 3.5–4 × 105 urine epithelial cells were
plated onto one well of a gelatin-coated 6-well plate 24 h before viral
transduction. The cells were transduced according to the manufacturer
protocol. Four days after transduction, cells were plated onto MEF feed-
er cells, and fed with iPS cell's medium supplemented with 30 ng/mL
freshly added bFGF (Thermo Fisher Scientific, USA). About 7 days after
plating, the first appearing colonies were picked for expansion into indi-
vidual iPS cell lines, transferred to Matrigel (BD Biosciences, USA)-coat-
ed plates and cultured with E8 medium (Thermo Fisher Scientific, USA).
confirmed pluripotency genes expression in all ADHD iPS cell lines
(Fig. 1a). The cells were plated onto 96-well plates (CellCarrier,
Perkin-Elmer, USA) and fixed with 4% paraformaldehyde for 15 min at
room temperature. Fixed cells were permeabilized with 0.3% Triton-X
and blocked with 2% BSA. Cells were then washed with PBS, incubated
with primary antibodies at 1:100 dilution, at 4 °C, overnight, in a
humid chamber. Primary antibodies were then washed with PBS and
cells were incubated with secondary antibodies at 1:400 dilutions at
room temperature for 1 h. After washing and incubation with DAPI for
5 min, cells were covered with glycerol; the plate was sealed with
AlumSeal CS film (Excel Scientific, USA) and stored at 4 °C until visuali-
zation. Images were acquired using the EVOS XL Cell Imaging System
(Thermo Fisher Scientific, USA) (Fig. 1a). The antibodies used in the ex-
periments are listed in Table 2.
104 J. Sochacki et al. / Stem Cell Research 17 (2016) 102–106

Fig. 2. Aneuploidy analysis of ADHD iPS cell lines. Analysis of chromosome copy number was carried out using low-pass whole genome sequencing. Snapshot of IGV Light Whole Genome
View screen depicts overviews of the derived iPS cell line chromosome sets. Dots correspond to sequencing tiles approximately 2 Mb long each. ADHD-4: MAPD: 0.130; Read count:
452,394; Total number of bases: 90.08 Mb; Total number of bases (AQ20): 79.36 Mb; % Bases (AQ20): 88.1; Mean coverage depth (fold): 0.03. ADHD-5: MAPD: 0.122; Read count:
476,642; Total number of bases: 91.93 Mb; Total number of bases (AQ20): 81.05 Mb; % Bases (AQ20): 88.2; Mean coverage depth (fold): 0.03. ADHD-10: MAPD: 0.138; Read count:
339,164; Total number of bases: 65.22 Mb; Total number of bases (AQ20): 57.82 Mb; % Bases (AQ20): 88.6; Mean coverage depth (fold): 0.021. MAPD, = median absolute pairwise
difference.

In vitro differentiation of iPS cell lines
The differentiation ability of ADHD iPS cell lines was analyzed by in
vitro embryonic body (EB) formation, according to a previously pub-
lished protocol (Itskovitz-Eldor et al. 2000). Briefly, 100 mm cell culture
dishes with ADHD iPS cell lines were treated with Collagenase IV (Ther-
mo Fisher Scientific, USA) for 30–40 min at 37 °C, washed with PBS and
seeded onto 60 mm culture dishes (Corning, USA). The cultures were
maintained on a horizontal shaker in DMEM supplemented with 20%
KSR, non-essential amino acids, L-glutamine, penicillin/streptomycin,
sodium pyruvate (all from Thermo Fisher Scientific, USA) and without
FGF to allow for formation of embryonic bodies (EBs) (Fig. 3a). After
7 days, EBs were seeded onto gelatin-coated 100 mm culture dishes
and grown for another 21 days with medium being changed every
other day. Subsequently, the cultures were harvested for total RNA iso-
lation using the GeneJET RNA Purification kit (Thermo Fisher Scientific,
USA), followed by RNA DNAse treatment and reverse transcription
using M-MLV (Thermo Fisher Scientific, USA). RT-PCR for the three
germ layer markers (Fig. 3b) was carried out using 0.5 μL of the reverse
transcription reaction in a total 10 μL reaction volume, with the follow-
ing parameters: denaturation 95 °C-15 s, Annealing 50 °C-15 s, elonga-
tion 72 °C-1 min (35 cycles). The PCR products were analyzed using
agarose gel (1%) electrophoresis. The primers used for PCR are listed
in Table 1. Part of the cultures was also plated onto 96-well plates
(CellCarrier, Perkin-Elmer, USA) to perform immunocytochemistry for
the markers of the three germ layers. Immunostaining was performed
as described above. Images were acquired using the Operetta System
(Perkin-Elmer, USA) (Fig. 3c).
Virus-free status of iPS cell lines
CytoTune®-iPS 2.0 Reprogramming Kit is considered a non-integra-
tive system. Nevertheless, it is necessary to confirm whether the
derived iPS cells contain any Sendai virus elements after the
reprogramming procedure. To confirm the absence of integration of
Sendai viral vectors, total RNA was isolated from ADHD iPSCs using
the GeneJET RNA Purification Kit (Thermo Fisher Scientific, USA).
Cells set-aside at the beginning of the reprogramming procedure
(passage 0) were used as positive controls for the presence of Sendai
RNA. The reverse transcription reactions were carried out using 1 μg
of DNAse-treated RNA and the M-MLV enzyme (Thermo Fisher Sci-
entific, USA). RT-PCR was carried out using 0.5 μL of the reverse tran-
scription reaction in a total 10 μL reaction volume, with the following
parameters: denaturation 95 °C-30 s, annealing 55 °C-30 s, elonga-
tion 72 °C-30 s (35 cycles). The PCR products were analyzed using
agarose gel (2%) electrophoresis (Fig. 1c). The primers used for PCR
are listed in Table 1.
Aneuploidy analysis of derived iPS cell lines
The ploidy of the ADHD iPS cell lines was confirmed by low-pass
whole-genome sequencing (Fig. 2). Genomic DNA was purified
from approximately 5 × 105 cells using QIAamp DNA Mini Kit
(Qiagen, USA). The sequencing library was constructed by the enzy-
matic fragmentation of 100 ng genomic DNA followed by the ligation
of sequencing adaptors using the Ion Xpress™ Plus Fragment Library
Kit (Thermo Fisher Scientific, USA). Ligated fragments were size se-
lected electrophoretically using a 2% E-Gel SizeSelect precast agarose
gel (Thermo Fisher Scientific, USA). Adaptor-mediated amplification
of the library was subsequently carried out (5 cycles) using the prim-
er mix supplied with the Ion Xpress™ Plus Fragment Library Kit
(Thermo Fisher Scientific, USA). The amplified library was quantitat-
ed by real time PCR using the Ion Library Quantitation Kit, diluted for
equalization and 24 pmol were pooled with other libraries for clonal
amplification using the Ion OneTouch system followed by library en-
richment performed on the Ion OneTouch ES (Thermo Fisher Scien-
tific, USA). Pooled libraries were sequenced using an Ion 316 Chip
 J. Sochacki et al. / Stem Cell Research 17 (2016) 102–106 105

Fig. 3. Trilineage differentiation analysis. A) In vitro embryonic body formation assay for pluripotency. B) Confirmation of the expression of germ layer markers by RT-PCR. Endoderm: AFP;
Mesoderm: MSX1; Ectoderm: Pax6. C) Immunofluorescence staining detection of germ layer markers. Endodermal: AFP, mesodermal: SMA and ectodermal: Nestin and TUJ1. Red/green:
germ layer markers. Blue: DAPI (nuclei). Magnification, 10×.

Table 1
Reverse transcription-PCR primers.
Target Primer sets
SeV Forward: GGA TCA CTA GGT GAT ATC GAG C⁎
Reverse: ACC AGA CAA GAG TTT AAG AGA TAT GTA TC⁎
KOS Forward: ATG CAC CGC TAC GAC GTG AGC GC
Reverse: ACC TTG ACA ATC CTG ATG TGG
Klf4 Forward: TTC CTG CAT GCC AGA GGA GCC C
Reverse: AAT GTA TCG AAG GTG CTC AA⁎⁎
c-Myc Forward: TAA CTG ACT AGC AGG CTT GTC G⁎⁎
Reverse: TCC ACA TAC AGT CCT GGA TGA TGA TG
GAPDH Forward: TTCGACAGTCAGCCGCATC
Reverse: GACTCCACGACGTACTCAGC
AFP Forward: AGA GTT GCT AAA GGA TAC CAG GA
Reverse: AGG CCA ATA GTT TGT CCT CAC
Pax6 Forward: AGA AAG AGT TTG AGA GAA CCC AT
Reverse: TCA TGT GTG TCT GCA TAT GTG G
MSX1 Forward: CCC TGG TGC TGT ACC CC
Reverse: GGT CCC TTC AAC CTA CCT T
⁎ Primer contains SeV genome sequences.
⁎⁎ Annealing of these primers to transgene-specific target sequences but not to endog-
enous genes sequences allows for the specific detection of the transgenes introduced by
the CytoTune® 2.0 Sendai reprogramming vectors.
on the Ion Personal Genome Machine using the 200 bp sequencing
chemistry Ion PGM™ Hi-Q (Thermo Fisher Scientific, USA). Aneu-
Product size ploidy analysis was carried using the Low-pass whole-genome aneu-
181 bp ploidy r. 0 workflow of the Ion Reporter v.5.0 suite. In order to
ascertain the accuracy of copy number calling ≥ 300,000 reads/sam-
528 bp
ple, median absolute pairwise difference (MAPD) ≤ 0.3 and a median
410 bp coverage depth ≥ 0.2 were set as minimum requirements.
532 bp
Acknowledgements
352 bp
301 bp The authors thank the lab-crew members for providing technical
support. Funds were provided by Brazilian Development Bank
468 bp (BNDES) (10.2.0051.1), Funding Authority for Studies ad Projects
(FINEP) (01.08.0657.00), National Council of Scientific and Technologi-
547 bp
cal Development (CNPq) (404687/2012-1) and Foundation for Re-
search Support in the State of Rio de Janeiro (FAPERJ) (E-26/210.812/
2014/E-26/210.890/2015/E-26/010.002983/2014). Fellowships from
Coordination for Improvement for Higher Education Personnel
(CAPES) (20092/2013-7), FAPERJ and CNPq.
106 J. Sochacki et al. / Stem Cell Research 17 (2016) 102–106

Table 2
Antibodies used for immunocytochemistry.

Antibody Company Catalog number Host Dilution

Sox2 Merck Millipore MAB5603 Rabbit 1:100

Oct3/4 Santa Cruz Biotechnology SC5279 Mouse 1:100
TRA-1-60 Merck Millipore MAB4360 Mouse 1:100
SSEA4 Merck Millipore MAB4304 Mouse 1:100
Nestin Sigma Aldrich N5413 Rabbit 1:100
TUJ1 Merck Millipore MAB1637 Mouse 1:100
AFP Santa Cruz Biotechnology SC15375 Rabbit 1:100
SMA Sigma Aldrich A2547 Mouse 1:100
Goat anti-rabbit Alexa Fluor® 488 Thermo Fisher Scientific A11008 Rabbit 1:400
Goat anti-mouse Alexa Fluor® 594 Thermo Fisher Scientific A11032 Mouse 1:400

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