An Induced Pluripotent Stem Cell Line GZHMCi004 A Derived Fro - 2021 - Stem Ce

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Stem Cell Research 53 (2021) 102322

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Stem Cell Research


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

An induced pluripotent stem cell line (GZHMCi004-A) derived from a fetus


with heterozygous G380R mutation in FGFR3 gene causing achondroplasia
Nan Li a, 1, Sheng Mou Lin b, 1, *, Yingting Li a, 1, Jimei Sun a, Luting Zhang a, Min Chen a, *
a
Department of Obstetrics and Gynecology, Department of Fetal Medicine and Prenatal Diagnosis, Key Laboratory for Major Obstetric Diseases of Guangdong Province,
The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
b
Department of Obstetrics and Gynecology, The University of Hong Kong – Shenzhen Hospital, Shenzhen 518053, China

A B S T R A C T

Achondroplasia (ACH; MIM #100800) is an autosomal dominant genetic disease caused by gain-of-function mutations in FGFR3 gene and results in short-limb
dwarfism. Here, we generated an induced pluripotent stem cell line GZHMCi004-A derived from umbilical cord blood mononuclear cells (UCBMCs) of a fetus
with heterozygous G380R mutation in FGFR3 gene. This iPSC line is a valuable in vitro model to study the pathological mechanism and the treatment of ACH.

(continued )
Resource Table:
Unique stem cell line GZHMCi004-A Unique stem cell line GZHMCi004-A
identifier identifier

Alternative name(s) of iPS80-1 Cell line repository/bank https://hpscreg.eu/cell-line/GZHMCi004-A


stem cell line Ethical approval This study was approved by the Research Ethics
Institution The Third Affiliated Hospital of Guangzhou Medical Committee of the Third Affiliated Hospital of
University Guangzhou Medical University ([2020]-093)
Contact information of Min Chen
distributor
Type of cell line iPSC
Origin human
Additional origin info Age: N/A 1. Resource utility
Sex: Female
Ethnicity if known: N/A The iPSC line GZHMCi004-A is an ideal cellular model to investigate
Cell Source Umbilical cord blood
the molecular mechanism and the pharmacotherapy of achondroplasia.
Clonality Clonal
Method of reprogramming Episomal vectors encoding OCT4, SOX2, KLF4, c-
MYC and BCL-XL 2. Resource details
Genetic Modification Yes
Type of Modification Hereditary mutation Achondroplasia (ACH; MIM #100800) is a rare autosomal dominant
Associated disease Achondroplasia
congenital disease representing the most common form of short-limb
Gene/locus Gene: FGFR3
Locus: 4p16.3 dwarfism in humans, which is caused by the gain-of-function mutation
Mutation: c.1138G > A (p.G380R) heterozygous in the fibroblast growth factor receptor 3 (FGFR3) gene (Horton et al.,
mutation 2007). Most patients with ACH have the heterozygous G380R mutation
Method of modification N/A
in FGFR3 gene, which affects bones development predominantly
Name of transgene or N/A
resistance
through endochondral ossification (Foldynova-Trantirkova et al., 2012;
Inducible/constitutive N/A Xie et al., 2020). Despite years of research, several aspects of FGFR3
system function in ACH remain unclear or controversial due to the lack of ideal
Date archived/stock date 20th February 2021 in vitro disease model. Here, we generated a human iPSC line
(continued on next column) GZHMCi004-A derived from a ACH fetus with heterozygous G380R

* Corresponding authors.
E-mail addresses: linsm@hku-szh.org (S.M. Lin), drchenmin2003@yahoo.com.hk, edchen99@gmail.com (M. Chen).
1
The first three authors contributed equally to this work.

https://doi.org/10.1016/j.scr.2021.102322
Received 4 March 2021; Accepted 27 March 2021
Available online 5 April 2021
1873-5061/© 2021 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/).
N. Li et al. Stem Cell Research 53 (2021) 102322

Table 1 3. Materials and methods


Characterization and validation.
Classification Test Result Data 3.1. Culture and reprogramming of UCBMCs
Morphology Photography Normal Fig. 1A
Phenotype Qualitative analysis: Positive Fig. 1B UCBMCs were isolated from the umbilical cord blood sample using
Alkaline phosphatase Ficoll-Hypaque (G&E Healthcare) and cultured in UCBMC medium,
staining composed of StemPro-34 SFM (ThermoFisher Scientific) supplemented
Qualitative analysis: Positive for Fig. 1D with 100 ng/mL SCF (Peprotech), 100 ng/mL FLT-3 (Peprotech), 20 ng/
Immunocytochemistry pluripotency
markers: SOX2,
mL IL-3 (Peprotech), 20 ng/mL IL-6 (Peprotech) and 2 mM L-Glutamine
OCT4, NANOG and (ThermoFisher Scientific). Six days later (day 0), 1 × 106 UCBMCs were
TRA-1–60 collected and electroporated with a mixture of episomal plasmids
Quantitative analysis: Positive for Fig. 1C expressing OCT4, SOX2, c-MYC, KLF4 and Bcl-XL as previously
RT-qPCR pluripotency
described (Su et al., 2016). Transfected cells were then seeded in 6-well
markers: NANOG,
OCT4 and SOX2 plate previously coated with MEFs in UCBMC medium. Two days after
Genotype Karyotype (G-banding) 46XX, Resolution Fig. 1 F electroporation, medium was changed with E6 medium (ThermoFisher
and resolution 550 Scientific) supplemented with 50 ng/mL FGF2 (Peprotech) and renewed
Identity Microsatellite PCR Not performed N/A every two days until day 8. The medium was then changed with mTeSR1
(mPCR) OR
STR analysis 21 loci tested, Available
medium (Stem Cell Technologies) and refreshed every other day. At day
100% matched with 18–22, individual iPSC colonies were manually picked and expanded in
authors mTeSR1 medium on plates coated with hESC-qualified Matrigel (BD
Mutation Sequencing heterozygous Fig. 1G Biosciences). The medium was changed every day and cells were
analysis (IF c.1138G > A (p.
passaged every 4–5 days using ReLeSRTM (Stem Cell Technologies) at
APPLICABLE) G380R) mutation in
FGFR3 gene 1:6 split ratio.
Southern Blot OR WGS Not performed N/A
Microbiology Mycoplasma Mycoplasma testing Fig. 1I 3.2. Alkaline phosphatase (AP) staining
and virology by PCR: Negative
Differentiation In vitro directed Expression of Fig. 1H
potential differentiation specific germ layer
The iPSCs grown in 6-well plate were fixed with 4% para­
markers, ectoderm: formaldehyde (Sigma) at room temperature for 20 min. AP staining was
PAX6 and Nestin; then performed using BCIP/NBT Alkaline Phosphatase Staining Kit
mesoderm: α-SMA; (Beyotime, China) according to the manufacture’s protocol at passage 4.
and endoderm:
SOX17
Donor screening HIV 1 + 2 Hepatitis B, Not performed N/A 3.3. Quantitative reverse transcription PCR (RT-qPCR)
(OPTIONAL) Hepatitis C
Genotype Blood group genotyping Not performed N/A Total RNA was extracted using Direct-zol RNA MiniPrep Regent
additional info HLA tissue typing Not performed N/A (Zymo Research) and then reverse transcribed with Hifair® III Reverse
(OPTIONAL)
Transcriptase (Yeasen Biotech Co., Ltd.). The qPCR was conducted on
CFX Connect (Bio-Rad) using Hieff Unicon® qPCR SYBR Green Master
(c.1138G > A) mutation in FGFR3 gene (Table 1). To establish the Mix (Yeasen Biotech Co., Ltd.) under conditions as follow: 95 ◦ C, 3 min;
GZHMCi004-A, UCBMCs were reprogrammed by episomal vectors 40 cycles of [95 ◦ C, 5 s; 60 ◦ C, 30 s]. Primers used are listed in Table 2.
encoding OCT4, SOX2, c-MYC, KLF4 and BCL-XL as previously described
(Su et al., 2016). The GZHMCi004-A iPSC line exhibited a normal 3.4. Karyotyping
morphology (Fig. 1A) and stained positive for alkaline phosphatase (AP,
Fig. 1B). Expression of endogenous pluripotent markers NANOG, OCT4 The G-banded karyotyping analysis of GZHMCi004-A at passage 8
and SOX2 was detected by RT-qPCR, which was comparable to the was performed at Shanghai ZhenHe Biotechnology Co, LTD, China.
positive control H9-hESC line (Fig. 1C). Immunofluorescence staining More than twenty well-divided metaphase spreads were selected
was also performed to confirm the expression of the pluripotent markers randomly for analyzing.
SOX2, OCT4, NANOG and TRA-1–60 (Fig. 1D). Karyotype analysis
showed that GZHMCi004-A had a normal diploid karyotype (46, XX) at 3.5. Mutation analysis
passage 8 (Fig. 1F). Heterozygous G380R (c.1138G > A) mutation in
FGFR3 gene in GZHMCi004-A was verified by Sanger sequencing Genomic DNA from cells was isolated using Genomic DNA Extraction
(Fig. 1G). In vitro directed differentiation was performed to assess the Kit (UnigeneDx, China). PCR was performed using Taq EXtra HotStart
differential potency of GZHMCi004-A. As shown in Fig. 1H, ReadyMix PCR Kit (KAPA) on PTC-100 Thermal Cycler (Bio-Rad) under
GZHMCi004-A could differentiated into all three germ layers, including conditions as follow: 94 ◦ C, 3 min; 30 cycles of [94 ◦ C, 25 s; 55 ◦ C, 15 s;
ectoderm (PAX6 and Nestin), mesoderm (α-SMA) and endoderm 72 ◦ C, 1 min]; 72 ◦ C, 3 min. PCR product was then sequenced by Sanger
(SOX17). The absence of exogenous reprogramming factors was sequencing to confirm the heterozygous G380R (c.1138G > A) mutation
confirmed by PCR at passage 12 as previously described (Fig. 1E) (Su in FGFR3 gene. Primers used are list in Table 2.
et al., 2016). GZHMCi004-A was negative for mycoplasma contamina­
tion by PCR (Fig. 1I). Finally, STR analysis showed that the genetic 3.6. Immunofluorescence staining
identity of GZHMCi004-A was the same as the parental UCBMCs (in­
formation available with the authors). The iPSCs grown on coverslips in 24-well plate were fixed with 4%
paraformaldehyde (Sigma) at room temperature for 20 min. Cells were
then treated with 0.2% Triton X-100 (Sigma) and 10% donkey serum
(ThermoFisher Scientific) in PBS for 1 h at room temperature for per­
meabilization and blocking. Primary antibodies were incubated over­
night at 4 ◦ C overnight. After washed with PBS three times, secondary
antibodies and DAPI (ThermoFisher Scientific) were incubated for 1 h at

2
N. Li et al. Stem Cell Research 53 (2021) 102322

Fig. 1. Characterization of GZHMCi004-A iPSC line.

room temperature. Both primary and secondary antibodies used in 3.9. Mycoplasma contamination test
Table 2 were diluted with 5% donkey serum in PBS. All images were
captured using DMi8 fluorescence microscope (Leica). Mycoplasma contamination was tested by PCR using GMyc-PCR
mycoplasma detection kit (Yeasen Biotech Co., Ltd.) according to the
3.7. In vitro directed differentiation manufacture’s instruction.

In vitro directed differentiation was performed using the STEMdiff™


Trilineage Differentiation Kit (STEMCELL Technologies) according to 3.10. Short tandem repeat (STR) analysis
the manufacturer’s protocol, and expression of the three germ layers’
markers were assayed using immunofluorescence staining. GZHMCi004-A and the parental UCBMCs were performed STR
analysis by Suzhou medpark medical technology Co., LTD. using 21 STR
3.8. Residual presence of episomal plasmids analysis loci (D19S433, D5S818, D21S11, D18S51, D6S1043, AMEL, D3S1358,
D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA,
Genomic DNA was isolated from GZHMCi004-A at passage 12 as D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA).
above. Then the absence of residual EBNA1 and OSW sequences from
episomal plasmids was confirmed by PCR as previously described (Su
et al., 2016). Plasmids were used as positive control, and H9-hESC was Declaration of Competing Interest
used as negative control. Primers used are listed in Table 2.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

3
N. Li et al. Stem Cell Research 53 (2021) 102322

Table 2
Reagents details.
Antibodies used for immunocytochemistry/flow-citometry

Antibody Dilution Company Cat # and RRID

Pluripotency Markers Mouse anti-OCT4 1:500 Santa Cruz Biotechnology Cat# sc-
5279, RRID:AB_628051
Goat anti-SOX2 1:500 R and D Systems Cat# AF2018, RRID:
AB_355110
Rabbit anti-NANOG 1:100 Abcam Cat# ab80892, RRID:
AB_2150114
Mouse anti-TRA-1–60 1:2000 Millipore Cat# MAB4360, RRID:
AB_2119183

Differentiation Markers Rabbit anti-PAX6 1:2000 BioLegend Cat# 901301, RRID:


AB_2565003
Mouse anti-Nestin 1:5000 Millipore Cat# MAB5326, RRID:
AB_2251134
Mouse anti-α-SMA 1:200 Sigma-Aldrich Cat# A5228,RRID:
AB_262054
Mouse anti-SOX17 1:200 R&D Systems Cat# MAB1924,RRID:
AB_2195646

Secondary antibodies Alexa Fluor 488 donkey anti-mouse IgG 1:1000 ThermoFisher Scientific Cat# A-21202,
RRID:AB_141607
Alexa Fluor 594 donkey anti-goat IgG 1:1000 ThermoFisher Scientific Cat# A-11058,
RRID: AB_2534105
Goat anti-mouse IgM (Heavy chain) Cross-Adsorbed 1:1000 ThermoFisher Scientific Cat# A-21042,
Secondary Antibody, Alexa Fluor 488 RRID:AB_141357
Alexa Fluor 488 donkey anti-rabbit IgG 1:1000 ThermoFisher Scientific Cat# A-
21,206, RRID:AB_2,535,792
Alexa Fluor 594 donkey anti-rabbit IgG 1:1000 ThermoFisher Scientific Cat# A-21207,
RRID: AB_141637

Primers

Target Forward/Reverse primer (5′ -3′ )

Pluripotency Markers SOX2/215 bp TGGACAGTTACGCGCACAT/


(RT-qPCR) CGAGTAGGACATGCTGTAGGT
NANOG/367 bp AACTGCATGCAGGACTGCAGAG/
TGAACCTCAGCTACAAACAGGTG
OCT4/408 bp AGAAGGATGTGGTCCGAGTGTG/
CCACCCTTTGTGTTCCCAATTCC

House-Keeping Genes GAPDH/197 bp GGAGCGAGATCCCTCCAAAAT/


(RT-qPCR) GGCTGTTGTCATACTTCTCATGG

FGFR3 mutation analysis FGFR3/310 bp ACGCCCATGTCTTTGCAGC/


(PCR) CAGAGAGGGCTCACACAGC

Episomal Plasmids EBNA1/244 bp TTTAATACGATTGAGGGCGTCT/


(PCR) GGTTTTGAAGGATGCGATTAAG
OSW/168 bp GGATTACAAGGATGACGACGA/
AAGCCATACGGGAAGCAATA

Acknowledgements References

This study was supported by the National Key Research and Devel­ Foldynova-Trantirkova, S., Wilcox, W.R., Krejci, P., 2012. Sixteen years and counting:
The current understanding of fibroblast growth factor receptor 3 (FGFR3) signaling
opment Program of China (2018YFC1004104) and the High Level- in skeletal dysplasias. Hum. Mutat. 33 (1), 29–41. https://doi.org/10.1002/
Hospital Program, Health Commission of Guangdong Province (HKU- humu.21636.
SZH201902017), P.R. China. We acknowledge the patient and her par­ Horton, W.A., Hall, J.G., Hecht, J.T., 2007. Achondroplasia. The Lancet 370 (9582),
162–172. https://doi.org/10.1016/S0140-6736(07)61090-3.
ents who participated in this study. Su, R.J., Neises, A., Zhang, X.B., 2016. Generation of iPS cells from human peripheral
blood mononuclear cells using episomal vectors. Methods Mol. Biol. 1357, 57–69.
Xie, Y., Su, N., Yang, J., Tan, Q., Huang, S., Jin, M., Ni, Z., Zhang, B., Zhang, D., Luo, F.,
Chen, H., Sun, X., Feng, J.Q., Qi, H., Chen, L., 2020. FGF/FGFR signaling in health
and disease. Signal Transduct. Target Ther. 5 (1), 181.

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