ORIGINAL ARTICLE Derivation of integration-free iPSCs from a Klinefelter syndrome patient T. Shimizu 1 • M. Shiohara 1 • T. Tai 1 • K. Nagao 1 • K. Nakajima 1 • H. Kobayashi 1, Received: 13 March 2015 / Accepted: 18 June 2015 / Published online: 3 July 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Purpose Klinefelter syndrome (KS) (47, XXY) is the most common sex chromosome abnormality in humans. KS is characterized by gynecomastia, tall stature, small testes, low testosterone levels, learning disabilities, and behavioral problems. KS is also associated with infertility due to non-obstructive azoospermia (NOA). The mecha- nism underlying NOA is still poorly understood, and although there is no current treatment, the use of microdissection testicular sperm extraction (micro-TESE) followed by in vitro fertilization can result in successful conception. The generation of induced pluripotent stem (iPS) cells derived from KS patients may be useful for studying the disease mechanism and identifying novel therapies. Methods Cells from a KS patient were transduced with Sendai viral vectors encoding four transcription factors, OCT4, SOX2, KLF4, and C-MYC, and the transduced cells were analyzed for in vitro and in vivo pluripotency. Results KS patient-derived iPS cells were successfully generated and shown to produce teratomas in the testes of SCID mice. In vitro differentiation of the iPS cells into cardiomyocyte-like cells was confirmed by the presence of clusters of beating cells. Conclusions KS patient-derived iPS cells that could dif- ferentiate into cardiomyocyte-like cells were established. Keywords Cardiomyocyte iPS Klinefelter syndrome Stem cells Testis Introduction Klinefelter syndrome (KS), or 47, XXY syndrome, is the most common sex chromosome abnormality found in humans, occurring in approximately 1 in 500–1000 live deliveries [1]. KS is characterized by gynecomastia, tall stature, small testes, low testosterone levels, learning dis- abilities, and behavioral problems. Affected men suffer from KS-associated progressive testicular failure (resulting in azoospermia or cryptozoospermia), small testes (5–7 cm 3 ), and low testosterone levels. In addition, indi- viduals with KS have an increased risk for various dis- eases, including diabetes, cardiovascular disease, and cancer, and their offspring have an increased risk for chromosomal abnormalities [1]. Thus, it is recommended that individuals with KS symptoms undergo prompt cytogenic evaluation. The KS karyotype arises spontaneously when paired X chromosomes fail to separate during the meiosis stage of oogenesis or spermatogenesis [2]. In addition, a small percentage ( \ 3 %) of X chromosome polysomy occurs during early divisions of the fertilized egg, while more than 10 % occurs as a result of postfertilization nondisjunction [3]. It is predicted that the aberrant expression of X chro- mosome-linked genes plays a role in the spermatogenesis defect seen in KS patients. However, the mechanism underlying the infertility of KS is still poorly understood, and the only treatment for the infertility is microdissection Electronic supplementary material The online version of this article (doi:10.1007/s12522-015-0213-9) contains supplementary material, which is available to authorized users. & H. Kobayashi [email protected]1 Department of Urology, Toho University School of Medicine, 6-11-1 Omori-Nishi, Ota-ku, Tokyo 143-8541, Japan 123 Reprod Med Biol (2016) 15:35–43 DOI 10.1007/s12522-015-0213-9
9
Embed
Derivation of integration-free iPSCs from a Klinefelter ...Derivation of integration-free iPSCs from a Klinefelter syndrome ... in azoospermia or cryptozoospermia), small testes (5–7
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
ORIGINAL ARTICLE
Derivation of integration-free iPSCs from a Klinefelter syndromepatient
T. Shimizu1 • M. Shiohara1 • T. Tai1 • K. Nagao1 • K. Nakajima1 • H. Kobayashi1,
Received: 13 March 2015 / Accepted: 18 June 2015 / Published online: 3 July 2015
� The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract
Purpose Klinefelter syndrome (KS) (47, XXY) is the
most common sex chromosome abnormality in humans.
KS is characterized by gynecomastia, tall stature, small
testes, low testosterone levels, learning disabilities, and
behavioral problems. KS is also associated with infertility
due to non-obstructive azoospermia (NOA). The mecha-
nism underlying NOA is still poorly understood, and
although there is no current treatment, the use of
Klinefelter syndrome (KS), or 47, XXY syndrome, is the
most common sex chromosome abnormality found in
humans, occurring in approximately 1 in 500–1000 live
deliveries [1]. KS is characterized by gynecomastia, tall
stature, small testes, low testosterone levels, learning dis-
abilities, and behavioral problems. Affected men suffer
from KS-associated progressive testicular failure (resulting
in azoospermia or cryptozoospermia), small testes
(5–7 cm3), and low testosterone levels. In addition, indi-
viduals with KS have an increased risk for various dis-
eases, including diabetes, cardiovascular disease, and
cancer, and their offspring have an increased risk for
chromosomal abnormalities [1]. Thus, it is recommended
that individuals with KS symptoms undergo prompt
cytogenic evaluation.
The KS karyotype arises spontaneously when paired X
chromosomes fail to separate during the meiosis stage of
oogenesis or spermatogenesis [2]. In addition, a small
percentage (\3 %) of X chromosome polysomy occurs
during early divisions of the fertilized egg, while more than
10 % occurs as a result of postfertilization nondisjunction
[3]. It is predicted that the aberrant expression of X chro-
mosome-linked genes plays a role in the spermatogenesis
defect seen in KS patients. However, the mechanism
underlying the infertility of KS is still poorly understood,
and the only treatment for the infertility is microdissection
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12522-015-0213-9) contains supplementarymaterial, which is available to authorized users.
Nucleic acids were stained with SYTOXR orange nucleic
acid stain (Molecular Probes).
In vitro pluripotency assessment
Confluent iPS cells in a 6-cm dish were harvested by
trypsinization and transferred to Poly (hydroxyethyl
methacrylate-co-methyl methacrylate; HEMA-MMA)-
coated six-well dishes in primate ES cell medium without
bFGF. The medium was changed every other day, and the
cells were maintained in floating culture for 8 days. The
cells were then placed on 0.1 % gelatin-coated six-well
dishes and incubated with ES medium for 8 days. After
embryoid body formation, we confirmed the cells’ ability
to differentiate in vitro by examining the expression of
differentiation markers by immunocytochemistry. The cells
were washed twice with PBS, fixed with 4 % (w/v)
paraformaldehyde for 20 min, permeabilized for 60 min
with PBS containing 0.1 % (v/v) Triton X-100, and then
blocked for 3 h with PBS containing 20 % donkey serum.
Anti-a-fetoprotein mouse IgG (R&D Systems), anti-a-smooth muscle actin mouse IgG (Dako), and anti-bIII-tubulin mouse IgG (Chemicon) were used to analyze the
expression of endodermal, mesodermal, and ectodermal
markers, respectively. The cells were then washed three
times with PBS containing 0.1 % (v/v) Triton X-100, fol-
lowed by incubation with an Alexa 488-conjugated anti-