Studies of Pluripotency in Embryonic Stem Cells and Induced Pluripotent Stem Cells

104 Pages·2014·5.24 MB·English

by  Xiaoyang Zhao (auth.)

Springer Theses
Recognizing Outstanding Ph.D. Research
Xiaoyang Zhao

Studies of
Pluripotency in
Embryonic Stem
Cells and Induced
Pluripotent Stem
Cells
Springer Theses
Recognizing Outstanding Ph.D. Research
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Xiaoyang Zhao
Studies of Pluripotency
in Embryonic Stem Cells
and Induced Pluripotent
Stem Cells
Doctoral Thesis accepted by
University of Chinese Academy of Sciences,
Beijing, China
1 3
Author Supervisor
Dr. Xiaoyang Zhao Prof. Qi Zhou
Institute of Zoology Institute of Zoology
Chinese Academy of Sciences Chinese Academy of Sciences
Beijing Beijing
China China
ISSN 2190-5053 ISSN 2190-5061 (electronic)
ISBN 978-94-017-8818-2 ISBN 978-94-017-8819-9 (eBook)
DOI 10.1007/978-94-017-8819-9
Springer Dordrecht Heidelberg New York London
Library of Congress Control Number: 2014933275
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Parts of this book have been published in the following articles:
Riaz, A., Zhao, X., Dai, X., Li, W., Liu, L., Wan, H., Yu, Y., Wang, L., Zhou, Q.
(2011). Mouse cloning and somatic cell reprogramming using electrofused blasto-
meres. Cell Res 21, 770–778. (Reproduced with Permission)
Zhao, X., Lv, Z., Li, W., Zeng, F., Zhou, Q. (2010). Production of mice using iPS
cells and tetraploid complementation. Nat Protoc 5(5), 963–971. (Reproduced
with Permission)
Liu, L., Luo, G.Z., Yang, W., Zhao, X., Zheng, Q., Lv, Z., Li, W., Wu, H.J., Wang,
L., Wang, X.J., et al. (2010). Activation of the imprinted Dlk1-Dio3 region cor-
relates with pluripotency levels of mouse stem cells. J Biol Chem 285, 19483–
19490. (Reproduced with Permission)
Zhao, X., Lv, Z., Liu, L., Wang, L., Tong, M., Zhou, Q. (2010). Derivation of
embryonic stem cells from Brown Norway rats blastocysts. J Genet Genomics 37,
467–473. (Reproduced with Permission)
Zhao, X.Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.L., Wang, X.,
Wang, L., et al. (2010). Efficient and rapid generation of induced pluripotent stem
cells using an alternative culture medium. Cell Res 20, 383–386. (Reproduced
with Permission)
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Supervisor’s Foreword
Pluripotent stem cells (including embryonic stem cells and induced pluripotent stem
cells) are promising cell resources for regenerative medicine. In 2004, Xiao-Yang
joined in my lab when we were focusing on reprogramming. For the following 6
years, he has focused on reprogramming of iPS cell generation, the derivation of the
ESC, and nuclear transfer embryonic stem cells. He found that mouse iPS cells are
fully reprogrammed, which could generate the iPS-all mice, the same as the counter-
part ESC, which is the most stringent test for pluripotent stem cells. It lets us know
that we may generate wonderful human iPS cells when we keep on the technique
revolution. After that, he also spent a lot of time to nfi d the difference between good
and bad quality iPS cells, and the molecular mechanism behind it. He and his col-
leagues found that the DMRs in the Dlk1-Dio3 cluster are aberrant hypermethyl-
ated in bad quality iPS cells, and the knockout serum replacement (KOSR) could
maintain the normal methylation pattern, while something in the FBS could induce
hypermethylation. He was also interested in the difference among the pluripotent
stem cells of mouse, rat, and primate. He derived the Brown Norway rat ESC lines
for the rfi st time, and tried to establish the naïve primate pluripotent stem cells. In
conclusion, Xiao-Yang wants to derive high quality mammalian pluripotent stem
cells, both in mouse and human, which will facilitate the mechanism study of pluri-
potency maintenance, and also the clinical application.
Beijing, February 2014 Prof. Qi Zhou
vii
Abstract
Stem cells have the ability to differentiate between all types of cells within the
body, and thus have great therapeutic potential in regenerative medicine for treat-
ing complicated disorders like Parkinson’s disease and spinal cord injury. There
are also many applications in drug development. However several roadblocks,
such as safety issues and low efficiency of pluripotent cell line derivation, need
to be resolved before their clinical application. This thesis focuses on these two
areas, and finds solutions to overcome their limitations.
The commonly used mouse pluripotent stem cells include embryonic stem cells
(ESC), nuclear transfer embryonic stem cells (ntES), and induced pluripotent stem
cells (iPSC), among others. These cells are studied and defined better in mouse
than in other species such as rat and human. It has always been an interesting topic
to find ways to transfer knowledge learned from mouse models to other species in
this area. Stem cell technology will be helpful for establishing disease models in
Brown Norway (BN) rats. Thus, following the derivation of mouse ESC, we have
successfully derived BN rat ESCs from blastocysts, and obtained chimera with
high contribution from these ESCs after blastocyst injection. In addition, we have
derived human ESC from discarded human embryos. These ESCs will provide
important resources to research on the significant differences between primate and
rodent ESC.
Induced pluripotent stem cells (iPSCs) are the result of a great new technol-
ogy to reprogram somatic cells. It has great potential in the field of regenerative
medicine since it can avoid immune rejection and face fewer ethical concerns.
However, safety issues need to be examined and the efficiency needs to be
improved before further application in the clinical settings. We performed a series
of experiments to find solutions to these questions.
First, we have established an efficient protocol to induce the iPSC from somatic
cells. We modified some aspects of the reprogramming process by using 20 %
Knockout Serum Replacement (KOSR) instead of the 15 % Fetal Bovine Serum
(FBS) in the induction medium, and achieved a 100-fold increase in derivation
efficiency for reprogramming Oct4-GFP mouse embryonic fibroblast (MEF) cells
to their relevant iPSC cells.
Secondly, we picked out iPSC clones on various days of post-viral infection
and established stable lines from each. We examined the gene expression pat-
terns and abilities of embryoid body formation to characterize these iPSC, and we
ix
x Abstract
produced chimeric animals by blastocyst injection. The iPSC chimeras exhibited
germline transmission. Next, we performed tetraploid complementation, the most
stringent assay to test pluripotency potential of iPSCs. The iPSCs gave rise to live,
full-term iPS mice. These iPS mice survived to adulthood and produced a subse-
quent generation of mice. The first iPS mouse, so far 24-months old, is still alive.
Thus, these iPS mice are the most important proof of the true pluripotency of the
iPSCs, showing that fully reprogrammed iPSC using the four “Yamanaka factors”
can be generated, and have similar developmental ability as ESCs. In addition to
MEFs, we also induced iPSCs from Neural Stem Cells (NSC) of 1-week-old mice
and mouse tail tip fibroblast (TTF) from 3 to 4-week-old mice and 8 to 12-week-
old mice. After performing the tetraploid complementation assay, we generated
iPS mice from NSC-iPSC and TTF-iPSC, and confirmed that adult cells can be
fully reprogrammed by Yamanaka factors, although TTF gave the lowest effi-
ciency to generate the iPSC.
We further explored the gene expression patterns of the 2n-iPSC and 4n-iPSC
(those only that produced chimeras or are tetraploid complementation competent,
respectively), and found no significant difference between them, except expression
levels of a cluster miRNA located in the chromosome 12 Dlk1-Dio3 region. The
sequencing results confirmed that the expression of the miRNA was repressed in
2n-iPSC, compared to ESC and 4n-iPSC. The iPSCs derived from the three-factor
iPSC (Oct4, Sox2, Klf4) showed similar results. The abnormal expression of the
Dlk1-Dio3 region was not corrected by nuclear transfer experiments. As this is a
conserved region in mammals, the gene expression in the Dlk1-Dio3 region might
serve as a good molecular marker for pluripotent stem cells.
In summary, we have derived ESC from several species, designed an efficient
system to generate iPSC, and reported the first iPS mice in the world, confirming
that somatic cells can be fully reprogrammed using the four Yamanaka factors. In
addition, we have found the Dlk1-Dio3 region to be a potential molecular marker
to separate the fully reprogrammed from partially reprogrammed iPSC. All these
results will help improve the safety of pluripotent stem cell in clinical applications
and increase the currently low efficiencies of their production.
Keywords ESC • iPSC • Pluripotency • Tetraploid  complementation • Dlk1-
Dio3 region