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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

For further volumes:

http://www.springer.com/series/8790
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accessible to scientists not expert in that particular field.
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

13
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)

Zhou, S., Ding, C., Zhao, X., Wang, E., Dai, X., Liu, L., Li, W., Liu, Z., Wan, H.,
Feng, C., et al. (2010). Successful generation of cloned mice using nuclear trans-
fer from induced pluripotent stem cells. Cell Res 20, 850–853. (Reproduced with
Permission)

Li, W., Zhao, X.Y., Wan, H.F., Zhang, Y., Liu, L., Lv, Z., Wang, X.J., Wang, L.,
Zhou, Q. (2011). iPS cells generated without c-Myc have active Dlk1-Dio3 region
and are capable of producing full-term mice through tetraploid complementation.
Cell Res 21, 550–553 (Reproduced with Permission)

Zhao, X.Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Wang, X., Wang,
L., Zeng, F., et al. (2010). Viable fertile mice generated from fully pluripotent iPS
cells derived from adult somatic cells. Stem Cell Rev 6, 390–397. (Reproduced
with Permission)

Zhao, X.Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.L., Ma,
Q.W., Wang, L., et al. (2009). iPS cells produce viable mice through tetraploid
complementation. Nature 461, 86–90. (Reproduced with Permission)

Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P.,
Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., Jaenisch, R. (2007). Treatment
of sickle cell anemia mouse model with iPS cells generated from autologous skin.
Science 318, 1920–1923 (Reproduced with Permission)

v
Adewumi, O., Aflatoonian, B., Ahrlund-Richter, L., Amit, M., Andrews, P.W.,
Beighton, G., Bello, P.A., Benvenisty, N., Berry, L.S., Bevan, S., Blum, B.,
Brooking, J., Chen, K.G., Choo, A.B., Churchill, G.A., Corbel, M., Damjanov,
I., Draper, J.S., Dvorak, P., Emanuelsson, K., Fleck, R.A., Ford, A., Gertow,
K., Gertsenstein, M., Gokhale, P.J., Hamilton, R.S., Hampl, A., Healy, L.E.,
Hovatta, O., Hyllner, J., Imreh, M.P., Itskovitz-Eldor, J., Jackson, J., Johnson,
J.L., Jones, M., Kee, K., King, B.L., Knowles, B.B., Lako, M., Lebrin, F.,
Mallon, B.S., Manning, D., Mayshar, Y., McKay, R.D., Michalska, A.E., Mikkola,
M., Mileikovsky, M., Minger, S.L., Moore, H.D., Mummery, C.L., Nagy, A.,
Nakatsuji, N., O'Brien, C.M., Oh, S.K., Olsson, C., Otonkoski, T., Park, K.Y.,
Passier, R., Patel, H., Patel, M., Pedersen, R., Pera, M.F., Piekarczyk, M.S.,
Pera, R.A., Reubinoff, B.E., Robins, A.J., Rossant, J., Rugg-Gunn, P., Schulz,
T.C., Semb, H., Sherrer, E.S., Siemen, H., Stacey, G.N., Stojkovic, M., Suemori,
H., Szatkiewicz, J., Turetsky, T., Tuuri, T., van den Brink, S., Vintersten, K.,
Vuoristo, S., Ward, D., Weaver, T.A., Young, L.A., Zhang, W. Characterization of
human embryonic stem cell lines by the International Stem Cell Initiative. Nature
Biotechnology 25, 803–816 (Reproduced with Permission)
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 find 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 first 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
Acknowledgments

I thank my supervisor, Prof. Qi Zhou, for his long-term help in guiding me in

all the projects and scientific experiments, and in supporting me in the projects I
wanted. He is also a good mentor in my life, taking care of my personal affairs and
helping me deal with problems.
I also thank Prof. Xiu-Jie Wang, Prof. Fan-Yi Zeng, and Prof. Jia-Hao Sha,
for their help in supervision and discussion. I need to thank Associated Prof. Liu
Wang, and all members in Prof. Qi Zhou’s lab. I also thank all professors in the
State Key Laboratory of Reproductive Biology, and the teachers in the Institute of
Zoology.
Finally, I thank my classmates, my friends, and my family for supporting me in
my research work.

xi
Contents

1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Progress in Somatic Cell Reprogramming. . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Nuclear Transfer of Somatic Cells. . . . . . . . . . . . . . . . . . . . 1
1.1.2 Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . 4
1.2 Research and Application of Stem Cells in Regenerative Medicine. . . 9
1.2.1 In Vitro Differentiation of Stem Cells . . . . . . . . . . . . . . . . . 11
1.2.2 Application of Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2 Establishment of ESC Lines Derived from Mice, Rats,

and Primate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.1 The Experimental Animals. . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.2 Discarded Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.3 Generation of Mouse Embryonic Stem Cells. . . . . . . . . . . . 29
2.2.4 Generation of Rat Embryonic Stem Cells . . . . . . . . . . . . . . 29
2.2.5 Generation of Human Embryonic Stem Cells. . . . . . . . . . . 30
2.2.6 Karyotype Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.7 Alkaline Phosphatase Staining. . . . . . . . . . . . . . . . . . . . . . . 30
2.2.8 Immunofluorescence Staining of Embryonic Stem Cells. . . 30
2.2.9 RT–PCR Detection of Gene Expression in Embryonic
Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2.10 Bisulfite Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2.11 Teratoma Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2.12 Blastocyst Injection (Chimeras and Tetraploid
Complementation). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.1 Production of Live Mouse Derived from Mouse ESC
with Tetraploid Embryo. . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.2 Establishment of Rat BN ESC. . . . . . . . . . . . . . . . . . . . . . . 33
2.3.3 Generation of Human ESC. . . . . . . . . . . . . . . . . . . . . . . . . . 35

xiii
xiv Contents

2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.4.1 Significant Difference Between Mouse and Rat ESCs . . . . 36
2.4.2 Difference Between Mouse ESC and Human ESC. . . . . . . 37
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3 Establishment of Highly Efficient Somatic Cell Reprogramming

System to Generate iPSC Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2.1 Experimental Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2.3 Virus Package and Transfection. . . . . . . . . . . . . . . . . . . . . . 44
3.2.4 iPSC Induction and Cell Lines Generation . . . . . . . . . . . . . 44
3.2.5 Flow Cytometry Analysis of iPSC. . . . . . . . . . . . . . . . . . . . 44
3.2.6 Karyotype Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.2.7 The Formation of Teratoma. . . . . . . . . . . . . . . . . . . . . . . . . 46
3.2.8 Diploid and Tetraploid Blastocyst Injection. . . . . . . . . . . . . 47
3.2.9 Southern Blot Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3.1 Induction of iPSC by Transfection of Four Factors. . . . . . . 47
3.3.2 Knockout Serum Replacement Improves iPSC
Induction Efficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.3.3 iPSC Retain Pluripotency. . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.4.1 KOSR Enhance Reprogramming Efficiency of iPSC . . . . . 49
3.4.2 Mechanism of the Enhancing Effect of KOSR
on iPSC Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

4 Pluripotency of iPSC and Underlining Mechanism. . . . . . . . . . . . . . . . 53

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2.1 Experimental Animal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2.3 Virus Packaging and Transfection. . . . . . . . . . . . . . . . . . . . 55
4.2.4 iPSC Induction and Establishment. . . . . . . . . . . . . . . . . . . . 55
4.2.5 Karyotype Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.6 Differentiation of iPSCs into Neural Stem Cells
and Myocardial Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.7 Teratoma Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.8 Diploid and Tetraploid Blastocyst Injection. . . . . . . . . . . . . 56
4.2.9 Microarray Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.10 Southern Blot Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.2.11 Simple Sequence Length Polymorphism. . . . . . . . . . . . . . . 57
Contents xv

4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3.1 Generation of Mouse Embryonic Fibroblast-Derived iPSC. . . 57
4.3.2 Production of Live iPSC Mouse with Tetraploid
Complementation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.3.3 Comparative Study of Gene Expression Between
MEF-iPSC and ESC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.3.4 Generation of Tail Tip Fibroblasts and Neural
Stem Cells-Derived iPSC. . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.3.5 iPS Mouse Generated from NSC-iPSC and TTF-iPSC. . . . . 63
4.3.6 Whole-Genome cDNA Analysis of iPSC. . . . . . . . . . . . . . . . 67
4.3.7 Differentiation of iPSC into Neurons and Cardiomyocytes. . . 67
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.4.1 Type of Donor Cells Influence iPSC Induction Efficiency. . . . 69
4.4.2 Pluripotent State of iPSC Inducted by Various Techniques. . . 72
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

5 Developmental Potential of Mouse iPSC. . . . . . . . . . . . . . . . . . . . . . . . . 75

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2.1 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2.3 Small RNA Microarray Analysis. . . . . . . . . . . . . . . . . . . . . . 77
5.2.4 GO Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
5.3.1 Deep Sequencing of microRNA (miRNA) Revealed
Its Role in Regulating Pluripotency. . . . . . . . . . . . . . . . . . . . 78
5.3.2 Dlk1-Dio3 Region as a Critical Marker to Identify
if iPSC Induced by Three Factors are Fully Reprogrammed. . . 81
5.3.3 Aberrant Expression of Dlk1-Dio3 Region Cannot
be Rescued by Nuclear Transfer. . . . . . . . . . . . . . . . . . . . . . . 84
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.4.1 Prediction of the Target Genes of miRNA. . . . . . . . . . . . . . . 84
5.4.2 Dynamic Changes of Dlk1-Dio3 Region During
Somatic Cell Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . 86
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

6 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Chapter 1
Introduction

The birth of the first cloned mammal “Dolly” and the establishment of human
embryonic stem cell lines were landmarks in regenerative biology, indicating that
mankind had achieved a huge breakthrough in somatic cell reprogramming and
stem cell research. Recently, with the emergence of new reprogramming strategy,
induced pluripotent stem (iPS) cells further promoted the development of these
two research fields. In this section, some latest advances in reprogramming and
stem cell research are briefly introduced, and the influence of these works on basic
research and clinical applications, as well as the perspective is discussed.

1.1 Progress in Somatic Cell Reprogramming

1.1.1 Nuclear Transfer of Somatic Cells

1.1.1.1 History of Somatic Cell Nuclear Transfer

As early as the 1930s, there had been some reports on somatic cell nuclear transfer
(SCNT). But only in 1952, the work of American scientists Briggs and King began
attracting public attention. They injected the nucleus of partially differentiated cells
of Rana pipiens into enucleated oocytes, and then found that the reconstructed
embryo developed into tadpoles as well as froglets (Briggs and King 1952). Soon
after, Gurdon et al. (1958) obtained sexually mature individuals of Xenopus lae-
vis by injecting single nuclei from fully differentiated somatic cell into enucleated
oocyte. From the 1970s to 1980s, Chinese scientists also made significant contri-
butions to this field of work by obtaining the first cloned fish in the world. The
above-mentioned works made people believe that enucleated oocyte plasma could
reprogram the differentiated nuclear into pluripotent state. However, it remains to
be proved whether this phenomenon exists for all species, as attempts to estab-
lish cloned mammals were not achieved for quite a long time. It was in 1981 that

X. Zhao, Studies of Pluripotency in Embryonic Stem Cells and Induced 1

Pluripotent Stem Cells, Springer Theses, DOI: 10.1007/978-94-017-8819-9_1,
© Springer Science+Business Media Dordrecht 2014
2 1 Introduction

Illmensee and Hoppe (1981) got cloned mouse through injecting the nucleus of
cells derived from inner cell mass (ICM) into enucleated zygote, but their exper-
iments cannot be repeated till now. Nevertheless, from then on, scientists started
to explore nuclear transfer in mammals and made a series of progresses. In 1997,
Wilmut, Campbell and colleagues obtained cloned embryos by transferring the
nucleus of adult sheep mammary gland cell into enucleated oocyte. After trans-
plantation, the embryos successfully developed into full-term animals, and thus
the famous cloned sheep “Dolly” came into this world. This was the first case
where nuclei of differentiated somatic cells could be reprogrammed by enucleated
oocyte plasma in mammals, but there was unclear about the source of the mam-
mary gland cell, since it could not be confirmed whether Dolly was derived from
somatic cells or from a few stem cells in the mammary gland. After that, on one
hand, researchers tried to get cloned animals in other species; and on the other
hand, they tested if any types of somatic cells could be reprogrammed.
In 1998, Wakayama and colleagues cloned mouse successfully. Their work was
truly a breakthrough in the field of SCNT. Then goat, calf, pig, and rat were cloned
successfully (Baguisi et al. 1999; Kato et al. 1998; Polejaeva et al. 2000; Zhou
et al. 2003). Compared to these species, mouse has more advantage as an experi-
mental model as adequate quantities of oocytes can be obtained for experiments.
Meanwhile, previous works in the transgenic technique, which started from the
1970s, made it convenient to obtain a number of labeled donor cells from mice for
nuclear transfer experiments.
Till now, 15 mammal species have been cloned successfully, which proves that
we can obtain cloned mammals by reprogramming somatic cells. Meanwhile, there
are no reports on reproductive clones from somatic cells of nonhuman primates.
Some researchers presume that the cause may be that there are no high-quality
oocytes from nonhuman primates for experiments.

1.1.1.2 Type of Donor Cell Influence Reprogramming Efficiency

Following Somatic Cell Nuclear Transfer

In 2002, using terminal differentiated lymphocytes as donor cells, Hochedlinger

et al. obtained pluripotent, nuclear transferred embryonic stem (ntES) cells. Via
tetraploid complementation, ntES cells could develop into cloned mice, which
were called “ES mice.” These findings demonstrated that the terminal differenti-
ated nucleus could be fully reprogrammed, although the efficiency was just 10 %.
In 2004, Eggan and co-workers proved that postmitotic, olfactory sensory neu-
rons could also be reprogrammed to a state of totipotency after nuclear transfer,
thus testifying that cloned animals could be achieved as long as the somatic cell
genome was integrate. This conclusion was confirmed by the experimental find-
ings of Li Jing Song et al. (Eggan et al. 2004; Li et al. 2004).
In these experiments, researchers found that the reprogramming efficiency of
donor cells declined gradually as they differentiated from embryonic cells into termi-
nally differentiated somatic cells. However, some others gave a different conclusion,
1.1 Progress in Somatic Cell Reprogramming 3

since it was difficult to get cloned animals from hematopoietic stem cells, whereas
terminally differentiated granuloblasts could be reprogrammed into individuals
(Sung et al. 2006). By contrast, subsequent reprogramming studies using induced
pluripotent stem cell (iPSC) technology demonstrated that the reprogramming effi-
ciency of terminally differentiated somatic cells was lower than partially differenti-
ated precursor cells and also it took a longer time for the fully matured cells to be
reprogrammed. The reasons for the inconsistent conclusions from these two repro-
gramming approaches may be that the cell cycle of donor cells and host oocytes
need to be elaborately coordinated (Eminli et al. 2009; Hochedlinger and Jaenisch
2007). With the development of nuclear transfer technique, we believe that the repro-
gramming efficiency of terminal differentiated cells may be as high as precursors
when some signaling pathways that inhibit reprogramming are blocked.

1.1.1.3 Mechanism of Somatic Cell Reprogramming

by Nuclear Transfer

The low reprogramming efficiency of nuclear transfer has led to ongoing research
to improve the methods. Previous studies show that a majority of the early embryo
arrests were caused by incomplete reprogramming, and many NT embryos were
abnormal in epigenetic patterns. Boiani et al. (2003) found that the expression
level of some important pluripotent genes, such as Oct4, was abnormal in cloned
embryos and the percentage of Oct4 positive cells in cloned embryos was lower
than that of the fertilized embryos. Later, it was found that there were no sig-
nificant differences between the gene expression and epigenetic pattern between
nuclear transfer embryonic stem cells (ntESC) derived from cloned embryos
and ESCs from fertilized embryos (Brambrink et al. 2006; Chang et al. 2009;
Wakayama et al. 2006). Although there was some deficiency in cloned preim-
plantation embryos, they could develop to the onset of gastrulation, but then were
arrested in gastrula, which was mainly due to the defects in trophoderm (Jouneau
et al. 2006). In 2010, Maruotti and colleagues isolated epiblast stem cells (EpiSC)
from cloned embryos, and found that there were no differences in gene expres-
sion pattern between EpiSC derived from controls and from the cloned embryos
with normal morphology. However, this was not true for EpiSC derived from
cloned embryos with abnormal morphology, although these EpiSC still maintain
the expression of some key genes (Maruotti et al. 2010). These findings indicate
that although cells in ICM of cloned embryos were less than those of normal
embryos, there were truly some pluripotent cells in ICM of cloned embryo; thus
we can establish ntES cell lines with high efficiency. However, once these embryos
derived from ntESC were transplanted into pseudopregnant mice, only a minority
may develop into cloned animals (Rideout et al. 2002).
So far, although we were aware that there were reprogramming factors in the
oocyte plasma, we knew little about the molecular mechanisms of how these fac-
tors worked. In 2007, Egli et al. found that there were also some reprogramming
factors in the cytoplasm of enucleated zygotes which were in the stage of mitosis.
4 1 Introduction

These factors were released from the nucleus when the karyotheca broke down
(Egli et al. 2007). Later, they proved that two-cell stage embryos also had repro-
gramming factors (Egli et al. 2009). In addition, Amjad et al. in our lab also veri-
fied the previous results (Riaz et al. 2011). As for the developmental abnormality
of cloned embryos, some researchers speculate that in the process of removing
the nucleus, a number of the reprogramming factors that reside in nucleus would
be lost, which lead to partial reprogramming and poor development of the cloned
embryos. The work of Yang et al. (2010) also supports the above assumption.
Because of the technical limitation and the abundance and complexity of proteins
within the nucleus of oocytes, factors involved in the initiation of the reprogram-
ming are still elusive.

1.1.2 Induced Pluripotent Stem Cells

Combining the great achievements in reprogramming and the findings based on the
study of embryonic stem cells, Japanese scientist Shinya Yamanaka and colleagues
established a new technique to reprogram somatic cells to pluripotent state by forc-
ing the overexpression of four factors (Oct4, Sox2, Klf4, and c-Myc). They named
it iPSC. This new technique immediately aroused the interest of scientists world-
wide, and stimulated a search wave for iPSCs (Takahashi and Yamanaka 2006).

1.1.2.1 Generation of iPSC

The early cell lines of iPSC established by Takahashi et al. maintained a limited
level of pluripotency. They formed teratomas that contained the derivatives of
three germ layers when injected subcutaneously into severe combined immuno-
deficiency (SCID) mice. Chimeric mice can be obtained through a diploid blasto-
cyst injection, although these chimeric embryos will die before birth. However, the
gene expression profile of these iPSC lines was significantly different from that of
embryonic stem cells. The expression of exogenous genes remained high, and the
promoter region of pluripotency marker Oct4 remained highly methylated. These
features indicated that the first generation iPSC are partially reprogrammed and
showed huge difference compared with mouse embryonic stem cells (Takahashi
and Yamanaka 2006).
In 2007, three laboratories independently improved on Yamanaka’s induction
methods to obtain iPSCs with more pluripotency. These new iPSC lines could
differentiate into various types of tissues and organs in chimeric mice, including
genital ridge. Similar to embryonic stem cells, the genome of these iPSCs showed
hypomethylation. Both their X chromosomes were activated in female iPSC and
exogenous genes were silenced. Expression levels of Oct4, Nanog, and other
important transcription factors, as well as their whole genome expression patterns
were similar to embryonic stem cells. These results showed that the new iPSC
1.1 Progress in Somatic Cell Reprogramming 5

lines met the common criteria of ESC. However, further analysis found that the
iPS mice could not be obtained by tetraploid complementation using these iPSCs.
As we know, the tetraploid complementation is the golden standard for the iden-
tification of ESC. The fact that iPSCs were not able to obtain iPS mice indicated
that these iPSCs were still partially reprogrammed, and there was a clear differ-
ence between embryonic stem cells and iPSCs (Maherali et al. 2007; Okita et al.
2007; Wernig et al. 2007).
Several months later, the laboratories of Yamanaka and Thomson reported the
successful establishment of human iPSC. These human iPSCs were pluripotent
cells, and they could form teratomas and differentiated to the derivatives of three
germ layers. Human iPSCs expressed pluripotent surface markers like SSEA-3,
Tra-1-60, and key pluripotent transcription factors like Oct4 and Nanog, and their
gene expression profiles were also similar to the ESC (Takahashi et al. 2007; Yu
et al. 2007).

1.1.2.2 Progress in Techniques for iPSC Reprogramming

Successful Induction of Nonviral and Nonintegrated iPSC

In the original induction system of iPSC, virus vectors were used to achieve high
transfection efficiency. This facilitated the generation of iPSCs, but also increased
genomic instability. People began to explore other ways to overexpress the four
reprogramming factors. In 2008, Hochedlinger and Yamanaka’s groups reported
to obtain nonintegrated iPSCs. Stadtfeld et al. used adenoviral vectors to obtain
noninsertion iPSCs, while Okita et al. obtained iPSCs through direct transfection
of plasmids. These were the first reports that nonintegrated iPSC could be estab-
lished independent of virus integration, and the insertion of the virus was not nec-
essary for iPSC reprogramming. However, these reports also showed that the virus
reprogramming system has its own advantages to get higher induction efficiency,
because it could maintain the expression of foreign genes at a high level for a rela-
tively longer time (Okita et al. 2008).
In 2009, Zhou et al. reported that they obtained mouse iPSC after about 1 month
of induction by supplying culture medium with proteins of the four transcription
factors (Oct4, Sox2, Klf4, and c-Myc), which were modified by adding nine argi-
nine-containing peptides to the c-terminal to form a transmembrane region, thus
giving them the ability to enter into the cell through cell membrane. Subsequently,
Kim et al. obtained human iPSC by the same method. Since there was no virus
insertion, the genome of protein-induced iPSC will be more stable, thus avoiding
the risk of genome mutation caused by virus insertion (Zhou et al. 2009).
Currently, the induction efficiency of iPSC using proteins is extremely low, so
it is critical to improve the induction efficiency. In addition, the long induction
time and the treatment of a variety of small molecules may cause mutations in the
genome of iPSC, so it is important to find new methods to lessen the induction
time (Kim et al. 2009a).
6 1 Introduction

Recently, Warren et al. obtained human iPSCs by transfecting mRNA into

somatic cells. With this technique they achieved the goal of nonviral, noninsertion
induction with comparatively high efficiency. But it is still unconfirmed whether
these iPSCs were completely reprogrammed, and the method needs to be further
certified in mice (Warren et al. 2010).

Somatic Cell Transdifferentiation

In 2008, Zhou et al. reported that they could transdifferentiate adult pancreatic
exocrine cells into beta cells in vivo by overexpression of three factors (Ngn3,
Pdx1, and Mafa). Their study demonstrated that it was possible to induce one type
of somatic cell to directly transdifferentiate into another type without intermedi-
ate dedifferentiation of somatic cell to pluripotent status. However the induction
efficiency is comparatively low, and transdifferentiation could only be achieved
in vivo (Zhou et al. 2008). In 2009, Takeuchi and Bruneau (2009) found that the
combination of Gata4, Tbx5, and Baf60c could be used to differentiate ectopic
mesodermal cells into cardiomyocytes. Later in 2010, Vierbuchen et al. found that
overexpression of three factors (Ascl1, Brn2, Myt1l) in vitro differentiates mouse
fetal fibroblasts and neonatal fibroblast cells into nerve cells, which can differenti-
ate into functional GABA neurons. In the same year, Leda and his fellows induced
the fetal fibroblast cells into functional cardiomyocytes, and confirmed that the
epigenetic imprinting of these myocardial cells were normal (Ieda et al. 2010).

Induction Efficiency of iPSC Reprogramming

It was found that the induction efficiency of iPSC was 0.01–0.5 % at the early
stage, which was comparatively lower than SCNT. To enhance the induction effi-
ciency, two common strategies—careful selection of donor cells and supplement-
ing of small molecule drugs could be used.
Mouse embryonic fibroblasts or tail tip fibroblasts (TTF) were first used as donor
cells to induce iPSC, while fibroblasts were commonly used as donor cells for SCNT.
These cells are believed to originate from the mesoderm and maintain the expres-
sion of c-Myc. Aasen et al. found that keratinocytes derived from the ectoderm were
more likely to be reprogrammed to iPSC. The reprogramming efficiency was 100
times higher than fibroblasts, and the time for clone formation was only half that of
fibroblasts (Aasen et al. 2008). When Kim et al. (2008) induced neural stem cells to
iPSC, they found it more efficient than fibroblasts, and they could use only two fac-
tors, Oct4 and Klf4, to obtain iPSCs efficiently. The following year, they further dem-
onstrated that using only one reprogramming factor, Oct4, they could also get iPSCs
(Kim et al. 2009b, c). These experiments showed that cells derived from the ecto-
derm could be reprogrammed into iPSC more easily. Although less reprogramming
factors were used and lower efficiency was observed in experiments to induce iPSCs
from hepatocytes and gastric cells (endoderm origin), Aoi et al. (2008) confirmed
that somatic cells derived from all three germ layers could be reprogrammed into
iPSCs. After this, up to ten more types of cells, including terminally differentiated
1.1 Progress in Somatic Cell Reprogramming 7

B lymphocytes and T lymphocytes, were selected as donor cells and successfully

induced into iPSCs (Hanna et al. 2009; Okita et al. 2008). In 2009, Eminli et al. sys-
tematically compared the iPSC induction efficiency among hematopoietic stem cells,
precursor cells, and terminally differentiated B and T cells in the hematopoietic sys-
tem. They found that reprogramming efficiency of stem cells was 300 times higher
than terminally differentiated cells. The more differentiated the donor cells, the lower
the efficiency of induction for iPSC, and the longer the time required for reprogram-
ming (Eminli et al. 2009). Therefore, the selection of donor cells, which could be
assessed easily, is an important issue in clinical applications in the future.
Small molecules that can promote the efficiency of reprogramming can be divided
into several categories: (1) drugs to promote demethylation or to increase the level of
acetylation; (2) drugs to suppress the signaling pathways, which promote the differ-
entiation of embryonic stem cells; (3) drugs to reduce the level of p53, which inhibits
the reprogramming, and so on. In 2008, Huangfu et al. found that deacetylase inhibitor
valproic acid (VPA) and DNA methylation inhibitors could significantly improve the
efficiency of iPSC induction. By replacing c-Myc with VPA, iPSC could be obtained
at the same efficiency as induced using the four factors. This result showed that VPA
could enhance the induction efficiency of iPSC (Huangfu et al. 2008a, b). Shi et al.
used neural precursor cells as donor cells, and they found that a supplement of G9a
histone methyltransferase inhibitor BIX-01294 could dramatically improve the induc-
tion efficiency of iPSC using Yamanaka’s four factors. In the presence of BIX-01294,
iPSC clone could also be observed even without the transfection of Oct4. Supplement
of MEK inhibitor PD0325901 could also enhance the reprogramming efficiency sig-
nificantly. With the addition of PD0325901, the efficiency of reprogramming was sig-
nificantly improved, even when only two factors, Oct4 and Klf4, were used (Shi et al.
2008a). In later studies, Shi et al. obtained iPS cells using BIX-01294 and BayK8644
from Oct4- and Klf4-transfected embryonic fibroblasts. This result shows that the
combined effects of these two factors could replace Sox2 and c-Myc (Shi et al. 2008b).
By adding the GSK3 inhibitor CHIR99021 and the MEK inhibitor PD0325901 to
neural stem cells transfected with the four factors, Silva et al. (2008) found that they
could transfer the partial reprogrammed iPSCs into completely reprogrammed iPSCs.
Ichida et al. (2009) found that TGF-Beta inhibitor could replace Sox2 to induce iPSCs.
It could indirectly upregulate the expression of nanog via inhibiting TGF-Beta sign-
aling pathway. Maherali et al. obtained the same results in the same year (Maherali
and Hochedlinger 2009). Li et al. reported that the combined effects of small mole-
cules could dramatically improve the efficiency of iPSC induction (Lin et al. 2009).
In 2008, Deng’s group at Peking University reported that the inhibition of P53 expres-
sion improved the induction efficiency of iPSC. Pei’s group reported that Vitamin C
improved the efficiency of iPSC induction partially by inhibiting the expression of
P53 (Esteban et al. 2010). Recently, Li et al. (2010b) reported that during the pro-
cess of iPSC induction, if the combination of HDAC inhibitor VPA, GSK3 antago-
nists CHIR99021, TGF-beta inhibitor 616452, and G9a histone methylation inhibitor
Tranylcypromine was used, only Oct4 was required to reprogram MEF to iPSC. These
results indicate that the combined effects of small molecule drugs can reduce the num-
ber of reprogramming factors needed for the induction of iPSC.
8 1 Introduction

Mechanism of Reprogramming by iPSC

Compared to SCNT, induction of iPSC takes a longer time, so it is relatively easier

for us to divide the process of iPSC induction into several stages so that we can do
some research work on the different stages of induction. Until now people could
only carry out some simple analysis on certain stages of the whole process, and
the molecular mechanisms of reprogramming are far from clear. Since there is no
systematic analysis of the entire process, we will give a brief introduction of some
related work in chronological order.
From 2007 to 2008, Hochedlinger’s group at Harvard University published a series
of articles depicting the reprogramming process from fibroblasts to iPSCs. First,
fibroblasts stopped to express fibroblast-specific marker Thy1, and turned to be par-
tial reprogrammed with positive SSEA1 and AP staining. Subsequently, pluripotency
factors were activated, telomerase activity was restored, and the X chromosome was
reactivated. In the late stage of iPSC induction, exogenous genes were gradually inac-
tivated, and finally successfully reprogrammed iPSC retained the true pluripotency
(Maherali et al. 2007). In 2008, Mikkelsen et al. found that the gene expressions and
histone modifications of partially reprogrammed fibroblasts showed significant dif-
ferences compared with fibroblasts and complete reprogrammed iPSCs. In 2009,
Sridharan et al. found that the co-binding sites of Oct4, Sox2, and Klf4 in partially
reprogrammed iPSCs showed some differences with those in completely repro-
grammed iPSCs or ESCs, while the binding sites of c-myc remained largely the same.
Since the previous three factors dominated the pluripotency of embryonic stem cells,
these findings indicated that the regulatory network associated with pluripotency may
not be correctly established in partially reprogrammed iPSCs (Sridharan et al. 2009).
We know that only a handful of cells can be transformed into pluripotent stem
cells in the conventional iPSC induction process, and some may speculate that
these iPSCs may be derived from a very small number of stem cells or precur-
sor cells that reside within donor cells. In 2009, five labs, including Yamanaka’s
group, independently found that decreased expression of P53 could improve the
induction efficiency of iPSCs. High expression of P53 activated P21 and other
downstream signaling pathways, so a lot of early iPSCs died of apoptosis, thereby
significantly reducing the induction efficiency (Hong et al. 2009; Kawamura et al.
2009; Utikal et al. 2009). Later, Hanna and his colleagues performed a series of
experiments on this issue. They found that if the enforced expression of exogenous
gene was extended to 8 weeks, most B lymphocyte precursor cells (92 %) could
be converted into iPSCs. This result showed that almost all somatic cells have the
potency to be reprogrammed to iPSCs. They further demonstrated that the effi-
ciency of iPSC induction could be improved by either decreasing the expression
of P53 or upregulating the level of Lin28. Their explanation was that these gene
modifications could shorten the cell cycle, which was inconsistent with previous
reports. This conflict may be caused by the different modes of reprogramming in
different types of somatic cells, but the promotion effect of nanog in iPSC induc-
tion was not related to cell cycle. Meanwhile, they found that the generation of
iPSC was a stochastic event, and there were no detectable differences between the
1.1 Progress in Somatic Cell Reprogramming 9

early and late reprogrammed somatic cells without excluding the possibility that
unknown differences may exist between those cells (Hanna et al. 2009). However,
Eminli et al. found that lower induction efficiency and longer induction time were
observed when inducing iPSCs from more differentiated donor cells. This indi-
cated that in the iPSC induction process, the precursor cells may be easily induced
in a relatively short period of time, so that random pattern and precursor cells
priority mode may exist at the same time in the iPSC induction process (Eminli
et al. 2009). In 2010, Li et al. (2010a) found that fetal fibroblasts from mesoderm
underwent MET (Mesenchymal-to-Epithelial Transition) in the process of iPSC
induction, and they confirmed that this process affected the induction efficiency.
Meanwhile, Samavarchi-Tehrani proved that BMP signaling promoted MET, thus
promoting the inducing efficiency of iPSC (Samavarchi-Tehrani et al. 2010).
All the above researches indicate that there were several barriers in MEF that
are needed to be overcome in the process of iPSC induction. This included the
inhibition of somatic cell-specific genes, the expression of stem cell-specific
genes, transition from partial reprogrammed cells into fully reprogrammed iPSC,
conversion of the proliferation mode from slow proliferation of somatic cells into
rapid proliferation of stem cells and immortalization, DNA demethylation, histone
acetylation, etc. To complete these key events, small related molecules may be
used to improve the induction efficiency.
In 2009, there was a report that activation-induced cytidine deaminase (AID)
could be used to induce pluripotency by binding to the promotor regions and ini-
tiating the demethylation of some pluripotent factors, like Oct4 and nanog. This
was similar to the demethylation role of AID played in the primordial germ stem
cells (PGC) formation (Unger et al. 2008). This result indicated that DNA demeth-
ylation process may be completed by a series of known or unknown demethylases
implying that there exist the same demethylation mechanisms in reprogramming
of iPSC as in the development.
However, there are still many unresolved issues on reprogramming mechanisms
of iPSC, which include whether Oct4 is the only truly reprogramming factor, how
the four factors transfer somatic cells from one steady state to another steady state,
are there any signaling pathways involved in reprogramming other than the clas-
sical four factors, and the factors that limit the reprogramming etc. These studies
provide a theoretical basis for the clinical application of iPSC. Upon elucidation of
these questions, the induction of iPSC will be safer, efficient, and rapid. It can also
promote the application of iPSC technology in clinical practice.

1.2 Research and Application of Stem Cells

in Regenerative Medicine

In 1981, two groups independently reported the establishment of embryonic stem

cells (ESCs) from ICM of mouse blastocysts, indicating the beginning of the new
ESC era. ESCs express key pluripotent transcription factors, including Oct4, Sox2,
10 1 Introduction

Nanog, Klf2, Klf4, Rex1, Esrrb, etc. It is alkaline phosphatase (AP)-positive and
maintains high telomerase activity. ESC can expand unlimitedly in vitro and dif-
ferentiate into various cell types of all three germ layers as well as germ cells. In
1998, based on his work to establish primate (monkey) ESCs, Dr. James Thomson
successfully established the first human ES cell line. Since then, ESC study has
truly found its way to link regenerative medicine.
In 1984, it was demonstrated that mouse ESCs could contribute to embryo
development and produce chimeric mice, and these chimeric mice could gen-
erate germline transmission mice by mating. Later, the first knockout mice were
generated by homologous recombination in 1989 and it was also proved that the
modified genome could be transmitted to the offspring. From 1989, thousands of
gene-targeted or transgenic mice were generated. The technique of knockout mice
significantly promotes research work on genome, especially for gene function stud-
ies. This useful technique can also be applied in other species, such as primates.
The ESC itself is a good in vitro model to study the mechanisms of self-
renewal and pluripotency. In 1988, LIF was identified as the key factor to support
self-renewal of ESCs. Ten years later, the transcription factor Oct4 was identi-
fied as a key pluripotent marker in ESCs. Since then, more and more factors were
identified, especially Oct4, Sox2, and Nanog complex. The transcriptional fac-
tors cooperate with each other or form complicated signaling network to regulate
the balance between self-renewal and differentiation in stem cells. In addition to the
genetic information, the epigenetic status also influences the self-renewal of stem
cells. ESCs have unique epigenetic modification, with hypomethylation genome,
and unique histone modification pattern, which is characterized by H3K4me3 and
H3K27me3 bilateral modification. The abnormal epigenetic modifications also
compromised the self-renewal of ESCs. In 2003, Qi-Long Ying and his colleagues
in Austin Smith lab reported that BMP4 signaling pathway inhibited the differen-
tiation of ESCs. Five years later, Austin Smith and their colleagues further identi-
fied that the GSK3b inhibitor CHIR99021 and ERK1/2 phosphorylation inhibitor
PD0325901 (2i) could sustain self-renewal of mouse ESC in a chemically defined
culture system. Using the 2i culture system, Qi-Long Ying and Austin Smith’s
groups successfully established germline-competent ESCs from rat blastocysts,
respectively, at the same time.
Human ESCs demonstrate distinct characteristics when compared with mouse
ESCs. Human ESCs exhibit several characteristics: (1) round-shaped colony with
clear margin, (2) can hardly be passaged as single cells, (3) with XaXi X chro-
mosome, and (4) can differentiate into trophectoderm cells. Human ESCs require
bFGF and Activin to maintain their self-renewal, while mouse ESCs rely on LIF
and BMP4. Why there are huge differences between human and mouse ESCs is
unclear. At present, the theory that those ESCs are actually in different stem cell
statuses is popular. Human ESCs are considered as “primed” pluripotent stem
cells, while mouse ESCs are regarded as “naïve” stem cells.
Both human iPSCs and mouse iPSCs share similar characteristics with their coun-
terpart ESCs. Because of their unique properties of infinite self-renewal and pluripo-
tency, ESCs and iPSCs can be used as in vitro models of embryogenesis, which greatly
1.2 Research and Application of Stem Cells 11

facilitates the research on embryology. In addition, scientists can induce ESCs to dif-
ferentiate into specialized somatic cells, and then those cells can be transplanted back
into animals to assess its potential clinical applications. Next we give a brief review of
the breakthroughs in in vitro differentiation of stem cells and their application.

1.2.1 In Vitro Differentiation of Stem Cells

1.2.1.1 Differentiation of Embryonic Stem Cells

Neural Differentiation

Mouse ESCs need LIF and bone morphogenetic protein (BMP) signals to maintain
self-renewal. The mouse ESCs differentiate when LIF or BMP signals are removed
(Ying et al. 2003a). Bain et al. were one of the pioneers in studying neural differen-
tiation of ESCs. They induced mouse ESCs to differentiate into neural cells, which
expressed a variety of neural markers, including BIII-tubulin, neurofilament M subu-
nit, subunits of Neurofilament L, glutamate receptor, the transcription factor Brn-3,
GFAP, etc. The functions of these cells had not been evaluated (Bain et al. 1995).
In 1996, Okabe et al. established the five-step method to induce mouse ESCs to
neural cells. In 1999, Brustle et al. successfully induced ESCs to differentiate into
precursors of oligodendrocytes and astrocytes in vitro. Those precursor cells could
establish axon connection with neurons, and restored the myelination in spinal cord
and brain in vivo in the rat model of human myelin disease (Brustle et al. 1999). In
2000, Lee’s group (2000) obtained enough CNS precursor cells from mouse ESCs,
and these CNS precursor cells were further amplified and differentiated into dopa-
minergic neurons, and finally differentiated into mature neurons. In the same year,
Kawasaki et al. found that using PA6 as feeder cells could induce mouse ESCs to
differentiate into dopaminergic neuron, while the supplement of BMP4 inhibited
neural differentiation. When these dopaminergic neurons were transplanted into
mouse brain, they could integrate into the striatum and maintained the expression
of dopaminergic markers (Kawasaki et al. 2000). In 2002, Kim and colleagues over-
expressed Nurr1 in mouse ESCs, and then the cells were used for neural induction.
They noticed that transduced ESCs could efficiently differentiate into functional
dopaminergic neurons, which could secrete dopamine. When transplanted into rat
model of Parkinson’s disease, the cells could integrate into the striatum and ame-
liorate the symptoms (Kim et al. 2002). In the same year, Wichterle et al. found that
treatment of embryoid bodies (EB) with retinoic acid (RA) could induce differentia-
tion of nerve cells of mesencephalon. When the cells were transplanted into chick
embryo, they could further specialize into motor neurons (Wichterle et al. 2002).
In 2003, Ying et al. established a new system for ESCs to differentiate to neural
cells. Mouse ESCs spontaneously differentiated into neural stem cells (Ying and
Smith 2003; Ying et al. 2003b), when cultured adherently on gelatin-coated sub-
strate in N2B27 medium (Ying and Smith 2003). Using this induction method, a
large quantity of neural stem cells with high purity could be obtained. These cells
12 1 Introduction

could be propagated for more than 20 passages with the supplement of FGF2 and
EGF, and still maintained the capacity to differentiate into neurons, astrocytes, and
oligodendrocytes (Conti et al. 2005; Glaser et al. 2007). In 2006, using this induc-
tion system, Lowell et al. (2006) found that Notch signaling could promote neural
commitment of mouse and human ESCs.
There is a great difference between human ESC and mouse ESC in the signaling
pathways that regulate self-renewal (Thomson et al. 1998). It has been noticed that
the most efficient induction condition used for moue ESCs does not always work
well for hESCs. In 2001, Zhang et al. found that hESC would aggregate to form EB
once feeder and FGF2 were removed from culture, and then neural precursor cells
were induced with the supplement of FGF2. After removing of FGF2, these neu-
ral precursors could further differentiate into neurons, astrocytes, and oligodendro-
cytes. When the neural precursor cells were transplanted into the brain of newborn
mice, they could integrate into various sites of the brain, and differentiate into neu-
rons and glial cells (Zhang et al. 2001). In 2004, Perrier et al. induced human ESCs
to differentiate into midbrain dopaminergic neurons, and soon, Li et al. treated the
human ESCs with RA to induce differentiation of hESC into neural precursor cells.
They found that mesencephalon derivatives could be induced only from Sox1+
neural precursors. Under the stimulation of Shh (Sonic Hedgehog), the mesenceph-
alon derivatives could further differentiate into motor neurons that were proved
to have the functional characteristics of motor neurons (Li et al. 2005). Yan et al.
(2005) first obtained Sox1-positive neural precursor cells, and then treated the cul-
ture with Shh and FGF8 to induce dopaminergic neurons that were demonstrated to
have electrophysiological activity and could secrete dopamine in vitro. In 2008, Li
et al. further improved the induction efficiency of motor neurons to 50 % by replac-
ing Shh with a small compound Purmorphamine to induce the differentiation of
human ESC into neural precursor cells of the ventral spinal cord. Yang et al. (2008)
induced differentiation of human ESC to dopaminergic neurons, which after trans-
plantation could ameliorate the symptom of rat model of Parkinson’s disease. Hu
et al. found that in the process of oligodendrocytes differentiation of human ESC,
Shh could promote the expression of the gene cluster depending on Shh signaling,
indicating that SHH have a conservative role in neural induction in vertebrates,
while this is not true for FGF2. FGF2 promotes the induction of oligodendrocyte
precursors cells in mouse (Oligodendrocyte Precursor Cells, OPC), while it inhibits
pre-OPC changing to OPC in humans (Hu et al. 2009).

Myocardial Differentiation

There has been a long research history of cardiomyocyte induction from ESCs. In
1985, Doetschman et al. detected myocardial cells using embryoid body culture. In
1996, Klug et al. transduced two genes that encoded the a-myosin and aminoglyco-
side phosphotransferase, respectively, into ESCs. By antibiotic resistance selection,
cardiocytes with high purity were obtained and subsequently transplanted into the
hearts of mice with malnutrition. The transplanted cells ultimately survived for more
than 7 weeks (Klug et al. 1996). Developmental biology studies show that BMP,
1.2 Research and Application of Stem Cells 13

WNT, and FGF signals are involved in heart development. In 2005, Yuasa et al.
(2005) found that the treatment of EBs derived from mouse ESC with Noggin, an
antagonist of BMP signal, could significantly enhance the induction efficiency for
cardiomyocytes. Later, Singh et al. (2007) found that Chibby, a WNT/beta-catenin
signal pathway antagonist, could raise the proportion of cardiomyocytes derived
from ESC, and inhibition of its expression by RNAi will hamper the induction of
cardiomyocytes. Kehat et al. (2001) found that after induction, 8.1 % of EBs derived
from human ESCs contained beating cardiomyocytes, which possessed the charac-
teristics of in vivo cardiomyocytes, according to biochemical analysis.

Other Line Age Commitment

ESCs have already been successfully induced into other cell types, including islet B
cells (Lumelsky et al. 2001), hematopoietic cells, (Palacios et al. 1995; Umeda et al.
2004), liver cells (Rambhatla et al. 2003), and so on. Here, we give a brief intro-
duction into recent breakthroughs in the induction of insulin-secreting B cells. Jiang
et al. (2007) induced endoderm cells from human ESC by supplying the culture with
Activin A. Subsequently, the cells were treated with all-trans RA to obtain islet pre-
cursor cells. In serum-free medium containing FGF2 and nicotine (nicotinamide),
the islet precursor cells differentiated into mature islet cells, which could survive for
6 weeks and relieved the symptoms of diabetes in mouse model after transplantation.

Differentiation of Nuclear Transfer-Derived ESCs

In 2001, Wakayama et al. isolated embryonic stem cells from blastocysts clones of
mouse granulose cells and tail tip fibroblasts. Upon neural induction, the nuclear
transfer-derived ESCs (ntESC) could be induced to dopaminergic and serotonergic
neurons. Chimeras could also be obtained by injecting ntESC into diploid blasto-
cysts, which certified the pluripotency of ntESC in vitro and in vivo (Wakayama et al.
2001). In 2003, Barberi et al. induced ESCs and ntESCs to differentiate into neural
cells. They obtained γ-aminobutyric acid (GABA), dopamine, serotonin, and motor
neurons, corresponding to forebrain, midbrain, hindbrain, and spinal cord origin.
These induced cells have an expression pattern and electrophysiological characteris-
tics similar to their in vivo counterparts. The transplantation of dopaminergic neurons
could alleviate symptoms in a mouse model of Parkinson’s disease (Barberi et al.
2003). Induction of ntESC differentiation is basically similar to ESCs, because ntESC
has developmental totipotency (Brambrink et al. 2006; Wakayama et al. 2006). There
are no reports yet on differences between these two types of pluripotent stem cells.

Differentiation of iPSCs

The technology of iPS overcomes ethical obstacles and issues of immune

rejection, thus iPSC was considered to have a wonderful application prospect
(Yamanaka 2007). Tremendous research work on induction of iPSC has been
conducted in just a few years.
14 1 Introduction

Wernig et al. induced iPSCs, which were derived from fibroblasts, to differenti-
ate into neural precursor cells, and then further into neurons and glial cells. When
transplanted into brains of fetal rats, iPS-derived neural cells integrated into the
brain tissue and differentiated into GABA and catecholaminergic neurons. Further
analysis indicated that the neurons showed electrophysiological activity. Neural
precursor cells derived from iPSC were sorted by flow cytometry to get rid of
undifferentiated pluripotent stem cells. On transplantation of the pure population
of neural precursors into the brains of rats, they can differentiate into dopaminer-
gic neurons and alleviate the symptom of rat model of Parkinson’s disease (Wernig
et al. 2008). Following the induction strategy of ESCs, Mauritz et al. induced dif-
ferentiation of iPSCs into cardiomyocytes for 24 days. They observed lower car-
diomyocyte induction efficiency and longer induction period for iPSCs compared
with ESCs. However, there were still 55 % of the EBs containing spontaneously
beating cardiomyocytes, and further gene expression study suggested that these
iPSC-derived cardiomyocytes possessed typical characteristics as in vivo cardi-
omyocytes. Narazaki et al. (2008) also successfully induced the iPSC to differ-
entiate into cardiomyocytes, and demonstrated that iPSCs are identical to ESCs
in their differentiation capacity, which indicated that we can get cardiomyocytes
through iPSC differentiation (Mauritz et al. 2008). These iPSC induction tech-
niques will be the foundation for clinical applications in regenerative medicine.
Besides myocardiocytes, Schenke-Layland et al. (2008) also acquired smooth
muscle cells, endothelial cells, and hematopoietic cells from iPSCs. Si-Tayeb et al.
(2010) induced mice iPSCs to differentiate to liver cells which could integrate into
the liver of fetal mice upon transplantation.
Buchholz et al. found no significant difference among retinal pigment epi-
thelial cells derived from human iPSCs, human ESCs, and in vivo counterparts.
This finding indicated that human iPSC possessed equal differentiation capacity
as human ESCs (Buchholz et al. 2009). Cai et al. (2010) induced the differentia-
tion of human iPSC into dopaminergic neurons. They found that dopaminergic
neurons derived from iPSCs showed no significant differences with that of human
ESC origin. On transplantation, precursors of dopaminergic neuron derived from
hESCs could differentiate into dopaminergic neurons and integrate into brain in a
rat model of Parkinson’s disease. Chambers et al. (2009) discovered that supple-
ment of Noggin and SB431542, two inhibitors of Smad signal, can dramatically
raise the efficiency of neural induction for both human ESCs and iPSCs.
Choi et al. induced human ESCs to differentiate into lin-CD34+ CD43+
CD45+ hematopoietic cells, and further into myeloid mononuclear cells. They
found no significant differences between human ESCs and iPSCs for hematopoi-
etic cell differentiation (Choi et al. 2009b). They also induced human iPSCs to dif-
ferentiate into hematopoietic and endothelial cells, and found that differences did
exist among various iPSC cell lines, but the induction shared the same transition
stages in the process of differentiation (Choi et al. 2009a).
Gai et al. induced differentiation of human iPSC into functional cardiomyocytes,
and showed no difference between iPSC-derived and ESC-derived cardiomyocytes,
according to RT-PCR and immunostaining results. They found that both immature
1.2 Research and Application of Stem Cells 15

and mature myocardiocytes existed in induction cultures of human iPSC and ESC
origin, and 5-azacytidine could promote cardiomyocyte induction, but dimethyl
sulfoxide showed no effect. The environment of low serum and BMP2 supplement
could slightly promote myocardial differentiation. These induced cardiomyocytes
showed response to drugs, suggesting their potential applications in the drug devel-
opment of cardiovascular diseases (Gai et al. 2009). Karumbayaram et al. (2009)
found that it has the same efficiency to induce motor neurons for human iPSCs and
ESCs. Furthermore, they found that these cells expressed typical markers of motor
neuron and were sensitive to electrical stimulation, indicating that iPSC-derived
motor neurons could be used as an in vitro model of neurological disorders.

1.2.2 Application of Stem Cells

1.2.2.1 Disease Model

Somatic cells derived from patients could be reprogrammed into pluripotent stem
cells using iPSCs technology, which is a new option in addition to nuclear transfer.
These iPSCs can then be induced into specific cell types or tissues in vitro. This in
vitro induction system of iPSCs could be used as a disease model for exploring the
pathogenesis of diseases and for drug screening. This in vitro model is also useful
for evaluating the improvement of symptoms after gene therapy (Daley et al. 2009).
To establish patient-specific iPSCs, Dimos et al. reprogrammed the somatic
cells of an 82-year-old man, who suffered from amyotrophic lateral sclerosis
(ALS), into iPSCs. These iPSCs were further induced into motor neurons in vitro.
Park and colleagues also generated iPSCs from somatic cells of patients with
inherited diseases, including adenosine deaminase deficiency-related severe com-
bined immunodeficiency (ADA-SCID), Shwachman–Bodian–Diamond syndrome
(SBDS), Gaucher disease type III (GD), DMD, BMD, Parkinson disease (PD),
Huntington disease (HD), Juvenile-onset, type 1 diabetes mellitus (JDM), and
down syndrome (DS). Patient-specific iPSCs were used as in vitro models to study
the pathogenesis of inherited diseases and so on (Park et al. 2008).
Lee reprogrammed somatic cells of patients suffering from the familial auto-
nomic (FD) into FD-iPSC. Then FD-iPSCs were induced to differentiate into
derivatives of three germ layers, including peripheral nerve cells. They found that
the gene expression of IKAKAP was lower than normal and this phenomenon was
also observed in FD patients, which was believed to be the cause of the disease.
After some analysis on FD-iPSC-derived cells, they found a series of deficiencies
in differentiation and migration of the cells, and they tried to reverse the defects
with drugs treatments (Lee et al. 2009).
Urbach et al reprogrammed fibroblasts from patients suffering Fragile X syndrome
(FX) disorders into FX-iPSC. They found that there is a significant difference in gene
expression between FX-iPSC and FX-ESC. This result indicates that iPSC and ESC
were different in epigenetic modifications (Urbach et al. 2010).
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