Journal of Clinical Gerontology & Geriatrics 2 (2011) 1e6

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Journal of Clinical Gerontology & Geriatrics

journal homepage: www.e-jcgg.com

Review article

Induced pluripotent stem cells and regenerative medicine

Yuh-Chi Chen, PhD a, b, y, Kung-Lin Tsai, MS c, d, y, Chia-Wei Hung, MD e, b, y, Dah-Ching Ding, MD, PhD f,
Lih-Hsin Chen, MS g, Yuh-Lih Chang, PhD a, g, Liang-Kung Chen, MD b, c, Shih-Hwa Chiou, MD, PhD a, d, g, *
a
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China
b
School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
c
Center for Geriatrics and Gerontology, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China
d
Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
e
Department of Neurology, Zhongxiao Branch, Taipei City Hospital, Taipei, Taiwan, Republic of China
f
Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital, Tzu Chi University, Hualien, Republic of China
g
Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, Republic of China

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

Article history: Stem cells, a special subset of cells derived from embryo or adult tissues, are known to present the
Received 12 November 2010 characteristics of self-renewal, multiple lineages of differentiation, high plastic capability, and long-term
Received in revised form maintenance. Recent reports have further suggested that neural stem cells (NSCs) derived from the adult
10 December 2010
hippocampal and subventricular regions possess the utilizing potential to develop the transplantation
Accepted 13 December 2010
strategies and to screen the candidate agents for neurogenesis, neuroprotection, and neuroplasticity in
neurodegenerative diseases. In this article, we review the roles of NSCs and other stem cells in neuro-
protective and neurorestorative therapies for neurological and psychiatric diseases. We show the
Keywords:
Stem cells
evidences that NSCs play the key roles involved in the pathogenesis of several neurodegenerative
Embryonic stem cell disorders, including depression, stroke, and Parkinson’s disease. Moreover, the potential and possible
Induced pluripotent stem cell utilities of induced pluripotent stem cells, reprogramming from adult fibroblasts with ectopic expression
Regenerative medicine of four embryonic genes, are also reviewed and further discussed. An understanding of the biophysiology
of stem cells could help us elucidate the pathogenicity and develop new treatments for neurodegener-
ative disorders. In contrast to cell transplantation therapies, the application of stem cells can further
provide a platform for drug discovery and small molecular testing, including Chinese herbal medicines.
In addition, the high-throughput stem cell-based systems can be used to elucidate the mechanisms of
neuroprotective candidates in translation medical research for neurodegenerative diseases.
Copyright Ó 2010, Asia Pacific League of Clinical Gerontology & Geriatrics. Published by Elsevier Taiwan
LLC. Open access under CC BY-NC-ND license.

1. Introduction of cells.1 ES cells are pluripotent stem cells derived from the inner cell
mass of mammalian blastocysts. They have remarkable abilities to
Stem cells are classified into three types according to their abilities proliferate indefinitely under appropriate in vitro culture system and
to differentiate. The first type is totipotent stem cells, which can be to differentiate into any cell types of all three germ layers.2,3 Since
implanted in the uterus of a living animal and give rise to a full isolation of human ES in 1998, ES cells have been regarded as
organism. The second type is pluripotent stem cells, such as embry- a powerful platform or tool for developmental studies, drug scre-
onic stem (ES) cells and induced pluripotent stem (iPS) cells. They can ening, diseases treatment, tissue repair engineering, and regenera-
give rise to every cell of an organism except extraembryonic tissues, tive medicine. However, two main limitations have impeded the
such as placenta. This limitation restricts pluripotent stem cells from application of ES cell-based therapy. First, ethical dilemma regarding
developing into a full organism. The third type is multipotent stem the human embryo donation and destruction. Second, ES cells are
cells. They are adult stem cells, which only generate specific lineages incompatible with the immune system of patients. To circumvent
these deficiencies, scientists worldwide have devoted to developing
* Corresponding author. Department of Medical Research and Education, Taipei a variety of reprogramming techniques to reverse somatic cells into
Veterans General Hospital, #201, Section 2, Shih-Pai Road, Taipei 112, Taiwan, a stem cell-like state.4 In 2006, Takahashi and Yamanaka5 published
Republic of China.
a landmark discovery that reprogramming of somatic cells back to
E-mail address: [email protected] (S.-H. Chiou).
y
Authors who contributed equally to this work. iPS cells could be achieved by retroviral transduction of four

2210-8335 Copyright Ó 2010, Asia Pacific League of Clinical Gerontology & Geriatrics. Published by Elsevier Taiwan LLC. Open access under CC BY-NC-ND license.
doi:10.1016/j.jcgg.2010.12.003
2 Y.-C. Chen et al. / Journal of Clinical Gerontology & Geriatrics 2 (2011) 1e6

pluripotency-associated transcription factorsdOct3/4, Sox2, c-Myc, reactivation. Another novel reprogramming technique using pig-
and Klf4. These iPS cells possessed morphological and molecular gyBac transposon was published.9,10,27 A polycistronic plasmid
features that resemble those of ES cells, as well as gave rise to tera- harboring four factors and piggyBac transposon was constructed
toma and germline-competent chimeras on injection into blasto- and integrated into the genome in the presence of piggyBac trans-
cysts. This amazing finding showed that cell fate could be posase. As the reprogramming process achieved, the inserted frag-
manipulated by certain genes and was recently honored by many ment was easily removed by reexpressing transposase. The
awards, including 2009’s Albert Lasker Basic Medical Research Award transposon-based method eliminates the use of virus, displays
and 2010’s International Balzan Prize. Since this astonishing report, equivalent efficiencies to retroviral transduction, excises integrated
iPS cells are now generated by various ways, including kinds of sequences without genome alteration, and therefore represents
exogenous genes delivery methods,6e10 choosing multiple somatic a landmark progress toward therapeutically relevant virus-free iPS
cell sources,11e15 and even by small compounds16 to improve the cells. To avoid introducing exogenous genetic materials, two
efficiency of the reprogramming process. amazing advances were reported. Zhou et al.28 demonstrated that
mouse fibroblasts could be fully reprogrammed by direct delivery of
2. Comparison of iPS cells with ES cells recombinant reprogramming proteins. In 2010, an impressive work
conducted by Warren et al.29 showed a strategy for reprogramming
Generally, fully reprogrammed iPS cells display numerous by administration of synthetic mRNAs that code for key factors and
properties similar to those of ES cells. First of all, iPS cells are created RNA-iPS cells. Both techniques are safer, simpler, and faster
morphologically identical to ES cells and show infinite proliferation approaches than the currently established genetic method.
and self-renewal abilities. Several molecular and functional assays
were set to evaluate the similarity of iPS cells to ES cells, including 4. Reactive oxygen species and stem cells differentiation
reactivation of self-renewal and pluripotency-associated genes,
telomerase activity, X chromosome, and stage-specific embryonic High efficiency of iPS cells reprogramming/differentiation is
surface antigens, suppression of somatic genes associated with cell required in clinical application. Many studies have reported that
of origin, silencing of exogenous factors, capabilities of in vitro reactive oxygen species (ROS) play a critical role in mediating iPS
differentiation, demethylation of promoters of pluripotency genes, cells or stem cells reprogramming/differentiation.30,31 Intracellular
and in vivo teratoma formation, chimera contribution, germline ROS serves as a second messenger in signaling transduction path-
transmission, and tetraploid complementation.7,17 A recent study ways. They are produced in vascular cells by a number of oxidases,
demonstrated that patient-specific iPS cells from dermal fibroblasts such as the NADPH oxidases and xanthine oxidase, lipoxygenases,
of patients with long QT syndrome can differentiate into functional cytochrome p450, and uncoupling of the mitochondrial respiratory
cardiac myocytes but still recapitulated the electrophysiological chain.32 iPS cells have similar function in stress defense mecha-
features of the disorder.18 Therefore, the major advantage of iPS nisms and mitochondrial regulation with human ES cells.33 Fran-
cells over ES cells is that iPS cells can be derived from a patient’s cisco et al.34 had revealed that high glucose promoted stem cell
own somatic cells, thereby avoiding immune rejection after trans- differentiation into cardiomyocyte by activating NADPH oxidase as
plantation and the ethical concerns raised by using ES cells. well as increasing intracellular ROS level. Ji et al.35 had reported
ROS-enhanced stem cell differentiation via mediating extracellular
3. Advances in reprogramming techniques signal-regulated kinase/c-Jun N-terminal kinase, P38 mitogen-
activated protein kinase, and protein kinase B. Furthermore, Varum
Based on their pluripotent capability of differentiating into any et al.36 had shown that attenuating the mitochondrial respiratory
functional cell type, iPS cells possess great potential for regenera- chain can increase pluripotency in human ES cells by facilitating
tive and therapeutic applications. However, the group led by Dr. intracellular ROS generation. Moreover, generation of ROS and the
Yamanaka also reported that these chimeras derived from mouse activities of antioxidant enzymes must be mainly manipulated to
iPS cells and their progeny often develop tumors mainly because of preserve the homeostasis of the intracellular redox status. Intra-
reactivation of c-Myc transgene.19 Thus, numerous approaches to cellular antioxidant enzymes, such as superoxide dismutase (SOD),
generate iPS cells with lower tumorigenicity have been established. catalase, and glutathione peroxidase, play an important role to
Several studies have shown that iPS cells generated without c-Myc mitigate oxidative stress, such as SODs protect against superoxide-
virus demonstrated reduced tumor incidence in chimeric mouse, mediated cytotoxicity by catalyzing O2 to form H2O2. SOD is inac-
but the efficiency of iPS creation is significantly reduced.20,21 To tivated by H2O2 formed by repressing of the superoxide anion.37
overcome this dilemma, Nakagawa et al.22 found another member Not only ROS level is activated but also intracellular antioxidant
of Myc, L-Myc, which possessed stronger activity to generate iPS enzymes are mediated during differentiation. Chen et al.38 had
cells and less tumorigenic activity. validated that intracellular antioxidant enzymes, mitochondrial
The use of genome-integrating retroviruses that are closely mass, as well as oxygen consumption rate were increased during
related with tumor formation was another major limitation of the differentiation in human mesenchymal stem cells.
original iPS cell generation techniques. Thus, reprogramming
strategies with nonintegrating systems seems to be solutions to 5. Clinical application of iPS cells
make iPS-based therapy feasible. In 2008, Stadtfeld et al.23 estab-
lished mouse iPS cells from fibroblasts and liver cells by non- 5.1. iPS cells in the diseases of central nervous system
integrating adenoviruses carrying four defined factors, suggesting
that insertional mutagenesis is not required for in vitro reprogram- The development of stem cell studies makes cell transplantation
ming. At the same time, Okita et al.24 successfully generated iPS cells a promising therapy for the diseases of central nervous system,
by transient transfection of two plasmids containing cDNAs including stroke, traumatic brain injury, hypoxic encephalopathy,
encoding four factors, eliminating transgenic integration by the use and degenerative disorders.39 Parkinson’s disease (PD) is the best
of retroviruses. More recently, Somers et al.25 and Carey et al.26 candidate for the cell replacement therapy because only one group
individually described a “stem cell cassette” or a polycistronic of cells are affected, which are dopaminergic neurons. The main
virus, a single lentiviral vector composed of all four factors, was able pathology of PD is cellular loss of the substantia nigra pars com-
to yield iPS with reduced insertional mutagenesis and viral pacta dopaminergic neurons that project to the striatum.40 Clinical
 Y.-C. Chen et al. / Journal of Clinical Gerontology & Geriatrics 2 (2011) 1e6 3

signs of PD, which include rest tremor, rigidity, and bradykinesia, Enhancement of effectiveness and eliminating adverse effects of
are evident when about 80% of striatal and 50% of nigral neurons this cell transplantation therapy required more extensive studies.
are lost.41 The first attempt of cell replacement therapy was to use
fetal mesencephalic tissue, and the results were successful in the 5.2. iPS cells in cardiovascular diseases
earliest reports.39,42,43 However, adverse effects and limitations
were revealed in the following studies, which included off-medi- In the aging population of a modern world, cardiovascular
cation dyskinesia,44e46 graft-induced inflammatory responses,47 diseases are major medical problems because they usually cause
and limited tissue availability.39 morbidity and mortality.1 The treatments of cardiovascular diseases
Graft-induced dyskinesia may be caused by unfavorable compo- include medication, surgical intervention, rehabilitation, exercise
sition of the fetal mesencephalic grafts. The fetal mesencephalic programs, and transplantation.61 There are several side effects,
tissue includes not only dopaminergic but also nondopaminergic complications, and limitations of transplantation therapy, such as
neurons.39 The exclusion of serotonin and g-aminobutyric acid immunological reaction, infection, and limited availability.62 A new
neurons and enrichment of substantia nigra dopaminergic neurons hope in cardiovascular regenerative medicine has been revealed
will decrease the occurrence of dyskinesia.47 Stem cells are ideal cell since Doetschman et al.63 successfully induced mouse ES cells
sources to achieve this goal. Recent evidence has shown that dopa- differentiating into cardiomyocytes in vitro in 1985. Many studies
minergic neurons derived from ES cells and bone marrow-derived had reported facilitated differentiation from ES cells or iPS cells into
neural progenitors are functional when grafted into parkinsonian cardiomyocytes, endothelial vascular cells, and smooth muscle
rats.48,49 Several methods are able to improve the effectiveness of cells.64,65 In animal models, cardiovascular regeneration therapy
midbrain dopaminergic neuron generation from stem cells, including markedly attenuated ventricular wall thinning as well as enhanced
manipulating transcription factor (e.g., Nurr1, Pitx3, or Lmx1a), contractility of cardiomyocytes postligation of the left anterior
coculture with astrocytes, and using fluorescence-activated cell descending artery,66 restored the function of heart and electric
sorting.47 The ability of deriving large quantities of correctly differ- stability after myocardial infarction,67 and enriched the formation
entiated dopamine neurons makes stem cells a good cell sources for of small capillaries and venules.68
transplantation in PD.
Cell replacement therapy is more complicated for stroke, brain 5.3. iPS cells in lung diseases
injury, and other degenerative diseases, such as Alzheimer’s
disease. The difficulties are because of variable cell types involved, Acute lung injury (ALI) is characterized by neutrophil accumu-
which include neurons, astrocytes, oligodendrocytes, and endo- lation in the lungs, interstitial edema, disruption of epithelial
thelial cells of blood vessels.50 ES cells have been demonstrated to integrity, and leakage of proteins into the alveolar space.69e72
have good developmental potential and significant survival rate Infection, associated with endotoxemia and blood loss are
after transplantation into the brain.51 Transplantation of ES cells frequent predisposing factors to the development of ALI69; and in
also recovered behavioral dysfunction induced by middle cerebral experimental settings, endotoxemia produces ALI. Neutrophils play
arterial occlusion in an animal model.52 However, the ethical a central role in this acute pulmonary inflammatory process as their
consideration, the limited availability, and the possibility of elimination can prevent the development of ALI.73 The neutrophils
immune rejection after transplantation restrict the accessibility of present in the lungs during ALI produce inflammatory mediators,
ES cells. including cytokines, such as interleukin-6 and macrophage
Because iPS cells are derived from the somatic cells, potential inflammatory peptide-2, and demonstrate increased activation of
immune rejection and ethical consideration can be avoided. transcriptional regulatory factors, including nuclear factor-kB
Recently, Wernig et al.53 demonstrated that neurons and glial cells (NF-kB).73e76
could be derived from iPS cells in vitro, and that transplantation of Binding elements for NF-kB are present in the enhancer/promoter
iPS cell-derived neurons into brain was able to improve behavior in regions of cytokine genes, such as interleukin-1b, macrophage
a rat model of PD. We also demonstrated an efficient method to inflammatory peptide-2, and tumor necrosis factor-a, as well as other
differentiate iPS cells into astrocyte-like and neuron-like cells, important immunoregulatory molecules, such as intercellular
which displayed functional electrophysiological properties. Our in adhesion molecule-1 and complement C4 protein.77 Inhibition of NF-
vivo study showed that direct injection of iPS cells into damaged kB activation prevents endotoxin-induced increases in proinfla-
areas of rat cortex significantly decreased the infarct size, improved mmatory cytokine expression in the lungs.76
the motor function, attenuated inflammatory cytokines, and iPS cell administration improved the impairment of pulmonary
mediated neuroprotection after middle cerebral artery occlusion. function in endotoxin-induced ALI, including airway resistance
Subdural injection of iPS cells with fibrin glue was as effective as the (enhanced pause), lung tidal volumes, and arterial partial oxygen
direct-injection method and provided a safer choice for cell pressure levels. Hypoxemia is the major symptom and sign of ALI,
replacement therapy.54 no matter whether in the mice model or in human cases. The effect
Teratoma or tumor formation is a major adverse effect of cell of iPS cell treatment was to rescue the hypoxemia, similar to
transplantation using ES or iPS cells.55 One of the methods to another study using a therapeutic agent in an animal model of lung
prevent teratoma/tumor formation is elimination of nonneural injury.78 A recent study found that transplantation of human ES
progenitors, which can be achieved by the elaboration of differ- cells abrogated bleomycin-induced lung injury in mice and restored
entiation protocols that allow maximal homogeneity of the trans- blood arterial oxygen saturation and lung tidal volume.79 Our study
plant56 or by cell sorting before transplantation.57 Exclusion of showed that the intravenous injection of iPS cells led to recovery of
poorly differentiated ES or iPS cells can also reduce the rate of the impairment of both airway resistance and lung tidal volume
teratoma or tumor formation.58 Some antioxidants may prevent induced by the instillation of endotoxin intratracheally. In a pre-
tumorigenesis after cell transplantation. Resveratrol, a natural vious mice model of early ALI, most changes in bronchoalveolar
polyphenol antioxidant, is demonstrated that it can inhibit tera- lavage suggestive of acute pulmonary irritation were compatible
toma formation in vivo.59 Our recent study also found that doco- with the changes in pulmonary function, such as airway resistance
sahexaenoic acid can inhibit teratoma formation in addition to (enhanced pause) and tidal volume.80 Thus, iPS cell therapy not
promoting dopaminergic differentiation in iPS cells in PD-like only abolished endotoxin-induced lung injury in mice but also
rats.60 It has been only two years since the development of iPS cells. improved the changes in pulmonary physiological function. This
4 Y.-C. Chen et al. / Journal of Clinical Gerontology & Geriatrics 2 (2011) 1e6

novel cellular therapy opened an era of cell-based transplantation number of iPS cells can be prepared in vitro. To date, iPS-derived
by overcoming the immune rejection and the ethical controversy strategies have been applied to four disease models, sickle cell
over the use of ES cells and mesenchymal stem cells. anemia, PD, hemophilia A, and acute myocardial infarction. How-
ever, there still exist several questions to be answered, such as what
5.4. iPS in liver diseases are the detailed molecular mechanisms of reprogramming? Can iPS
cells be generated solely by chemical compounds like epigenetic
Liver diseases and liver injuries are common health problems modifier without DNA transduction? How to improve the yield of
throughout the world. The loss of functional liver tissue after injury iPS? In addition, to provide replacement cells for therapy, a new cell
will activate a wound healing process aimed to repair and restore by lineage switching or direct conversion from a normal somatic
the integrity of the injured liver. Intense or uncontrollable insults cell should also be considered.96 In conclusion, the iPS techniques
could efface the healing response and result in end-stage liver open a new era for stem cell research and offer promising oppor-
disease, which is irreversibly associated with liver failure. Curr- tunities for patient-specific pluripotent cell-based regenerative
ently, orthotopic liver transplantation is the most effective therapy medicine.
for acute and chronic liver failure. However, it is limited by shortage
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