1. www.biochemj.org
Biochem. J. (2010) 428, 11–23 (Printed in Great Britain)
11
doi:10.1042/BJ20100102
REVIEW ARTICLE
Emerging use of stem cells in regenerative medicine
Adrian K. K. TEO*† and Ludovic VALLIER*1
Stem cells represent a unique opportunity for regenerative
medicine to cure a broad number of diseases for which current
treatment only alleviates symptoms or retards further disease
progression. However, the number of stem cells available has
speedily increased these past 10 years and their diversity presents
new challenges to clinicians and basic scientists who intend to use
them in clinics or to study their unique properties. In addition, the
recent possibility to derive pluripotent stem cells from somatic
cells using epigenetic reprogramming has further increased the
clinical interest of stem cells since induced pluripotent stem cells
could render personalized cell-based therapy possible. The present
review will attempt to summarize the advantages and challenges
of each type of stem cell for current and future clinical applications
using specific examples.
INTRODUCTION
infarction, and genetic diseases like myopathy, where a specific
subtype of cells are not functional. The number of diseases that
could be cured using stem cells is extremely broad. Thus the
present review will only focus on specific examples of current
applications which illustrate the recent progress towards the use
of stem cells in clinics.
Human stem cells can be classified into three broad categories in
accordance with their origin and their capacity of differentiation.
Somatic stem cells are multipotent stem cells which originate from
differentiated organs (adult or foetal). They can reside specifically
in in vivo niches (endogenous somatic stem cells) and sometimes
can be isolated and expanded in vitro (exogenous somatic stem
cells). The third category (pluripotent stem cells) includes ESCs
which are derived from embryos at the blastocyst stage and
hiPSCs [human iPSCs (induced pluripotent stem cells)] which
are generated from reprogrammed somatic cells. In the present
review, we will examine the properties of each stem cell category
and discuss their advantages for clinical application by describing
relevant examples. In addition, we will define their drawbacks and
the necessary progress for their future clinical applications.
During the past decade, stem cells have become a major focus for
translational medicine. These cell types combine two properties
that make them uniquely attractive for regenerative medicine. A
stem cell is defined as a cell type which has the property to selfrenew while maintaining the capacity to differentiate into diverse
cell types. Stem cells are therefore different from progenitor cells
which can differentiate into mature cell types but are incapable of
self-renewing, or somatic cells which are capable of proliferating
but unable to differentiate. By combining these properties of selfrenewal and differentiation, stem cells could allow the production
of an infinite quantity of cell types with a clinical interest. Stem
cells are also becoming increasingly attractive for basic research
since they can be expanded in vitro while maintaining their native
properties, allowing basic studies which are impossible with
primary tissue cultures or biopsy material. Consequently, specific
stem cells such as ESCs (embryonic stem cells) have become
a reference model for understanding key molecular mechanisms
which control cell fate choice and organ differentiation.
Importantly, stem cells are already present in clinics especially
HSCs (haematopoietic stem cells), which have been successfully
used for more than 40 years in bone marrow transplantations to
treat diverse blood disorders such as leukaemia [1]. Future clinical
applications of stem cells concern a broad number of degenerative
diseases (i.e. disease in which one cell type or part of an organ
fails) which could potentially be treated using stem-cell-based
therapy. This includes major metabolic diseases such as T1D
(Type 1 diabetes), caused by the destruction of insulin-secreting
β-cells [2], diverse brain and myelin disorders in which specific
neural cells are targeted such as MS (multiple sclerosis), PD
(Parkinson’s disease) or HD (Huntington’s disease), heart disease,
where some cardiac cells need to be replaced upon myocardial
Key words: cell replacement therapy, human, regenerative
medicine, stem cell.
TOTIPOTENCE, PLURIPOTENCE AND MULTIPOTENCE
The clinical use of a specific type of stem cell is ultimately defined
by its capacity to generate a broad number of cell types. This
capacity of differentiation can be divided into three categories.
Totipotent stem cells are capable of differentiating into all adult
and embryonic tissues, including extra-embryonic tissues such
as trophectoderm. Embryos at the one and two cell stages and
GCs (germ cells) are likely to be the only cells which display this
capacity naturally in an embryonic environment [3]. So far, there is
no known stem cell in vitro which has no restriction in its capacity
of differentiation. The major issue here is to define the assays and
Abbreviations used: AFP, α foetal protein; ALB, albumin; AT, adipose tissue; BM, bone marrow; CK, cytokeratin; DE, definitive endoderm; EGF, epidermal
growth factor; ESC, embryonic stem cell; FD, familial dysautonomia; FGF, fibroblast growth factor; GC, germ cell; GvHD, graft versus host disease; hESC,
human ESC; HNF, hepatocyte nuclear factor; HSC, haematopoietic stem cell; IGF1, insulin-like growth factor 1; iPSC, induced pluripotent stem cell; hiPSC,
human iPSC; MAPC, multipotent adult progenitor cell; mESC, mouse ESC; MSC, mesenchymal stromal cell; NSC, neural stem cell; OPC, oligodendrocyte
progenitor cell; PD, Parkinson’s disease; SCF, stem cell factor; SCNT, somatic cell nuclear transfer; SGL, subgranular layer; SMA, spinal muscular atrophy;
SVZ, subventricular zone; T1D, Type 1 diabetes; TGF, transforming growth factor; UCB, umbilical cord blood.
1
To whom correspondence should be addressed (email lv225@cam.ac.uk).
c The Authors Journal compilation c 2010 Biochemical Society
Biochemical Journal
*Laboratory for Regenerative Medicine, University of Cambridge, West Forvie Building, Forvie Site, Robinson Way, Cambridge CB2 0SZ, U.K., and †Institute of Medical Biology,
A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648
2. 12
A. K. K. Teo and L. Vallier
Table 1 Types of pluripotent stem cells derived from embryos and the
germline
mEpiSC, mouse epiblast stem cell; m(h)EGC, mouse (human) embryonic GC; mGSC, multipotent
germline stem cell; PGC, primordial GC.
Type of pluripotent stem cell
Source of derivation
Reference
mESC
Primate ESC
hESC
Pre-implantation embryo
Pre-implantation embryo
Pre-implantation embryo
Morula and late blastocyst stage embryo
Single blastomere
Parthenogenetic embryo
Post-implantation embryo
PGC
Neonatal and adult mouse testis
Cultured human PGCs
[5,6]
[176,177]
[111,178]
[179,180]
[181]
[182–184]
[185,186]
[187,188]
[189,190]
[191]
mEpiSC
mEGC
mGSC
hEGC
standards that would allow the definition of totipotency of human
stem cells in vitro. Obvious ethical considerations preclude the
use of chimaera formation and germline transmission in humans
(see below) and the development of in vitro assays to demonstrate
that one cell type can differentiate into all the cell types existing
in the human body will be extremely challenging.
Pluripotent stem cells are capable of differentiating into the
derivatives of the three germ layers (ectoderm, mesoderm and
endoderm) and GCs. However, they are not totipotent as they
cannot differentiate into certain cell types especially extraembryonic tissues such as trophectoderm. Pluripotent stem cells
have been derived from embryos at different developmental
stages and from the adult germline [4] (Table 1). Pluripotency
is currently defined by specific assays. The most commonly used
assay in non-human species is germline transmission. In this case,
pluripotent stem cells are injected into embryos at the blastocyst
stage and the resulting chimaeric embryo is re-transplanted into
a pseudo-pregnant female to continue its development. The
offspring is chimaeric in all the tissues, including the germline,
which is validated by the transmission of the genetic background
originating from the injected stem cells to the next generation.
The best example of pluripotent stem cells fulfilling this criteria
remains to be mESCs (mouse ESCs) [5,6], which can differentiate
into various tissues in vitro and can contribute to the germline
in vivo after blastocyst injection. However, mESCs are unable
to differentiate into trophectoderm in the absence of genetic
modifications and thus are defined as pluripotent stem cells.
Concerning hESCs (human ESCs), their pluripotent state has
been demonstrated by a broad number of studies showing that
they can differentiate into a large number of cell types including
the trophectoderm and germ cells [7,8]. However, germline
transmission assays are unfeasible for obvious ethical reasons
and pluripotency in human cells is usually demonstrated by their
capacity to form teratomas. Indeed, pluripotent stem cells can
form teratomas containing derivatives of the three germ layers
once injected into an immunodeficient host. However, this assay
is not quantitative and relies on histological analyses. Therefore
the lack of proper standards to define pluripotency in human stem
cells remains an issue which could explain the current confusion
regarding the pluripotent status of somatic stem cells such as
MAPCs (multipotent adult progenitor cells) [9,10].
Multipotent stem cells are able to differentiate into all the cell
types constituting an organ. The most prominent example remains
HSCs, which are capable of differentiating into all the cell types of
the haematopoietic system. Two types of assays are currently
used to demonstrate the multipotent state of stem cells. The first
is to demonstrate that one stem cell can reconstitute an
c The Authors Journal compilation c 2010 Biochemical Society
entire organ. For instance, HSCs can reconstitute an entire
haematopoietic system after transplantation into sub-lethally
irradiated animals. However, this type of assay is difficult to
perform in humans since they require the destruction of the
targeted organ and also require clonal analysis which can be
technically challenging. Indeed, adult stem cells often require
a complex environment to survive and to self-renew in, and this
niche can be difficult to mimic in vitro. The second type of assay
is based on lineage tracing experiments in which the stem cell
of interest is genetically tagged and hence marks its progeny.
Thus, using this approach, one can follow the origin of a cell
type in a specific organ. The existence of gut stem cells [11] and
skin stem cells [12] has been characterized using this approach.
Multipotent stem cells have been identified in a broad number
of organs in rodents. However, the existence and functionality
of their human counterpart remains in some cases controversial
since the lack of appropriate technology renders their isolation
and thus characterization extremely challenging.
SOMATIC STEM CELLS
Endogenous somatic stem cells
Somatic stem cells are multipotent and can usually differentiate
into all the cell types constituting their organ of origin. Some
somatic stem cells such as hepatic progenitor cells proliferate
very slowly and can be activated by specific conditions such
as stress response or injury [13]. Other somatic stem cells
such as vascular progenitor cells are proliferating constantly
and are in high numbers [14]. They are often localized in a
specific niche that is essential to maintain their property of
self-renewal and differentiation [15]. Ultimately, their normal
function could be to maintain a constant number of cells in
an organ. Somatic stem cells have been reported to be found
in the brain [16,17], haematopoietic system, skin [18,19], heart
[20,21], muscle [22,23], lung [24,25], intestine [26] and liver
[27,28] (Figure 1 and Table 2). They represent the ideal target for
regenerative medicine since their mobilization would not require
invasive methods or immunosuppressive treatments.
For instance, a broad number of studies have focused on
activating endogenous NSCs (neural stem cells) to replace missing
neural cells [29,30]. The use of FGF2 (fibroblast growth factor 2),
EGF (epidermal growth factor) [31], TGFα (transforming growth
factor α) [32], VEGF (vascular endothelial growth factor) [33,34],
SCF (stem cell factor) [35], glucocorticoids and IGF1 (insulin-like
growth factor 1) [36] could possibly activate neuronal replacement
in neurodegenerative diseases and also in stroke injury. Another
example would be the activation of in vivo muscle repair using
growth factors [37]. IGF1 has been strongly linked to promote
cellular recruitment and muscular repair [38]. Together with the
inhibition of the NF-κB (nuclear factor κB) pathway [39] and
modulation of TNFα (tumour necrosis factor α) function [40],
muscular atrophy could be prevented [41].
However, the clinical use of endogenous somatic stem cells
is currently limited for diverse reasons. Their existence remains
controversial in several organs especially in humans where lineage
tracing or reconstitution experiments are impossible. Therefore
their identification and characterization is difficult and past
experience has shown that results obtained in animal models might
not be translated directly to humans. This limits the study of the
mechanisms controlling the self-renewal and differentiation of
endogenous stem cells, which is necessary for the development
of future clinical treatments. Two controversial examples concern
the pancreas and liver. Using lineage tracing techniques in
the mouse, it has been demonstrated that pancreatic β-cells
3. Emerging use of stem cells in regenerative medicine
Table 2
13
Types of adult stem cells reportedly found
Type of adult stem cell
Location found
Expandable in vitro ?
Used in clinical trials?
HSC
MSC
Muscle satellite stem cell
NSC
Limbal stem cell
Skin stem cell
Gut stem cell
Lung stem cell
Cardiac stem cell
Liver stem cell
Pancreatic stem cell
BM, placenta, UCB and peripheral blood
BM, amniotic fluid, placenta, AT and UCB
Between basement membrane and cell membrane (sarcolemma) of muscle fibre
SVZ of lateral ventricles and SGL of dentate gyrus of the hippocampus in the brain
Corneoscleral limbus
Basal layer of epidermis and base of hair follicles
Crypts
Between airways (bronchioles) and alveoli
Heart
Canals of Hering at the terminal bile ductules
Pancreatic ducts
Yes
Yes
No
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
Yes
Going to start
Going to start
No
No
No
No
No
No
Figure 1
Endogenous and exogenous somatic stem cells for cell-based therapy
Endogenous adult stem cells (SC) can be activated with the use of drugs or growth factors to prompt regeneration in tissues or organs in vivo . Exogenous adult stem cells can be expanded in ex
vivo cultures and then transplanted into the human body for cell replacement or to stimulate regeneration via paracrine actions. Stem cells which remain to be fully characterized are indicated by (?).
‘regenerate’ via self-replication of existing β-cells and not from
stem cells [42,43]. However, recent reports have contradicted
these observations by showing that β-cells can be made de
novo from islet progenitor cells expressing Ngn3 and residing
in the pancreatic duct [44]. Nevertheless, most of these results
were obtained in animal models of injury in which the pancreas
is induced to regenerate after duct ligation, and recent studies
have not been able to repeat these experiments [45]. Therefore
it remains to be demonstrated that this model is physiologically
relevant and that a pool of pancreatic stem cells can be identified
in normal conditions, especially in humans.
Hepatic oval cells/hepatic progenitors represent another
controversial adult stem cell [46,47]. These liver-specific stem
cells originally identified in rodents could be used to make
hepatocytes for cell-based therapy of liver failure and liver
metabolic diseases [48–50]. They are bipotent (i.e. they are
capable to differentiate into duct cells or hepatocytes) as shown
by the expression of bile duct markers [CK (cytokeratin)-7,
CK-19 and OV-6], and hepatocyte markers [AFP (α foetal protein)
and ALB (albumin)]. They also express HNFs (hepatocyte nuclear
factors) which mark hepatic progenitors during embryonic liver
development [51]. In humans, they are localized in the canals of
Hering at the terminal bile ductules [52] and are reportedly activated in disease conditions [53,54]. However, they are extremely
rare (if in existence) and possibly inaccessible [52]. There have
been reports of the isolation of hepatic progenitor cells from the
adult human liver [55–57]. However, the lack of definitive markers
and the dissimilarity between human and rodents renders the
identification of a common hepatic progenitor cell difficult [58].
These two examples illustrate the difficulty of translating
observations obtained in animal models directly to humans [59–
61]. Indeed, stem cells in distant species might have different
functions and activity [62,63]. Another major drawback is
that endogenous somatic stem cells could be targeted by the
diseases, explaining why endogenous somatic stem cells appear
to be incapable or insufficient in stopping the progression of
c The Authors Journal compilation c 2010 Biochemical Society
4. 14
A. K. K. Teo and L. Vallier
degenerative diseases. Finally, the use of endogenous stem
cells could be precluded in genetic diseases whereby stem cells
continue to produce progeny carrying the same phenotype.
Consequently, genetic correction will be necessary, implicating
the use of gene therapy approaches or complex manipulations
in vitro. In conclusion, mobilization of endogenous somatic stem
cells represents the ultimate objective of regenerative medicine.
This approach is the most ideal, ‘simple’ and natural as we
are simply prompting and activating our innate body system to
repair diseased tissues. There would not be complicated problems
such as immunological response to exogenous foreign cells or
ethical issues related to ESCs. However, their clinical use will
require better identification and characterization to develop new
approaches allowing their mobilization in vivo, especially in a
diseased environment.
Exogenous somatic stem cells
Exogenous somatic stem cells are derived in vitro from diverse
organs. They have a high capacity of proliferation and can be
maintained in vitro for a prolonged period of time. Exogenous
somatic stem cells are commonly recognized to exhibit lineagerestricted multipotency. However, several reports have suggested
that somatic stem cells could be pluripotent since they can
differentiate into a broad number of cell types unrelated to
their organ of origin [64]. In addition, controversial reports
have suggested that these cells could participate in germ layer
formation [64]. However, these results have been difficult to
replicate and the current consensus is that adult somatic stem
cells have a restricted capacity of differentiation compared with
those of ESCs. Furthermore, exogenous somatic stem cells often
represent an in vitro state which has no equivalence in vivo.
However, their use in regenerative medicine is promising since
they are easy to isolate and can be expanded in vitro. In addition,
immunosuppressive treatments are not required due to autologous
transplantations, making them ideal candidates for clinical trials.
The two best examples are NSCs [65,66] and multipotent MSCs
(mesenchymal stromal cells) [67,68] which are currently being
used in several clinical trials.
NSCs are progenitor cells located in the SVZ (subventricular
zone) of the lateral ventricles or SGL (subgranular layer) of the
dentate gyrus of the hippocampus in the brain. NSCs have been
cultured in vitro as cell clumps known as ‘neurospheres’ [69,70].
The neurospheres can be passaged several times, demonstrating
their capacity of self-renewal. They are also multipotent since
they can differentiate into neurons and glia [71,72]. Therefore
NSCs represent a unique source of cells for clinical application,
and several clinical trials are currently testing their efficiency
[73]. One of them is conducted by StemCells Inc. (San Francisco,
CA, U.S.A.) and its objective is to use NSCs for the treatment
of Batten’s disease, a lysosomal storage disorder. In addition,
several trials have used neuronal cells for stroke patients [74,75].
ReNeuron (Guildford, U.K.) is currently proposing to transplant
myc-immortalized hNSCs (human NSCs) [76] derived from
human foetal tissue into stroke patients. Therefore the first clinical
applications of NSCs have started. However, several challenges
have to be solved before larger trials could start, including better
isolation and expansion methods for NSCs, as well as prevention
of tumour formation after transplantation [77].
MSCs are fibroblast-like cells which can be isolated from
diverse sources including BM (bone marrow) [78], amniotic
fluid [79], placenta [80,81], AT (adipose tissue) [82,83] and
UCB (umbilical cord blood) [84]. MSCs are mainly identified
by their adherent property, differentiation potential, and cellsurface markers such as CD73, CD90 and CD105 [67,85,86].
c The Authors Journal compilation c 2010 Biochemical Society
However, their capacity of differentiation in vivo and in vitro
is limited as they are not pluripotent. Besides MSCs, several
related types of cells including MAPCs were isolated from the
BM [9,10]. MSCs are multipotent [87] and have been reported
to be able to differentiate into osteoblasts, adipocytes [88],
chondrocytes [89,90], and epithelial [91], endothelial [92] and
neural cells [93]. It should be noted that MSCs derived from
different tissues may exhibit slightly different propensities and
capacities to differentiate into specific cell types. For example,
MSCs from synovial tissue may be more effective in repairing
cartilage defects than MSCs from other tissues [94]. MSCs selfrenew in vitro but not indefinitely since they do senesce after
20 population doublings and ultimately lose their potential to
differentiate [95]. More importantly, they do tend to generate
abnormal karyotypes in vitro as their genomes are highly unstable
[96]. This could represent a major limitation for their use in cellbased therapy.
Nonetheless, the interest in MSCs for clinical applications
resides less in their capacity of differentiation than in their
paracrine actions on tissue repair [97]. Indeed, MSCs express
low levels of MHC class II molecules and thus should be weakly
immunogenic, suggesting that immunosuppressive treatment
might not be required. In addition, they have been shown to be
able to modulate the immune response of cells in vitro and to
limit inflammation [98]. Hence they could be used for preventing
graft rejections and GvHD (graft versus host disease). MSCs
were first used in clinical trials as a complementary treatment for
breast cancer patients [99]. Thereafter they were demonstrated
further to be beneficial against GvHD [100]. On the basis of
these successes, a broad number of studies are currently being
performed to define their efficacy to treat Crohn’s disease [101],
ALS (amyotrophic lateral sclerosis) [102] and heart diseases
[103]. In particular for heart therapy, autologous BM-MSCs have
been used in controlled clinical trials [104]. Importantly, MSCs do
not repopulate damaged tissues and cells quickly disappear after
engraftment in vivo. Despite this, small but positive effects on
cardiac performance have been observed, and this clinical benefit
could be achieved through immune modulation and increased
angiogenesis.
MSCs could also potentially be used to treat liver diseases.
BM-MSCs apparently are able to differentiate into hepatocytelike cells without cell fusion [105,106]. UCB-MSCs [107] and
AT-MSCs [108] can also be differentiated into hepatocyte-like
cells. hMSC (human MSC)-derived hepatocytes have recently
been reported to be able to restore liver function [109]. MSCs are
now being used in clinical trials for end-stage liver disease [110]
and initial results appear promising. However, long-term recovery
of liver function remains to be demonstrated and further studies
will be required to understand the mechanisms by which MSCs
can restore liver function. In summary, the mechanisms by which
MSCs can achieve their immunomodulatory and detoxification
functions, as well as paracrine secretions, remain to be defined.
Further basic studies will be extremely helpful in validating their
real function in vivo and the mechanisms by which they can
alleviate so many symptoms. However, their interest for clinical
application cannot be denied and the broad number of clinical
trials which are currently performed with MSCs should rapidly
provide some information regarding their real efficacy and longterm effect.
The examples described above illustrate the advantages
of somatic stem cells for clinical applications. These cells
are relatively easy to isolate and they do not require
immunosuppressive treatment. In addition, they can be used to
treat a broad number of diseases without major risks. All of
these aspects greatly simplify their use in a clinical context
5. Emerging use of stem cells in regenerative medicine
and thus make them attractive for clinical trials. However, an
increasing amount of data are suggesting that even a short
period of time in culture in vitro can modify the genetic and
epigenetic characteristics of somatic stem cells. The consequences
of these modifications on cell proliferation and differentiation
after transplantation and after a prolonged period of time
in vivo will have to be examined carefully. Finally, the mechanisms
involving somatic stem cells in tissue repair and/or in modulating
immune response are not always fully understood and additional
basic studies are required to fully define the clinical potential of
these different cell types.
EMBRYONIC STEM CELLS
hESCs are pluripotent stem cells derived from the inner
cell mass of embryos at the blastocyst stage [111].
Their embryonic origin confers upon them the capacity to
proliferate indefinitely in vitro while maintaining the capacity
to differentiate into derivatives of the three germ layers from which
all adult organs are derived. Therefore hESCs represent a unique
opportunity for regenerative medicine since they could be used
to generate an infinite quantity of cells with a clinical interest.
However, the clinical application of these unique cell types is
currently limited by two challenges: the difficulty to generate
fully functional cell types and also the safety issue associated
with teratoma formation.
Difficulty to generate fully functional cell types from hESCs
A major focus of the past 10 years has been the development
of robust protocols to differentiate hESCs into homogenous
populations of cell types with a clinical interest. Consequently,
a broad number of protocols have been developed to generate
diverse cell types, including retina cells [112,113], dopaminergic
neurons [114], motor neurons [115,116], OPCs (oligodendrocyte
progenitor cells) [117], cardiomyocytes [118,119], pancreatic βcells [120] and hepatocytes [121–124] (Figure 2 and Figure 3).
These methods mark a clear progress towards the generation of
fully functional cell types. They have benefited from the lessons
learnt from mESCs as well as from the knowledge developed
by developmental biology studies. Indeed, past experiences have
shown that following a natural path of development in vitro
remains the best way to generate functional cell types from
pluripotent stem cells of embryonic origin. Therefore most of the
protocols developed to drive differentiation of hESCs respect key
stages of embryonic development. However, this methodology
also implies that generation of fully mature and functional
cells from hESCs will require an extended period of time of
in vitro culture, mimicking a complex environment constantly
changing over time. Therefore such an approach is presented
with unprecendented challenges which would require a strong
collaboration between stem cell biologists and developmental
biologists. Generation of pancreatic β-cells is a good example
to illustrate the progress and challenges regarding the generation
of clinically relevant cells from hESCs.
The Edmonton Protocol has proven that islet transplantation via
the hepatic portal vein can correct insulin deficiency in diabetic
patients [125]. However, the lack of cadaveric donors severely
limits the number of patients that could benefit from this therapy.
Generating pancreatic cells from hESCs represent a unique
opportunity to bypass this limitation. However, the resulting
β-cells would have to synthesize and store insulin, and also
maintain blood glucose in a physiological range when challenged
with sporadic food intake. Past experiences [126,127] have
15
demonstrated that strict adherence to the pancreas developmental
process is essential for the production of pancreatic cells from
ESCs [2,128,129]. Accordingly, a robust method of differentiation
should respect four major stages: (i) formation of DE (definitive
endoderm) [130,131], (ii) formation of PE (pancreatic endoderm),
(iii) formation of endocrine progenitors, and (iv) maturation of
endocrine progenitors into fully functional hormone-secreting
cells. Several methods are now available to efficiently generate
DE cells from hESCs and to further differentiate the resulting
cells into pancreatic progenitors. However, the specification of
these progenitors toward the endocrine pathway as well as the
production of fully mature β-cells remains a challenge.
Indeed, the most advanced protocol for the differentiation
of hESCs into pancreatic β-cells allows the production of
multi-hormonal immature pancreatic endocrine cells which lack
Nkx6.1 and MafA co-expression. Despite this major limitation,
the pancreatic progenitors generated using this approach can
differentiate into fully functional pancreatic cells in vivo after
transplantation into immuno-deficient mice and the resulting
β-cells can re-establish euglycaemia in a mouse model of
diabetes [120]. These results are extremely encouraging since
they provide the first demonstration that stem-cell-based therapy
could be used to treat diabetes. However, partially differentiated
pancreatic progenitors can easily be contaminated by pluripotent
cells which further increase the risk of teratoma formation
associated with hESCs (see below). In addition, non-chemically
defined culture conditions containing FBS (foetal bovine serum)
are still used, increasing the difficulty for such approaches to
be approved for clinical trials. Finally, any cell-based therapy
approach will have to address specific problems associated with
the auto-immune disease involved in T1D. Therefore the results
obtained with hESCs are very promising and they demonstrate
the potential of pluripotent stem cells to develop new treatment
for diabetes. However, more efforts are still required to generate
fully functional β-cells from human pluripotent stem cells and to
demonstrate that pancreatic progenitors can be used safely in a
clinical context.
Importantly, the challenges described above for pancreatic
cells are shared with many other cell types. For example, HSCs
generated from hESCs resemble those of the yolk sac [132,133]
and thus have a limited capacity of repopulation in immunodeficient animals [134]. In addition, hESC-derived hepatocytes
appear to have an embryonic identity since they have limited
capacity to colonize the adult liver and they usually display limited
functionality when compared with their in vivo counterparts [121–
124]. Therefore developing robust protocols of differentiation
remains a major challenge to deliver the clinical promises of
hESCs. However, this issue can be solved by directly using the
progenitors already available as shown with pancreatic cells (see
above) and/or by increasing our understanding of the mechanisms
controlling organ maturation. This last approach is currently used
successfully for a large number of cell types and especially with
neural cells.
Indeed, protocols currently available to generate functional
neurons, retina cells or OPCs have strongly benefited from
knowledge generated by developmental biologists concerning
neural tube patterning and neuronal specification [135,136].
Therefore these protocols generally respect key stages of
development and require extended periods of time of in vitro
culture (between 45 days and up to 3 months) to generate
functional cell types. In addition, the availability of animal
models for specific neurodegenerative diseases and the existence
of robust in vitro functional tests allowed validation of the
cells generated. For example, transplantation of ESC-derived
dopaminergic neurons appeared to restore motor function in
c The Authors Journal compilation c 2010 Biochemical Society
6. 16
Figure 2
A. K. K. Teo and L. Vallier
Human pluripotent stem cells for cell-based therapy
hESCs can be derived from the inner cell mass of the human blastocyst, whereas hiPSCs can be derived via reprogramming of human somatic cells. These human pluripotent stem cells can be
differentiated into clinically useful cell types, such as neural cells, retinal cells, cardiomyocytes, hepatocytes and pancreatic β-cells, and be used for cell replacement therapy.
animal models of PD [114], and studies have shown that
hESCs can be differentiated into retinal pigment cells which
can restore retinal structure and function in animal models of
retinal degeneration [137]. Finally, OPCs generated from hESCs
have been transplanted into animal models of spinal cord injury
and the transplanted cells appear not only to recolonize the
scarred area, but also to promote the recruitment of endogenous
stem cells, which ultimately results in restoration of mobility
[117,138]. These exciting preliminary findings led to the first
hESC-based clinical trial for treatment of spinal cord injuries by
Geron Corporation (Menlo Park, CA, U.S.A.). This clinical trial,
despite being put on hold at the moment, represents an important
step towards clinical applications of human pluripotent stem cells
and the outcome will bring essential information concerning
the feasibility of stem-cell-based therapy for a large number of
diseases.
Safety issue with hESCs
Safety issues continue to be the main limitation for the use
of hESCs in clinics, and teratoma formation represents one
of the most difficult challenges (Figure 4). Indeed, hESCs are
naturally tumourous by definition. hESC lines are considered
to be pluripotent based on the capacity to form teratomas
once injected into an immunodeficient host. Therefore there
is a risk of tumour formation if the transplanted cells are
contaminated by residual pluripotent stem cells. Importantly,
several reports have shown that this risk is increased when
early progenitors are used [120,139] and that methods producing
heterogeneous populations of cells favour contamination by
undifferentiated cells. Hence teratoma formation can easily be
avoided using robust methods of differentiation allowing the
generation of a homogenous population of fully differentiated
cells. A second cause of teratoma formation could be more
c The Authors Journal compilation c 2010 Biochemical Society
problematic. Differentiated cells generated from ESCs could have
the capacity to dedifferentiate into pluripotent stem cells when
grown in a specific environment in vivo. However, there is no
evidence that fully differentiated cells generated from hESCs can
be reprogrammed in vivo or in vitro without overexpression of
pluripotency factors. Systematic studies regarding the occurrence
of teratoma formation after transplantation of hESC-derived
somatic cells could be necessary to completely rule out such a
hypothesis.
Graft overgrowth represents another issue associated with
progenitors generated from hESCs. Indeed, the injection of early
progenitors in the adult environment can cause uncontrolled
proliferation of these progenitors, resulting in a tumour-like
structure incompatible with the function of the transplanted
organ. For example, a recent report has described neuronal
progenitors generated from hESCs to continue proliferating after
transplantation in the brain, leading to abnormal growth [140].
This issue illustrates once again the importance of generating
fully differentiated cell types from hESCs. The issue of tumour
formation associated with hESCs is also heightened by their
relative karyotypic instability [141]. Indeed, hESCs are prone
to acquire chromosomal translocations when grown over a
prolonged period of time in culture. This is a common feature
with any primary cell culture [142], which can be solved by
karyotyping hESCs regularly and by maintaining robust culture
conditions [143]. However, the most robust method currently
available to expand hESCs involves co-culture with feeder cells
and serum, which are not always fully compatible with clinical
applications.
To conclude, the risk of tumour formation by hESCs remains
an issue, but it can be easily controlled by using methods of
differentiation allowing the generation of homogenous population
of fully differentiated cells. Importantly, technical solutions
can be used to guarantee the absence of pluripotent stem
7. Emerging use of stem cells in regenerative medicine
Figure 3
17
An example of an hESC differentiation protocol
Directed differentiation of hESCs to hepatic progenitors using fully defined culture conditions [175]. hESCs were differentiated into endoderm using activin, BMP4 (bone morphogenetic protein 4),
FGF2 and the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Hepatic specification was then initiated with FGF10, RA (retinoic acid) and an inhibitor of the activin/nodal receptor SB431542.
Addition of FGF4, HGF and EGF resulted in maturation of the hepatic progenitors expressing some markers of mature hepatocytes, such as ALB, AFP, AAT (α 1 -antitrypsin) and CK18.
Figure 4
Roadmap from human stem cells to cell replacement therapy
Some selection criteria for the type of human stem cells (SCs) and the methodology of differentiation prior to approval for clinical use. Thereafter the end-products can be used for cell replacement
therapy or for drug testing. FDA, Federal Drug Administration.
c The Authors Journal compilation c 2010 Biochemical Society
8. 18
A. K. K. Teo and L. Vallier
cells in transplanted cells. Methods of purification allowing
transplantation of homogenous populations of differentiated
cells are currently available and are developed by several
laboratories [144–147]. This can be achieved with FACS using
specific cell-surface markers for pluripotent stem cells and/or for
differentiated cells. hESCs can also be engineered to contain a
selection gene which can be used to avoid the presence of any
residual undifferentiated cells [148]. Finally, encapsulation of
differentiated cells when compatible with their functionality can
also be an advantageous solution. Therefore there are multiple
solutions to increase the safety of cells generated from hESCs,
which should accelerate their use in clinic.
HUMAN-INDUCED PLURIPOTENT STEM CELLS
Pluripotent stem cells can also be generated by reprogramming
somatic cells using either SCNT (somatic cell nuclear transfer)
or overexpression of pluripotency factors. However, SCNT
has never been performed successfully in humans and the
difficulty in obtaining human oocytes remains a major limitation.
The recent possibility to generate hiPSCs from somatic cells
by overexpressing pluripotency factors such as Oct4, Sox2,
Klf4 and c-Myc represents a more immediate opportunity for
regenerative medicine. Indeed, this approach can be used to
generate hiPSCs from a large number of patients of different
age and gender [149,150]. In addition, a broad number of
somatic cells can be reprogrammed by the four factors approach,
including skin fibroblasts [151], keratinocytes [152], NSCs
[153], B-lymphocytes [154], pancreatic β-cells [155], hepatocytes
and intestinal cells [156]. Therefore hiPSCs could enable
the production of patient-specific cell types which are fully
immune-compatible with the original donor, thereby avoiding the
use of immunosuppressive treatment during cell-based therapy
(Figure 2). In addition, hiPSCs can be generated from somatic
cells isolated from patients suffering from diverse diseases. Then,
the resulting ‘diseased’ hiPSCs can be differentiated into the cell
type targeted by the disease and thus provide in vitro models to
perform large-scale studies impossible with primary cell culture or
with biopsy material. Two recent studies have shown that hiPSCs
can be used to study SMA (spinal muscular atrophy) and FD
(familial dysautonomia) [156a,156b]. In both studies, ‘diseased’
hiPSCs were differentiated into neuronal cells which then display
phenotype characteristic of cells from patients with SMA or FD.
Therefore these culture systems provide, for the first time, an
in vitro model to study these complex diseases and also to screen
pharmaceutical compounds in vitro using relevant tissues. Hence
hiPSCs represent a unique opportunity to develop new treatments
for a large number of diseases which are lacking relevant in vitro
models (Figure 4).
Limitations to in vivo use
The use of hiPSCs in vivo is currently limited by several issues.
hiPSCs are commonly generated using viruses overexpressing the
factors necessary to achieve epigenetic reprogramming. Therefore
the resulting hiPSCs contain multiple copies of each transgene
in their genome and these insertions can provoke mutations
in key genes, such as oncogenes or cell-cycle regulators. This
phenomenon has been well described in gene therapy clinical
trials for SCID (severe combined immunodeficiency) [157].
Indeed, several patients developed leukaemia after insertion of
the lentiviruses in proto-oncogene LMO2 (LIM domain only
2) [158,159]. Therefore the use of viruses to generate hiPSCs
represents a major limitation for their in vivo use. A solution
could be to sequence the entire genome of each hiPSC to select
c The Authors Journal compilation c 2010 Biochemical Society
lines that only integrate viruses in regions of the genome devoid of
genes. Alternatively, a large number of groups are now developing
non-integrative methods to generate hiPSCs, including the use
of transitory transfection of episomal viruses [160–162]. The
development of small molecules represents the ultimate solution
to the problems associated with genetic modifications and several
reports have shown that c-Myc and Sox2 could be replaced
respectively, by molecules such as the histone modifier inhibitor
BIX-01294 [163] and the TGFβ receptor inhibitor SB431542
[164]. However, all of these methods have been used on a limited
number of somatic cells of embryonic origin and more work is
required to demonstrate that they can be used with a broad number
of adult patients.
Generation of hiPSCs also require the use of c-Myc and/or the
inhibition of the p53 pathway, both of which present safety issues.
Indeed, studies in the mouse have shown that reactivation of the
exogenous c-Myc transgene can induce tumour formation [165].
Recent reports have also shown that inhibition of the p53 pathway
strongly increases the efficiency of epigenetic reprogramming and
thus suggests that the main pathway protecting cells against DNA
damage needs to be turned off to allow the generation of hiPSCs
[166–171]. The consequences on the genomic integrity of hiPSCs
remain to be demonstrated, but inhibition of p53 could increase
the karyotypic instability already observed with hESCs. Finally,
the pluripotent status of hiPSCs confers upon them the capacity
to form teratomas in vivo and this safety issue, which is shared
with hESCs, could be aggravated in hiPSCs. Indeed, a recent
report has shown that the capacity of iPSCs to form teratomas
can vary in function of their origin [172]. This phenomenon could
be explained by the persistence of undifferentiated cells after
differentiation, suggesting that some iPSC lines are more difficult
to differentiate than others. Alternatively, differentiated cells
generated from iPSCs could re-acquire a pluripotent status after
transplantation in vivo. Therefore further studies are necessary to
fully demonstrate that differentiated cells generated from iPSCs
are epigenetically stable over a prolonged period of time in vitro
and in vivo.
Generation of fully differentiated cells
hiPSCs and hESCs share many characteristics, including their
capacity of differentiation, the expression of pluripotency markers
and their dependence on activin/nodal signalling to maintain
their pluripotent status [173]. These functional similarities also
imply that hiPSCs and hESCs suffer from the same limitations,
which include the risk of teratoma formation (see above),
variability in their capacity to differentiate and difficulty in
generating fully functional cells. Moreover, incomplete epigenetic
reprogramming, epigenetic memory, transgene insertion and
genetic instability could worsen the variability between hiPSCs
and thus render its use almost impossible for personalized therapy.
Indeed, each hiPSC line could require the development of a
specific method of differentiation. Consequently, further studies
are needed to definitively demonstrate that hiPSCs generated from
a broad number of patients can all be efficiently differentiated
into one particular cell type using a unique protocol. In addition,
the absence of robust protocols to generate fully differentiated
cells from hiPSCs is currently limiting the development of
in vitro models of disease. Indeed, the cells generated from
diseased hiPSCs display features of embryonic progenitors which
cannot be used to mimic disease only occurring in the adult
[174]. Therefore much more effort will be required to deliver
the clinical promises of hiPSCs. However, this major objective
could be achieved very rapidly since hiPSCs have become the
main focus of many laboratories and they will plainly benefit
9. Emerging use of stem cells in regenerative medicine
from the experience accumulated over the past 10 years with
hESCs.
CONCLUSIONS AND PERSPECTIVES
The increasing types of stem cells available represent a unique
opportunity for regenerative medicine as they will offer new
treatment for a broad number of diseases which cannot be fully
cured by drug-based therapy. Importantly, stem cells are already
reaching the clinic as shown by the large number of clinical trials
using diverse types of stem cells. However, stem cells present
new challenges that will need to be solved to fully realise their
clinical promises. The broad diversity of stem cells available is
now offering a large choice for clinicians and a universal stem
cell for ‘all the diseases’ is unlikely to exist. Therefore, the
‘right’ stem cell will have to be used for the ‘right’ disease. In
addition, safety could block or delay a large number of clinical
applications. Indeed, the necessity to demonstrate that a stem
cell is not harmful represents a necessary hurdle, especially with
pluripotent stem cells capable of forming teratomas. Therefore
regulatory agencies will have to define new criteria to evaluate
the risk associated with specific stem cells and their differentiated
progeny. This process is often delayed by the difficulty to define
what is an acceptable risk/benefit with stem-cell-based therapy. Is
teratoma an acceptable risk compared with a terminal disease? In
addition, novel animal models are urgently needed to reinforce
the work currently performed in rodents. Indeed, the mouse
presents clear advantages but also important limitations. Their
size is not relevant for human treatment and mouse models
rarely reproduce, in full, the human disease. Finally, monitoring
functionality of human cells in a fully immunosuppressed xenoenvironment present obvious issues. Therefore the use of large
animals or non-human primates will be necessary to validate the
functionality and safety of the cells generated from hESCs/hiPSCs
and also other stem cells. To conclude, clinical applications of
stem cells are becoming a reality and future applications will
continue to require a strong collaboration between basic scientists,
clinicians, regulatory agencies and, more importantly, patients,
whose support is essential.
ACKNOWLEDGEMENTS
We thank Dr Tamir Rashid for helpful discussions, Ms Stephanie Brown for proof-reading
prior to submission, and Dr Nicholas Hannan for providing the immunostaining figures.
FUNDING
A. K. K. T. is supported by A*STAR Graduate Academy of A*STAR (Agency for Science,
Technology and Research), Singapore, and L.V. is supported by the Medical Research
Council (U.K.).
REFERENCES
1 Burt, R. K., Loh, Y., Pearce, W., Beohar, N., Barr, W. G., Craig, R., Wen, Y., Rapp, J. A.
and Kessler, J. (2008) Clinical applications of blood-derived and marrow-derived stem
cells for nonmalignant diseases. JAMA, J. Am. Med. Assoc. 299, 925–936
2 Baetge, E. E. (2008) Production of β-cells from human embryonic stem cells. Diabetes
Obes. Metab. 10 (Suppl. 4) 186–194
3 Seydoux, G. and Braun, R. E. (2006) Pathway to totipotency: lessons from germ cells.
Cell 127, 891–904
4 Yu, J. and Thomson, J. A. (2008) Pluripotent stem cell lines. Genes Dev. 22,
1987–1997
5 Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci.
U.S.A. 78, 7634–7638
19
6 Evans, M. J. and Kaufman, M. H. (1981) Establishment in culture of pluripotential cells
from mouse embryos. Nature 292, 154–156
7 Clark, A. T., Bodnar, M. S., Fox, M., Rodriquez, R. T., Abeyta, M. J., Firpo, M. T. and
Pera, R. A. (2004) Spontaneous differentiation of germ cells from human embryonic
stem cells in vitro . Hum. Mol. Genet. 13, 727–739
8 Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka, T. P. and
Thomson, J. A. (2002) BMP4 initiates human embryonic stem cell differentiation to
trophoblast. Nat. Biotechnol. 20, 1261–1264
9 Aranguren, X. L., McCue, J. D., Hendrickx, B., Zhu, X. H., Du, F., Chen, E., Pelacho, B.,
Penuelas, I., Abizanda, G., Uriz, M. et al. (2008) Multipotent adult progenitor cells
sustain function of ischemic limbs in mice. J. Clin. Invest. 118, 505–514
10 Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D.,
Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M. et al. (2002)
Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49
11 van der Flier, L. G. and Clevers, H. (2009) Stem cells, self-renewal, and differentiation
in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260
12 Blanpain, C. and Fuchs, E. (2009) Epidermal homeostasis: a balancing act of stem cells
in the skin. Nat. Rev. Mol. Cell. Biol. 10, 207–217
13 Gaudio, E., Carpino, G., Cardinale, V., Franchitto, A., Onori, P. and Alvaro, D. (2009)
New insights into liver stem cells. Digest. Liver Dis. 41, 455–462
14 Pacilli, A. and Pasquinelli, G. (2009) Vascular wall resident progenitor cells: a review.
Exp. Cell Res. 315, 901–914
15 Yin, T. and Li, L. (2006) The stem cell niches in bone. J. Clin. Invest. 116, 1195–1201
16 Doetsch, F. (2003) A niche for adult neural stem cells. Curr. Opin. Genet. Dev. 13,
543–550
17 Lederer, C. W. and Santama, N. (2008) Neural stem cells: mechanisms of fate
specification and nuclear reprogramming in regenerative medicine. Biotechnol. J. 3,
1521–1538
18 Bickenbach, J. R. and Grinnell, K. L. (2004) Epidermal stem cells: interactions in
developmental environments. Differentiation 72, 371–380
19 Bickenbach, J. R., Stern, M. M., Grinnell, K. L., Manuel, A. and Chinnathambi, S.
(2006) Epidermal stem cells have the potential to assist in healing damaged tissues. J.
Investig. Dermatol. Symp. Proc. 11, 118–123
20 Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., Kasahara,
H., Rota, M., Musso, E., Urbanek, K. et al. (2003) Adult cardiac stem cells are
multipotent and support myocardial regeneration. Cell 114, 763–776
21 Messina, E., De Angelis, L., Frati, G., Morrone, S., Chimenti, S., Fiordaliso, F., Salio,
M., Battaglia, M., Latronico, M. V., Coletta, M. et al. (2004) Isolation and expansion of
adult cardiac stem cells from human and murine heart. Circ. Res. 95, 911–921
22 Martin, C. M., Russell, J. L., Ferdous, A. and Garry, D. J. (2006) Molecular signatures
define myogenic stem cell populations. Stem Cell Rev. 2, 37–42
23 Shi, X. and Garry, D. J. (2006) Muscle stem cells in development, regeneration, and
disease. Genes Dev. 20, 1692–1708
24 Neuringer, I. P. and Randell, S. H. (2004) Stem cells and repair of lung injuries. Respir.
Res. 5, 6
25 Neuringer, I. P. and Randell, S. H. (2006) Lung stem cell update: promise and
controversy. Monaldi Arch. Chest Dis. 65, 47–51
26 Casali, A. and Batlle, E. (2009) Intestinal stem cells in mammals and Drosophila . Cell
Stem Cell 4, 124–127
27 Kung, J. W. and Forbes, S. J. (2009) Stem cells and liver repair. Curr. Opin. Biotechnol.
20, 568–574
28 Ochiya, T., Yamamoto, Y. and Banas, A. (2009) Commitment of stem cells into
functional hepatocytes. Differentiation 79, 65–73
29 Cameron, H. A., Hazel, T. G. and McKay, R. D. (1998) Regulation of neurogenesis by
growth factors and neurotransmitters. J. Neurobiol. 36, 287–306
30 Androutsellis-Theotokis, A., Rueger, M. A., Mkhikian, H., Korb, E. and McKay, R. D.
(2008) Signaling pathways controlling neural stem cells slow progressive brain
disease. Cold Spring Harb. Symp. Quant. Biol. 73, 403–410
31 Kuhn, H. G., Winkler, J., Kempermann, G., Thal, L. J. and Gage, F. H. (1997) Epidermal
growth factor and fibroblast growth factor-2 have different effects on neural progenitors
in the adult rat brain. J. Neurosci. 17, 5820–5829
32 Fallon, J., Reid, S., Kinyamu, R., Opole, I., Opole, R., Baratta, J., Korc, M., Endo, T. L.,
Duong, A., Nguyen, G. et al. (2000) In vivo induction of massive proliferation, directed
migration, and differentiation of neural cells in the adult mammalian brain. Proc. Natl.
Acad. Sci. U.S.A. 97, 14686–14691
33 Jin, K., Zhu, Y., Sun, Y., Mao, X. O., Xie, L. and Greenberg, D. A. (2002) Vascular
endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo . Proc.
Natl. Acad. Sci. U.S.A. 99, 11946–11950
34 Cao, L., Jiao, X., Zuzga, D. S., Liu, Y., Fong, D. M., Young, D. and During, M. J. (2004)
VEGF links hippocampal activity with neurogenesis, learning and memory. Nat. Genet.
36, 827–835
c The Authors Journal compilation c 2010 Biochemical Society
10. 20
A. K. K. Teo and L. Vallier
35 Jin, K., Mao, X. O., Sun, Y., Xie, L. and Greenberg, D. A. (2002) Stem cell factor
stimulates neurogenesis in vitro and in vivo . J. Clin. Invest. 110, 311–319
36 Aberg, M. A., Aberg, N. D., Hedbacker, H., Oscarsson, J. and Eriksson, P. S. (2000)
Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat
hippocampus. J. Neurosci. 20, 2896–2903
37 Rosenthal, N., Santini, M. P. and Musaro, A. (2006) Growth factor enhancement of
cardiac regeneration. Cell Transplant. 15 (Suppl. 1), S41–S45
38 Mourkioti, F. and Rosenthal, N. (2005) IGF-1, inflammation and stem cells: interactions
during muscle regeneration. Trends Immunol. 26, 535–542
39 Mourkioti, F. and Rosenthal, N. (2008) NF-κB signaling in skeletal muscle: prospects
for intervention in muscle diseases. J. Mol. Med. 86, 747–759
40 Chen, S. E., Gerken, E., Zhang, Y., Zhan, M., Mohan, R. K., Li, A. S., Reid, M. B. and Li,
Y. P. (2005) Role of TNF-α signaling in regeneration of cardiotoxin-injured muscle.
Am. J. Physiol. Cell Physiol. 289, C1179–C1187
41 Acharyya, S., Villalta, S. A., Bakkar, N., Bupha-Intr, T., Janssen, P. M., Carathers, M., Li,
Z. W., Beg, A. A., Ghosh, S., Sahenk, Z. et al. (2007) Interplay of IKK/NF-κB signaling
in macrophages and myofibers promotes muscle degeneration in Duchenne muscular
dystrophy. J. Clin. Invest. 117, 889–901
42 Dor, Y., Brown, J., Martinez, O. I. and Melton, D. A. (2004) Adult pancreatic β-cells are
formed by self-duplication rather than stem-cell differentiation. Nature 429,
41–46
43 Teta, M., Rankin, M. M., Long, S. Y., Stein, G. M. and Kushner, J. A. (2007) Growth and
regeneration of adult β cells does not involve specialized progenitors. Dev. Cell 12,
817–826
44 Xia, B., Zhan, X. R., Yi, R. and Yang, B. (2009) Can pancreatic duct-derived progenitors
be a source of islet regeneration? Biochem. Biophys. Res. Commun. 383, 383–385
45 Solar, M., Cardalda, C., Houbracken, I., Martin, M., Maestro, M. A., De Medts, N.,
Xu, X., Grau, V., Heimberg, H., Bouwens, L. and Ferrer, J. (2009) Pancreatic exocrine
duct cells give rise to insulin-producing β cells during embryogenesis but not after
birth. Dev. Cell 17, 849–860
46 Fausto, N. (2004) Liver regeneration and repair: hepatocytes, progenitor cells, and
stem cells. Hepatology 39, 1477–1487
47 Shafritz, D. A., Oertel, M., Menthena, A., Nierhoff, D. and Dabeva, M. D. (2006) Liver
stem cells and prospects for liver reconstitution by transplanted cells. Hepatology 43,
S89–S98
48 Malhi, H., Irani, A. N., Gagandeep, S. and Gupta, S. (2002) Isolation of human
progenitor liver epithelial cells with extensive replication capacity and differentiation
into mature hepatocytes. J. Cell Sci. 115, 2679–2688
49 Wang, X., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M. and Grompe, M.
(2003) The origin and liver repopulating capacity of murine oval cells. Proc. Natl. Acad.
Sci. U.S.A. 100 (Suppl. 1), 11881–11888
50 Dan, Y. Y., Riehle, K. J., Lazaro, C., Teoh, N., Haque, J., Campbell, J. S. and Fausto, N.
(2006) Isolation of multipotent progenitor cells from human fetal liver capable of
differentiating into liver and mesenchymal lineages. Proc. Natl. Acad. Sci. U.S.A. 103,
9912–9917
51 Nagy, P., Bisgaard, H. C. and Thorgeirsson, S. S. (1994) Expression of hepatic
transcription factors during liver development and oval cell differentiation. J. Cell Biol.
126, 223–233
52 Dan, Y. Y. and Yeoh, G. C. (2008) Liver stem cells: a scientific and clinical perspective.
J. Gastroenterol. Hepatol. 23, 687–698
53 Lowes, K. N., Brennan, B. A., Yeoh, G. C. and Olynyk, J. K. (1999) Oval cell numbers in
human chronic liver diseases are directly related to disease severity. Am. J. Pathol.
154, 537–541
54 Roskams, T., Yang, S. Q., Koteish, A., Durnez, A., DeVos, R., Huang, X., Achten, R.,
Verslype, C. and Diehl, A. M. (2003) Oxidative stress and oval cell accumulation in
mice and humans with alcoholic and nonalcoholic fatty liver disease. Am. J. Pathol.
163, 1301–1311
55 Herrera, M. B., Bruno, S., Buttiglieri, S., Tetta, C., Gatti, S., Deregibus, M. C., Bussolati,
B. and Camussi, G. (2006) Isolation and characterization of a stem cell population from
adult human liver. Stem Cells 24, 2840–2850
56 Schmelzer, E., Wauthier, E. and Reid, L. M. (2006) The phenotypes of pluripotent
human hepatic progenitors. Stem Cells 24, 1852–1858
57 Schmelzer, E., Zhang, L., Bruce, A., Wauthier, E., Ludlow, J., Yao, H. L., Moss, N.,
Melhem, A., McClelland, R., Turner, W. et al. (2007) Human hepatic stem cells from
fetal and postnatal donors. J. Exp. Med. 204, 1973–1987
58 Theise, N. D., Saxena, R., Portmann, B. C., Thung, S. N., Yee, H., Chiriboga, L., Kumar,
A. and Crawford, J. M. (1999) The canals of Hering and hepatic stem cells in humans.
Hepatology 30, 1425–1433
59 Bhindi, R., Witting, P. K., McMahon, A. C., Khachigian, L. M. and Lowe, H. C. (2006)
Rat models of myocardial infarction. Pathogenetic insights and clinical relevance.
Thromb. Haemostasis 96, 602–610
c The Authors Journal compilation c 2010 Biochemical Society
60 DiBernardo, A. B. and Cudkowicz, M. E. (2006) Translating preclinical insights into
effective human trials in ALS. Biochim. Biophys. Acta 1762, 1139–1149
61 Rivera, J. and Tessarollo, L. (2008) Genetic background and the dilemma of translating
mouse studies to humans. Immunity 28, 1–4
62 Hanley, N. A., Hanley, K. P., Miettinen, P. J. and Otonkoski, T. (2008) Weighing up
β-cell mass in mice and humans: self-renewal, progenitors or stem cells? Mol. Cell.
Endocrinol. 288, 79–85
63 Ausoni, S. and Sartore, S. (2009) From fish to amphibians to mammals: in search of
novel strategies to optimize cardiac regeneration. J. Cell Biol. 184, 357–364
64 Kuci, S., Kuci, Z., Latifi-Pupovci, H., Niethammer, D., Handgretinger, R., Schumm, M.,
Bruchelt, G., Bader, P. and Klingebiel, T. (2009) Adult stem cells as an alternative
source of multipotential (pluripotential) cells in regenerative medicine. Curr. Stem Cell
Res. Ther. 4, 107–117
65 Gage, F. H. (2000) Mammalian neural stem cells. Science 287, 1433–1438
66 Goldman, S. A. and Windrem, M. S. (2006) Cell replacement therapy in neurological
disease. Philos. Trans. R. Soc. London Ser. B 361, 1463–1475
67 Bernardo, M. E., Locatelli, F. and Fibbe, W. E. (2009) Mesenchymal stromal cells. Ann.
N.Y. Acad. Sci. 1176, 101–117
68 Prockop, D. J. (2009) Repair of tissues by adult stem/progenitor cells (MSCs):
controversies, myths, and changing paradigms. Mol. Ther. 17, 939–946
69 Morshead, C. M., Reynolds, B. A., Craig, C. G., McBurney, M. W., Staines, W. A.,
Morassutti, D., Weiss, S. and van der Kooy, D. (1994) Neural stem cells in the adult
mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.
Neuron 13, 1071–1082
70 Reynolds, B. A. and Weiss, S. (1992) Generation of neurons and astrocytes from
isolated cells of the adult mammalian central nervous system. Science 255,
1707–1710
71 Song, H., Stevens, C. F. and Gage, F. H. (2002) Astroglia induce neurogenesis from
adult neural stem cells. Nature 417, 39–44
72 Song, H. J., Stevens, C. F. and Gage, F. H. (2002) Neural stem cells from adult
hippocampus develop essential properties of functional CNS neurons. Nat. Neurosci.
5, 438–445
73 Taupin, P. (2006) Therapeutic potential of adult neural stem cells. Recent Pat. CNS
Drug Discov. 1, 299–303
74 Kondziolka, D., Wechsler, L., Goldstein, S., Meltzer, C., Thulborn, K. R., Gebel, J.,
Jannetta, P., DeCesare, S., Elder, E. M., McGrogan, M. et al. (2000) Transplantation of
cultured human neuronal cells for patients with stroke. Neurology 55, 565–569
75 Meltzer, C. C., Kondziolka, D., Villemagne, V. L., Wechsler, L., Goldstein, S., Thulborn,
K. R., Gebel, J., Elder, E. M., DeCesare, S. and Jacobs, A. (2001) Serial [18 F]
fluorodeoxyglucose positron emission tomography after human neuronal implantation
for stroke. Neurosurgery 49, 586–591
76 Choi, C. Q. (2007) A stroke for stem cells. Sci. Am. 296, 8–9
77 Amariglio, N., Hirshberg, A., Scheithauer, B. W., Cohen, Y., Loewenthal, R.,
Trakhtenbrot, L., Paz, N., Koren-Michowitz, M., Waldman, D., Leider-Trejo, L. et al.
(2009) Donor-derived brain tumor following neural stem cell transplantation in an
ataxia telangiectasia patient. PLoS Med. 6, e1000029
78 Friedenstein, A. J., Petrakova, K. V., Kurolesova, A. I. and Frolova, G. P. (1968)
Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and
hematopoietic tissues. Transplantation 6, 230–247
79 De Coppi, P., Bartsch, Jr, G., Siddiqui, M. M., Xu, T., Santos, C. C., Perin, L.,
Mostoslavsky, G., Serre, A. C., Snyder, E. Y., Yoo, J. J. et al. (2007) Isolation of
amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100–106
80 In ‘t Anker, P. S., Scherjon, S. A., Kleijburg-van der Keur, C., de Groot-Swings, G. M.,
Claas, F. H., Fibbe, W. E. and Kanhai, H. H. (2004) Isolation of mesenchymal stem cells
of fetal or maternal origin from human placenta. Stem Cells 22, 1338–1345
81 Chien, C. C., Yen, B. L., Lee, F. K., Lai, T. H., Chen, Y. C., Chan, S. H. and Huang, H. I.
(2006) In vitro differentiation of human placenta-derived multipotent cells into
hepatocyte-like cells. Stem Cells 24, 1759–1768
82 Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., Alfonso,
Z. C., Fraser, J. K., Benhaim, P. and Hedrick, M. H. (2002) Human adipose tissue is a
source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295
83 Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., Benhaim, P.,
Lorenz, H. P. and Hedrick, M. H. (2001) Multilineage cells from human adipose tissue:
implications for cell-based therapies. Tissue Eng. 7, 211–228
84 Bieback, K., Kern, S., Kluter, H. and Eichler, H. (2004) Critical parameters for the
isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22,
625–634
85 Tyndall, A. and Uccelli, A. (2009) Multipotent mesenchymal stromal cells for
autoimmune diseases: teaching new dogs old tricks. Bone Marrow Transplant. 43,
821–828
86 Karp, J. M. and Leng Teo, G. S. (2009) Mesenchymal stem cell homing: the devil is in
the details. Cell Stem Cell 4, 206–216
11. Emerging use of stem cells in regenerative medicine
87 Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D.,
Moorman, M. A., Simonetti, D. W., Craig, S. and Marshak, D. R. (1999) Multilineage
potential of adult human mesenchymal stem cells. Science 284, 143–147
88 in ‘t Anker, P. S., Noort, W. A., Scherjon, S. A., Kleijburg-van der Keur, C.,
Kruisselbrink, A. B., van Bezooijen, R. L., Beekhuizen, W., Willemze, R., Kanhai, H. H.
and Fibbe, W. E. (2003) Mesenchymal stem cells in human second-trimester bone
marrow, liver, lung, and spleen exhibit a similar immunophenotype but a
heterogeneous multilineage differentiation potential. Haematologica 88, 845–852
89 Sekiya, I., Colter, D. C. and Prockop, D. J. (2001) BMP-6 enhances chondrogenesis in
a subpopulation of human marrow stromal cells. Biochem. Biophys. Res. Commun.
284, 411–418
90 Indrawattana, N., Chen, G., Tadokoro, M., Shann, L. H., Ohgushi, H., Tateishi, T.,
Tanaka, J. and Bunyaratvej, A. (2004) Growth factor combination for chondrogenic
induction from human mesenchymal stem cell. Biochem. Biophys. Res. Commun.
320, 914–919
91 Sueblinvong, V., Loi, R., Eisenhauer, P. L., Bernstein, I. M., Suratt, B. T., Spees, J. L.
and Weiss, D. J. (2008) Derivation of lung epithelium from human cord blood-derived
mesenchymal stem cells. Am. J. Respir. Crit. Care Med. 177, 701–711
92 Yue, W. M., Liu, W., Bi, Y. W., He, X. P., Sun, W. Y., Pang, X. Y., Gu, X. H. and Wang,
X. P. (2008) Mesenchymal stem cells differentiate into an endothelial phenotype,
reduce neointimal formation, and enhance endothelial function in a rat vein grafting
model. Stem Cells Dev. 17, 785–793
93 Greco, S. J. and Rameshwar, P. (2007) Enhancing effect of IL-1α on neurogenesis from
adult human mesenchymal stem cells: implication for inflammatory mediators in
regenerative medicine. J. Immunol. 179, 3342–3350
94 Koga, H., Muneta, T., Nagase, T., Nimura, A., Ju, Y. J., Mochizuki, T. and Sekiya, I.
(2008) Comparison of mesenchymal tissues-derived stem cells for in vivo
chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit. Cell
Tissue Res. 333, 207–215
95 Digirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R. and Prockop, D. J.
(1999) Propagation and senescence of human marrow stromal cells in culture: a
simple colony-forming assay identifies samples with the greatest potential to propagate
and differentiate. Br. J. Haematol. 107, 275–281
96 Li, H., Fan, X., Kovi, R. C., Jo, Y., Moquin, B., Konz, R., Stoicov, C., Kurt-Jones, E.,
Grossman, S. R., Lyle, S. et al. (2007) Spontaneous expression of embryonic factors
and point mutations in aged mesenchymal stem cells: a model of age-related
tumorigenesis in mice. Cancer Res. 67, 10889–10898
97 Togel, F., Weiss, K., Yang, Y., Hu, Z., Zhang, P. and Westenfelder, C. (2007)
Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the
recovery from acute kidney injury. Am. J. Physiol. Renal Physiol. 292, F1626–F1635
98 Spitkovsky, D. and Hescheler, J. (2008) Adult mesenchymal stromal stem cells for
therapeutic applications. Minim. Invasive Ther. Allied Technol. 17, 79–90
99 Koc, O. N., Gerson, S. L., Cooper, B. W., Dyhouse, S. M., Haynesworth, S. E., Caplan,
A. I. and Lazarus, H. M. (2000) Rapid hematopoietic recovery after coinfusion of
autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in
advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18,
307–316
100 Le Blanc, K., Rasmusson, I., Sundberg, B., Gotherstrom, C., Hassan, M., Uzunel, M.
and Ringden, O. (2004) Treatment of severe acute graft-versus-host disease with third
party haploidentical mesenchymal stem cells. Lancet 363, 1439–1441
101 Bocelli-Tyndall, C., Bracci, L., Spagnoli, G., Braccini, A., Bouchenaki, M., Ceredig, R.,
Pistoia, V., Martin, I. and Tyndall, A. (2007) Bone marrow mesenchymal stromal cells
(BM-MSCs) from healthy donors and auto-immune disease patients reduce the
proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro .
Rheumatology 46, 403–408
102 Mazzini, L., Ferrero, I., Luparello, V., Rustichelli, D., Gunetti, M., Mareschi, K., Testa,
L., Stecco, A., Tarletti, R., Miglioretti, M. et al. (2009) Mesenchymal stem cell
transplantation in amyotrophic lateral sclerosis: a Phase I clinical trial. Exp, Neurol.,
doi:10.1016/j.expneurol.2009.08.007
103 Psaltis, P. J., Zannettino, A. C., Worthley, S. G. and Gronthos, S. (2008) Concise
review: mesenchymal stromal cells: potential for cardiovascular repair. Stem Cells 26,
2201–2210
104 Yi, B. A., Wernet, O. and Chien, K. R. (2010) Pregenerative medicine: developmental
paradigms in the biology of cardiovascular regeneration. J. Clin. Invest. 120, 20–28
105 Schwartz, R. E., Reyes, M., Koodie, L., Jiang, Y., Blackstad, M., Lund, T., Lenvik, T.,
Johnson, S., Hu, W. S. and Verfaillie, C. M. (2002) Multipotent adult progenitor cells
from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest.
109, 1291–1302
106 Sato, Y., Araki, H., Kato, J., Nakamura, K., Kawano, Y., Kobune, M., Sato, T., Miyanishi,
K., Takayama, T., Takahashi, M. et al. (2005) Human mesenchymal stem cells
xenografted directly to rat liver are differentiated into human hepatocytes without
fusion. Blood 106, 756–763
21
107 Hong, S. H., Gang, E. J., Jeong, J. A., Ahn, C., Hwang, S. H., Yang, I. H., Park, H. K.,
Han, H. and Kim, H. (2005) In vitro differentiation of human umbilical cord
blood-derived mesenchymal stem cells into hepatocyte-like cells. Biochem. Biophys.
Res. Commun. 330, 1153–1161
108 Banas, A., Teratani, T., Yamamoto, Y., Tokuhara, M., Takeshita, F., Quinn, G., Okochi, H.
and Ochiya, T. (2007) Adipose tissue-derived mesenchymal stem cells as a source of
human hepatocytes. Hepatology 46, 219–228
109 Banas, A., Teratani, T., Yamamoto, Y., Tokuhara, M., Takeshita, F., Osaki, M., Kato, T.,
Okochi, H. and Ochiya, T. (2009) Rapid hepatic fate specification of adipose-derived
stem cells and their therapeutic potential for liver failure. J. Gastroenterol. Hepatol. 24,
70–77
110 Kharaziha, P., Hellstrom, P. M., Noorinayer, B., Farzaneh, F., Aghajani, K., Jafari, F.,
Telkabadi, M., Atashi, A., Honardoost, M., Zali, M. R. and Soleimani, M. (2009)
Improvement of liver function in liver cirrhosis patients after autologous mesenchymal
stem cell injection: a phase I-II clinical trial. Eur. J. Gastroenterol. Hepatol. 21,
1199–1205
111 Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J.,
Marshall, V. S. and Jones, J. M. (1998) Embryonic stem cell lines derived from human
blastocysts. Science 282, 1145–1147
112 Klimanskaya, I., Hipp, J., Rezai, K. A., West, M., Atala, A. and Lanza, R. (2004)
Derivation and comparative assessment of retinal pigment epithelium from human
embryonic stem cells using transcriptomics. Cloning Stem Cells 6, 217–245
113 Lund, R. D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B., Girman,
S., Bischoff, N., Sauve, Y. and Lanza, R. (2006) Human embryonic stem cell-derived
cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells 8, 189–199
114 Takagi, Y., Takahashi, J., Saiki, H., Morizane, A., Hayashi, T., Kishi, Y., Fukuda, H.,
Okamoto, Y., Koyanagi, M., Ideguchi, M. et al. (2005) Dopaminergic neurons generated
from monkey embryonic stem cells function in a Parkinson primate model. J. Clin.
Invest. 115, 102–109
115 Wichterle, H., Lieberam, I., Porter, J. A. and Jessell, T. M. (2002) Directed
differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397
116 Li, X. J., Du, Z. W., Zarnowska, E. D., Pankratz, M., Hansen, L. O., Pearce, R. A. and
Zhang, S. C. (2005) Specification of motoneurons from human embryonic stem cells.
Nat. Biotechnol. 23, 215–221
117 Nistor, G. I., Totoiu, M. O., Haque, N., Carpenter, M. K. and Keirstead, H. S. (2005)
Human embryonic stem cells differentiate into oligodendrocytes in high purity and
myelinate after spinal cord transplantation. Glia 49, 385–396
118 Dai, W., Field, L. J., Rubart, M., Reuter, S., Hale, S. L., Zweigerdt, R., Graichen, R. E.,
Kay, G. L., Jyrala, A. J., Colman, A. et al. (2007) Survival and maturation of human
embryonic stem cell-derived cardiomyocytes in rat hearts. J. Mol. Cell. Cardiol. 43,
504–516
119 Caspi, O., Huber, I., Kehat, I., Habib, M., Arbel, G., Gepstein, A., Yankelson, L., Aronson,
D., Beyar, R. and Gepstein, L. (2007) Transplantation of human embryonic stem
cell-derived cardiomyocytes improves myocardial performance in infarcted rat hearts.
J. Am. Coll. Cardiol. 50, 1884–1893
120 Kroon, E., Martinson, L. A., Kadoya, K., Bang, A. G., Kelly, O. G., Eliazer, S., Young, H.,
Richardson, M., Smart, N. G., Cunningham, J. et al. (2008) Pancreatic endoderm
derived from human embryonic stem cells generates glucose-responsive
insulin-secreting cells in vivo . Nat. Biotechnol. 26, 443–452
121 Chen, Y., Soto-Gutierrez, A., Navarro-Alvarez, N., Rivas-Carrillo, J. D., Yamatsuji, T.,
Shirakawa, Y., Tanaka, N., Basma, H., Fox, I. J. and Kobayashi, N. (2006) Instant
hepatic differentiation of human embryonic stem cells using activin A and a deleted
variant of HGF. Cell Transplant. 15, 865–871
122 Cai, J., Zhao, Y., Liu, Y., Ye, F., Song, Z., Qin, H., Meng, S., Chen, Y., Zhou, R., Song, X.
et al. (2007) Directed differentiation of human embryonic stem cells into functional
hepatic cells. Hepatology 45, 1229–1239
123 Duan, Y., Catana, A., Meng, Y., Yamamoto, N., He, S., Gupta, S., Gambhir, S. S. and
Zern, M. A. (2007) Differentiation and enrichment of hepatocyte-like cells from human
embryonic stem cells in vitro and in vivo . Stem Cells 25, 3058–3068
124 Oertel, M. and Shafritz, D. A. (2008) Stem cells, cell transplantation and liver
repopulation. Biochim. Biophys. Acta 1782, 61–74
125 Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E., Warnock, G. L.,
Kneteman, N. M. and Rajotte, R. V. (2000) Islet transplantation in seven patients with
type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N.
Engl. J. Med. 343, 230–238
126 Rajagopal, J., Anderson, W. J., Kume, S., Martinez, O. I. and Melton, D. A. (2003)
Insulin staining of ES cell progeny from insulin uptake. Science 299, 363
127 Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R. and McKay, R. (2001)
Differentiation of embryonic stem cells to insulin-secreting structures similar to
pancreatic islets. Science 292, 1389–1394
c The Authors Journal compilation c 2010 Biochemical Society
12. 22
A. K. K. Teo and L. Vallier
128 Madsen, O. D. (2005) Stem cells and diabetes treatment. APMIS 113, 858–875
129 Docherty, K., Bernardo, A. S. and Vallier, L. (2007) Embryonic stem cell therapy for
diabetes mellitus. Semin. Cell Dev. Biol. 18, 827–838
130 D’Amour, K. A., Agulnick, A. D., Eliazer, S., Kelly, O. G., Kroon, E. and Baetge, E. E.
(2005) Efficient differentiation of human embryonic stem cells to definitive endoderm.
Nat. Biotechnol. 23, 1534–1541
131 Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy, M., Woo, S., Fehling,
H. J. and Keller, G. (2004) Development of definitive endoderm from embryonic stem
cells in culture. Development 131, 1651–1662
132 Wang, L. (2006) Endothelial and hematopoietic cell fate of human embryonic stem
cells. Trends Cardiovasc. Med. 16, 89–94
133 Bhatia, M. (2007) Hematopoiesis from human embryonic stem cells. Ann. N.Y. Acad.
Sci. 1106, 219–222
134 Bhatia, M. (2007) Hematopoietic development from human embryonic stem cells.
Hematology Am. Soc. Hematol. Educ. Program, 11–16
135 Caviness, Jr, V. S., Nowakowski, R. S. and Bhide, P. G. (2009) Neocortical
neurogenesis: morphogenetic gradients and beyond. Trends Neurosci. 32, 443–450
136 Tanaka, E. M. and Ferretti, P. (2009) Considering the evolution of regeneration in the
central nervous system. Nat. Rev. Neurosci. 10, 713–723
137 Vugler, A., Lawrence, J., Walsh, J., Carr, A., Gias, C., Semo, M., Ahmado, A., da Cruz,
L., Andrews, P. and Coffey, P. (2007) Embryonic stem cells and retinal repair. Mech.
Dev. 124, 807–829
138 Sharp, J., Frame, J., Siegenthaler, M., Nistor, G. and Keirstead, H. S. (2010) Human
embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve
recovery after cervical spinal cord injury. Stem Cells 28, 152–163
139 Basma, H., Soto-Gutierrez, A., Yannam, G. R., Liu, L., Ito, R., Yamamoto, T., Ellis, E.,
Carson, S. D., Sato, S., Chen, Y. et al. (2009) Differentiation and transplantation of
human embryonic stem cell-derived hepatocytes. Gastroenterology. 136, 990–999
140 Aubry, L., Bugi, A., Lefort, N., Rousseau, F., Peschanski, M. and Perrier, A. L. (2008)
Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro
and in quinolinic acid-lesioned rats. Proc. Natl. Acad. Sci. U.S.A. 105, 16707–16712
141 Hanson, C. and Caisander, G. (2005) Human embryonic stem cells and chromosome
stability. APMIS 113, 751–755
142 Cervantes, R. B., Stringer, J. R., Shao, C., Tischfield, J. A. and Stambrook, P. J. (2002)
Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc.
Natl. Acad. Sci. U.S.A. 99, 3586–3590
143 Mitalipova, M. M., Rao, R. R., Hoyer, D. M., Johnson, J. A., Meisner, L. F., Jones, K. L.,
Dalton, S. and Stice, S. L. (2005) Preserving the genetic integrity of human embryonic
stem cells. Nat. Biotechnol. 23, 19–20
144 Choi, K. D., Vodyanik, M. A. and Slukvin, II. (2009) Generation of mature human
myelomonocytic cells through expansion and differentiation of pluripotent stem
cell-derived lin-CD34+CD43+CD45+ progenitors. J. Clin. Invest. 119, 2818–2829
145 Fong, C. Y., Peh, G. S., Gauthaman, K. and Bongso, A. (2009) Separation of SSEA-4
and TRA-1-labelled undifferentiated human embryonic stem cells from a
heterogeneous cell population using magnetic-activated cell sorting (MACS) and
fluorescence-activated cell sorting (FACS). Stem Cell Rev. Rep. 5, 72–80
146 Choo, A. B., Tan, H. L., Ang, S. N., Fong, W. J., Chin, A., Lo, J., Zheng, L., Hentze, H.,
Philp, R. J., Oh, S. K. and Yap, M. (2008) Selection against undifferentiated human
embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1.
Stem Cells 26, 1454–1463
147 Tan, H. L., Fong, W. J., Lee, E. H., Yap, M. and Choo, A. (2009) mAb 84, a cytotoxic
antibody that kills undifferentiated human embryonic stem cells via oncosis. Stem
Cells 27, 1792–1801
148 Schuldiner, M., Itskovitz-Eldor, J. and Benvenisty, N. (2003) Selective ablation of
human embryonic stem cells expressing a “suicide” gene. Stem Cells 21, 257–265
149 Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and
Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts
by defined factors. Cell 131, 861–872
150 Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S.,
Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R. et al. (2007) Induced pluripotent stem
cell lines derived from human somatic cells. Science 318, 1917–1920
151 Lowry, W. E., Richter, L., Yachechko, R., Pyle, A. D., Tchieu, J., Sridharan, R., Clark,
A. T. and Plath, K. (2008) Generation of human induced pluripotent stem cells from
dermal fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 105, 2883–2888
152 Aasen, T., Raya, A., Barrero, M. J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R.,
Bilic, J., Pekarik, V., Tiscornia, G. et al. (2008) Efficient and rapid generation of induced
pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26, 1276–1284
153 Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo, M. J.,
Ruau, D., Han, D. W., Zenke, M. and Scholer, H. R. (2008) Pluripotent stem cells
induced from adult neural stem cells by reprogramming with two factors. Nature 454,
646–650
c The Authors Journal compilation c 2010 Biochemical Society
154 Hanna, J., Markoulaki, S., Schorderet, P., Carey, B. W., Beard, C., Wernig, M.,
Creyghton, M. P., Steine, E. J., Cassady, J. P., Foreman, R. et al. (2008) Direct
reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell
133, 250–264
155 Stadtfeld, M., Brennand, K. and Hochedlinger, K. (2008) Reprogramming of pancreatic
β cells into induced pluripotent stem cells. Curr. Biol. 18, 890–894
156 Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T. and
Yamanaka, S. (2008) Generation of pluripotent stem cells from adult mouse liver and
stomach cells. Science 321, 699–702
156a Lee, G., Papapetrou, E. P., Kim, H., Chambers, S. M., Tomishima, M. J., Fasano, C. A.,
Ganat, Y. M., Menon, J., Shimizu, F., Viale, A. et al. (2009) Modelling pathogenesis and
treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406
156b Ebert, A. D., Yu, J., Rose, Jr, F. F., Mattis, V. B., Lorson, C. L., Thomson, J. A. and
Svendsen, C. N. (2009) Induced pluripotent stem cells from a spinal muscular atrophy
patient. Nature 457, 277–280
157 Cassani, B., Montini, E., Maruggi, G., Ambrosi, A., Mirolo, M., Selleri, S., Biral, E.,
Frugnoli, I., Hernandez-Trujillo, V., Di Serio, C. et al. (2009) Integration of retroviral
vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID
patients treated with gene therapy. Blood 114, 3546–3556
158 Hacein-Bey-Abina, S., Garrigue, A., Wang, G. P., Soulier, J., Lim, A., Morillon, E.,
Clappier, E., Caccavelli, L., Delabesse, E., Beldjord, K. et al. (2008) Insertional
oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin.
Invest. 118, 3132–3142
159 Howe, S. J., Mansour, M. R., Schwarzwaelder, K., Bartholomae, C., Hubank, M.,
Kempski, H., Brugman, M. H., Pike-Overzet, K., Chatters, S. J., de Ridder, D. et al.
(2008) Insertional mutagenesis combined with acquired somatic mutations causes
leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118,
3143–3150
160 Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. and Yamanaka, S. (2008) Generation
of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953
161 Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I. I. and Thomson, J. A.
(2009) Human induced pluripotent stem cells free of vector and transgene sequences.
Science 324, 797–801
162 Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. and Hochedlinger, K. (2008) Induced
pluripotent stem cells generated without viral integration. Science 322, 945–949
163 Shi, Y., Desponts, C., Do, J. T., Hahm, H. S., Scholer, H. R. and Ding, S. (2008)
Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4
with small-molecule compounds. Cell Stem Cell 3, 568–574
164 Ichida, J. K., Blanchard, J., Lam, K., Son, E. Y., Chung, J. E., Egli, D., Loh, K. M.,
Carter, A. C., Di Giorgio, F. P., Koszka, K. et al. (2009) A small-molecule inhibitor of
tgf-β signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5,
491–503
165 Okita, K., Ichisaka, T. and Yamanaka, S. (2007) Generation of germline-competent
induced pluripotent stem cells. Nature 448, 313–317
166 Kawamura, T., Suzuki, J., Wang, Y. V., Menendez, S., Morera, L. B., Raya, A., Wahl,
G. M. and Belmonte, J. C. (2009) Linking the p53 tumour suppressor pathway to
somatic cell reprogramming. Nature 460, 1140–1144
167 Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Okita, K.
and Yamanaka, S. (2009) Suppression of induced pluripotent stem cell generation by
the p53-p21 pathway. Nature 460, 1132–1135
168 Li, H., Collado, M., Villasante, A., Strati, K., Ortega, S., Canamero, M., Blasco, M. A.
and Serrano, M. (2009) The Ink4/Arf locus is a barrier for iPS cell reprogramming.
Nature 460, 1136–1139
169 Marion, R. M., Strati, K., Li, H., Murga, M., Blanco, R., Ortega, S., Fernandez-Capetillo,
O., Serrano, M. and Blasco, M. A. (2009) A p53-mediated DNA damage response limits
reprogramming to ensure iPS cell genomic integrity. Nature 460, 1149–1153
170 Utikal, J., Polo, J. M., Stadtfeld, M., Maherali, N., Kulalert, W., Walsh, R. M., Khalil, A.,
Rheinwald, J. G. and Hochedlinger, K. (2009) Immortalization eliminates a roadblock
during cellular reprogramming into iPS cells. Nature 460, 1145–1148
171 Banito, A., Rashid, S. T., Acosta, J. C., Li, S., Pereira, C. F., Geti, I., Pinho, S., Silva,
J. C., Azuara, V., Walsh, M. et al. (2009) Senescence impairs successful
reprogramming to pluripotent stem cells. Genes Dev. 23, 2134–2139
172 Miura, K., Okada, Y., Aoi, T., Okada, A., Takahashi, K., Okita, K., Nakagawa, M.,
Koyanagi, M., Tanabe, K., Ohnuki, M. et al. (2009) Variation in the safety of induced
pluripotent stem cell lines. Nat. Biotechnol. 27, 743–745
173 Vallier, L., Touboul, T., Brown, S., Cho, C., Bilican, B., Alexander, M., Cedervall, J.,
Chandran, S., Ahrlund-Richter, L., Weber, A. and Pedersen, R. A. (2009) Signaling
pathways controlling pluripotency and early cell fate decisions of human induced
pluripotent stem cells. Stem Cells 27, 2655–2666
174 Saha, K. and Jaenisch, R. (2009) Technical challenges in using human induced
pluripotent stem cells to model disease. Cell Stem Cell 5, 584–595
13. Emerging use of stem cells in regenerative medicine
175 Touboul, T., Hannan, N., Corbineau, S., Martinez, A., Martinet, C., Branchereau, S.,
Mainot, S., Strick-Marchand, H., Pedersen, R., Santo, J. D. et al. (2009) Generation of
functional hepatocytes from human embryonic stem cells under chemically defined
conditions which recapitulate liver development. Hepatology, doi: 10.1002/hep.23506
176 Thomson, J. A., Kalishman, J., Golos, T. G., Durning, M., Harris, C. P., Becker, R. A.
and Hearn, J. P. (1995) Isolation of a primate embryonic stem cell line. Proc. Natl.
Acad. Sci. U.S.A. 92, 7844–7848
177 Thomson, J. A., Kalishman, J., Golos, T. G., Durning, M., Harris, C. P. and Hearn, J. P.
(1996) Pluripotent cell lines derived from common marmoset (Callithrix jacchus )
blastocysts. Biol. Reprod. 55, 254–259
178 Bongso, A., Fong, C. Y., Ng, S. C. and Ratnam, S. (1994) Isolation and culture of inner
cell mass cells from human blastocysts. Hum. Reprod. 9, 2110–2117
179 Stojkovic, M., Lako, M., Stojkovic, P., Stewart, R., Przyborski, S., Armstrong, L., Evans,
J., Herbert, M., Hyslop, L., Ahmad, S. et al. (2004) Derivation of human embryonic
stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells
22, 790–797
180 Strelchenko, N., Verlinsky, O., Kukharenko, V. and Verlinsky, Y. (2004) Morula-derived
human embryonic stem cells. Reprod. Biomed. Online 9, 623–629
181 Klimanskaya, I., Chung, Y., Becker, S., Lu, S. J. and Lanza, R. (2006) Human embryonic
stem cell lines derived from single blastomeres. Nature 444, 481–485
182 Lin, G., OuYang, Q., Zhou, X., Gu, Y., Yuan, D., Li, W., Liu, G., Liu, T. and Lu, G. (2007)
A highly homozygous and parthenogenetic human embryonic stem cell line derived
from a one-pronuclear oocyte following in vitro fertilization procedure. Cell Res. 17,
999–1007
183 Mai, Q., Yu, Y., Li, T., Wang, L., Chen, M. J., Huang, S. Z., Zhou, C. and Zhou, Q.
(2007) Derivation of human embryonic stem cell lines from parthenogenetic
blastocysts. Cell Res. 17, 1008–1019
23
184 Revazova, E. S., Turovets, N. A., Kochetkova, O. D., Kindarova, L. B., Kuzmichev, L. N.,
Janus, J. D. and Pryzhkova, M. V. (2007) Patient-specific stem cell lines derived from
human parthenogenetic blastocysts. Cloning Stem Cells 9, 432–449
185 Brons, I. G., Smithers, L. E., Trotter, M. W., Rugg-Gunn, P., Sun, B., Chuva de Sousa
Lopes, S. M., Howlett, S. K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R. A. and
Vallier, L. (2007) Derivation of pluripotent epiblast stem cells from mammalian
embryos. Nature 448, 191–195
186 Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., Mack, D. L.,
Gardner, R. L. and McKay, R. D. (2007) New cell lines from mouse epiblast
share defining features with human embryonic stem cells. Nature 448,
196–199
187 Matsui, Y., Zsebo, K. and Hogan, B. L. (1992) Derivation of pluripotential
embryonic stem cells from murine primordial germ cells in culture. Cell 70,
841–847
188 Resnick, J. L., Bixler, L. S., Cheng, L. and Donovan, P. J. (1992) Long-term
proliferation of mouse primordial germ cells in culture. Nature 359,
550–551
189 Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba,
S., Kato, T., Kazuki, Y., Toyokuni, S. et al. (2004) Generation of pluripotent stem cells
from neonatal mouse testis. Cell 119, 1001–1012
190 Guan, K., Nayernia, K., Maier, L. S., Wagner, S., Dressel, R., Lee, J. H., Nolte, J., Wolf,
F., Li, M., Engel, W. and Hasenfuss, G. (2006) Pluripotency of spermatogonial stem
cells from adult mouse testis. Nature 440, 1199–1203
191 Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M., Littlefield, J. W., Donovan, P. J.,
Blumenthal, P. D., Huggins, G. R. and Gearhart, J. D. (1998) Derivation of pluripotent
stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. U.S.A.
95, 13726–13731
Received 15 January 2010/4 March 2010; accepted 9 March 2010
Published on the Internet 28 April 2010, doi:10.1042/BJ20100102
c The Authors Journal compilation c 2010 Biochemical Society