Overview Articles
From One-Cell to Tissue:
Reprogramming, Cell
Differentiation and Tissue
Engineering
DONGHUI ZHANG AND WEI JIANG
Adult somatic cells can be reprogrammed into pluripotent stem cells, which can indefinitely proliferate in a dish and differentiate into almost all
the cell types that constitute the human body. These cells hold great promise for cell therapy and regenerative medicine, as well as drug screening
and disease modeling. Theoretically, we can create a source of versatile, therapeutic cells that could be genetically matched to any patient.
These pluripotent stem cells can be differentiated into desired cell types and eventually used to reconstruct a tissue or an organ with insights
gained from tissue engineering. Inspired by the pioneering work in stem cells, particularly the remarkable reprogramming technique, the fields
of regenerative medicine and tissue engineering have advanced rapidly in the past several years. Here recent progress in the fields of induced
pluripotent stem cells and tissue engineering is reviewed, with a focus on applications in medicine.
Keywords: stem cells, tissue engineering, cell differentiation, cell reprogramming, induced pluripotent stem cells (iPSCs)
P
luripotent stem cells, which include embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs), have an extraordinary capacity for self-renewal and
for differentiation into multiple lineages. The first human
ESC line was established in 1998 using fresh or frozen
cleavage-stage human embryos produced by in vitro fertilization (IVF) for clinical purposes (Thomson et al. 1998). This
finding provided a useful platform to study human development and cell differentiation. Scientists have made important
discoveries with this platform (Murry and Keller 2008);
however, the ethical issues surrounding the use of human
embryos and human eggs have greatly limited further study.
Furthermore, the different genetic or epigenetic backgrounds
of individual ESC lines endow them with different behavior
patterns in terms of lineage differentiation bias (Osafune
et al. 2008). Unfortunately, the few human ESC lines that
are already established cannot meet the challenges of disease
modeling from patient biopsy samples (Grskovic et al. 2011).
The development of the iPSC technique in 2006 addressed
these issues and revolutionized the field of stem cell research.
Making autologous pluripotent stem cells
In 2006, Takahashi and Yamanaka (2006) reported a
remarkable finding: They introduced 24 genes into mouse
fibroblasts, and, surprisingly, the fibroblast cells started to
express a pluripotent ESC-specific marker reporter. They
then narrowed the number of introduced genes down to
four (Oct3/4, Sox2, Klf4, and c-Myc) and further demonstrated that the fibroblast was “reprogrammed” to the naive
pluripotent state by germ-line transmission assay (Takahashi
and Yamanaka 2006). This technique was quickly adapted
to create human iPSC lines by many groups including
Yamanaka’s (Takahashi et al. 2007). Since the establishment
of the technique, more and more data have suggested that
iPSCs and ESCs share very similar characteristics in terms
of self-renewal and their ability to differentiate into multiple lineages. Because of the great interest in and promise
of cell therapy and regenerative medicine, many scientists
have been working extensively with iPSCs (for a review, see
Gonzalez et al. 2011, Han et al. 2010) and have made great
progress: They have extended the iPSC technique to different cell types and patient samples (Park et al. 2008), as well
as to other species including rhesus monkey (Liu et al. 2008);
they have reduced the number of factors needed down to
only one (Oct4) by using neural stem cells as starting material (Kim JB et al. 2009); they have identified new factors that
improve the proportion of treated cells that are successfully
reprogrammed from the original 0.02% to almost 100%
BioScience 65: 468–475. © The Author(s) 2015. Published by Oxford University Press on behalf of the American Institute of Biological Sciences. All rights
reserved. For Permissions, please e-mail: [email protected].
doi:10.1093/biosci/biv016
Advance Access publication 4 March 2015
468 BioScience • May 2015 / Vol. 65 No. 5
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Overview Articles
of immune rejection when iPSCs were
transplanted into mouse recipients with
Virus vector
the same genetic background. They
Growth factor
Nonintegrated
also found abnormal gene expression
mRNA, proteins
in some iPSCs and their derivatives,
which might be because of random tranSmall molecule
Ectoderm
IPSC
scriptional activation during reprogramSmall molecule
ming process, and that transplantation
Nuclear transfer
of the autologous-derived iPSCs induced
a T-cell-dependent immune response
(Zhao et al. 2011). Interestingly, another
Mesoderm
ESC
Somatic cell
research group repeated the same experiment but failed to observe any similar
Transdifferentiation /lineage reprogramming
immune rejection (Guha et al. 2013).
The inconsistency might be due to differences in the quality of generated iPSCs.
Endoderm
Although it remains to be clarified in
future studies, this conflict has raised the
Figure 1. An illustration of making multiple-lineage differentiated cells
possibility of problematic immunogenicfrom somatic cells. Somatic cells can be reprogrammed into either induced
ity even with transplantation of autolopluripotent stem cells (iPSCs), by delivering reprogramming factors via DNA
gous cells.
vector (including viral vector and nonintegrated vectors), mRNA or proteins,
Somatic cells can be reprogrammed
or reprogramming chemicals, or embryonic stem cells by nuclear transfer.
into a pluripotent state either with tranThe reprogrammed iPSCs and ESCs can be directed to all the three germ layer
scription factors (i.e., using the strategy
lineages. As an alternative, somatic cells can be directly induced to other lineages developed by Yamanaka and his colby overexpressing key lineage master genes (for a review, see Graf 2011).
leagues) or by nuclear transfer (figure 1).
Researchers have therefore also explored
the potential of nuclear transfer to generate autologous plu(Rais et al. 2013). In addition, because genetic manipulation
ripotent stem cells. Ethical objections have sharply limited
via the introduction of recombinant DNA into cells poses
such research, but recently three independent studies have
potential risks in clinical applications, many strategies have
suggested that nuclear transfer can indeed be used to generate
been explored to overcome such risks (figure 1). Of note,
human pluripotent stem cells. Soon after the establishment
direct delivery of synthetic modified mRNA (Warren et al.
of a technique for producing human ESCs by nuclear trans2010) has been shown to achieve reprogramming in human
fer (Tachibana et al. 2013), other groups rapidly successfully
cells; even more exciting, a combination of a handful of
repeated the protocol using fibroblasts from healthy adults
simple chemical compounds has been reported to repro(Chung et al. 2014) and an adult patient with type 1 diabegram mouse fibroblast into iPSCs, which could eliminate
tes (Yamada et al. 2014). In addition, two research groups
the need to use potentially oncogenic factors (Hou et al.
comprehensively compared the somatic mutations and gene
2013). Although these methods still require optimization
expression levels in a panel of human iPSC lines with those
and need to be independently demonstrated in human cells,
in ESC lines created via either IVF or somatic cell nuclear
it is reasonable to believe that robust non-DNA mediated
transfer. They performed genome-wide sequencing of DNA
reprogramming will be realistic in the near future.
methylation, copy number variations and transcriptome in
Even though iPSCs are quite similar to ESCs, there are
those lines. Interestingly, although Ma and colleagues found
a few notable differences that should be addressed before
iPSCs retained more residual DNA methylation patterns
iPSCs can find clinical applications. One concern is that the
typical of parental somatic cells and were therefore not as
cells have been subjected to genetic manipulation. However,
good as ESCs for cell replacement therapies (Ma et al. 2014),
this risk might be overcome either by developing vectors that
Johannesson and colleagues (2014) reported both nuclear
introduce factors into cells without integrating new genetic
transfer ESCs and iPSCs exhibited comparable genetic and
material into cellular DNA or by non-DNA-mediated reproepigenetic defects. Those confusions that should be clarified
gramming, as was discussed above. Another major concern
in future studies indicate current reprogramming technique
is the immunogenicity. Because iPSCs made from a patient
needs further improvement and that nuclear transfer might
have the same genetic background as that individual, we
be an alternative to a certain extent.
would expect there to be no severe immune rejection
upon transplantation of autologous iPSC-derivatives back
Lineage differentiation from pluripotent stem cells
to the patient. However, mouse studies indicate that this
Reprogrammed iPSCs show exceptional potential to differconfidence might not be well founded. Zhao and colentiate into multiple cell lineages and provide a promising
leagues (2011) reported no obvious graft formation because
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Overview Articles
source of specialized cells for cell therapy of degenerative
diseases such as Parkinson’s disease (because of the lack of
dopamine-generating neurons) and type 1 diabetes mellitus
(because of the loss of insulin-producing pancreatic beta
cells). Most important, autologous iPSC-derived functional
cells cause minimal immune rejection in principle when
transplanted back to patients. In addition, through differentiation of human iPSCs, the reprogramming technique provides a source of cells suitable for drug screening and safety
testing that avoids the use of primary human cells, which
are expensive and of which the supply is limited. Therefore,
lineage differentiation has become one of the top focuses in
the field of stem cell and tissue engineering.
During the past decade, many strategies have been
explored to guide the direct differentiation of iPSCs and
ESCs. At first, scientists simply removed the self-renewal
and let them spontaneously form “embryoid bodies”—a
self-assembling aggregate mimicking many of the hallmarks
of early embryonic development—when suspended in a culture medium. Their aim was for the cells to spontaneously
differentiate into all three germ layers. Indeed, many cell
types could be detected in the mixed cell population generated using this method, but the efficiency of production of
any particular desired cell type was extremely low (ItskovitzEldor et al. 2000). Researchers also cocultured human
ESCs with differentiated mouse cell types or physically
neighboring supporting cells with the idea that differentiated or surrounding cells might provide factors that would
amount to a “code” able to direct cell lineage (Mummery et
al. 2003). This strategy is not applicable to clinical purposes,
however. To further improve the differentiation efficiency,
some research groups have designed new approaches that
involve genetic manipulation of key transcription factors to
induce human ESCs to differentiate directly into desired cell
types. However, these differentiation methods may restrict
the use of the resulting cells in transplantation therapy
because the methods use exogenous DNA vectors to modify
the ESC genome. It is, therefore, very important to develop
better methods for the induction of human ESCs, ones that
result in a high efficiency of differentiation and increased
cell purity without the introduction of modifications to the
genome. Scientists have to this end applied insights from
basic research and developmental biology and developed
new stepwise strategies. They have developed ways to apply
growth factors and small chemical inhibitors and agonists in
a stepwise manner to achieve direct differentiation in a way
that mimics in vivo development. Multiple cell lineages have
been successfully generated this way (Williams et al. 2012),
such as the heart lineage, as is evidenced by the recent report
that human ESC-derived cardiomyocytes show certain celltypical functions when grafted into the heart of a nonhuman
primate (Chong et al. 2014).
Despite these fruitful efforts in direct cell differentiation, there are some important problems that remain to be
addressed before we can expect clinical applications. First,
the differentiation efficiency still does not reach 100%, so
470 BioScience • May 2015 / Vol. 65 No. 5
as well as desired cell types we always also acquire some
undefined cells. This complicates the functional testing of
the cells and presents safety complications. One solution
would be to continue to optimize our differentiation protocols and improve differentiation efficiency; alternatively, we
could find effective ways to enrich and purify the desired
cells. Several groups have recently identified surface markers for the pancreatic cell lineage (Jiang et al. 2011, Kelly
et al. 2011) and for cardiomyocytes (Dubois et al. 2011) in
human ESC/iPSCs differentiation system. Furthermore, we
know that human pluripotent stem cells have unique surface
markers. So positive and negative selection regimes could in
principle be combined to enrich and purify the desired cells
and eliminate the potential risk of proliferation of undesired
pluripotent cells.
Second, there are notable differences between different
human ESC and iPSC lines (Osafune et al. 2008). Some
scientists have recognized and risen to this challenge and
attempted to find biomarkers that will allow them to predict
the lineage differentiation bias. Recent successes include
the discovery of the role of the miR-371-3 cluster in neural
differentiation (Kim H et al. 2011) and of the role of WNT3
in endoderm lineage differentiation (Jiang et al. 2013). A
simple measurement of the expression level of such biomarkers in a panel of undifferentiated pluripotent stem cell
lines could reveal which line has the highest neural or endodermal lineage differentiation capacity, which would greatly
facilitate the later differentiation process.
For clinical and drug screening applications, large numbers of differentiated cells are necessary. Usually, billions of
terminally differentiated cells are required for a successful
individual transplantation, such as islet transplantation in
type 1 diabetes patients. Generating such large numbers of
differentiated cells is an emerging challenge. One option
is to expand iPSCs/ESCs at the pluripotent stage, at which
stage they can self-renew in an unlimited way. For example,
researchers at ViaCyte used an adherent culture format to
provide a virtually unlimited starting resource of pluripotent cells, and then manufactured differentiated cells in a
dynamic rotational suspension culture system. Eventually,
they optimized the procedure and performed numerous
scaled differentiation experiments that reproducibly generated populations of defined composition that were highly
enriched for pancreatic cell lineages (Schulz et al. 2012).
An alternative strategy is to expand progenitor cells at
the intermediate stage, without differentiation, using customized conditions in which the iPSC/ESC-derived cells
retain some proliferation capacity. A group from Harvard
University recently reported an expansion of more than a
million-fold of human endodermal cells with full retention of their developmental potential. This was achieved
by coculture of the cells with organ-matched mesenchyme
(Sneddon et al. 2012). An independent group established
endodermal progenitor cell lines from human ESC/iPSCs,
which allowed an expansion of almost 1016-fold, whereas
the morphology and gene expression pattern characteristic
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of endoderm were maintained (Cheng et al. 2012). More
important, clonally derived endodermal progenitor cells
could be differentiated into numerous endodermal lineages,
including glucose-responsive pancreatic beta cells, hepatocytes, and intestinal epithelia, upon manipulation of their
culture conditions in vitro or transplantation into mice
(Cheng et al. 2012). Taken together, these results demonstrate that expansion of progenitors offers an efficient way
of producing large numbers of differentiated cells from stem
cells at relatively low cost. The approach could therefore find
wide application in regenerative biology.
Functional maturation requires three-dimensional
structure
The strategy of using cellular signal activators and inhibitors discovered through basic research in developmental
biology has made possible huge achievements in terms of
generating multiple cell types via human ESC/iPSC differentiation. However, the approach hit a bottleneck when
attempts were made to use it for the extremely complicated problem of developing complex tissues and organs.
Organogenesis relies on collaborative signals from the
surrounding tissues. Epithelium–mesenchyme, epithelium–
endothelium, and endothelium–mesenchyme interactions
occur simultaneously and synergistically determine the
maintenance, regionalization, differentiation, and maturation of cells through patterned supportive or inhibitory
effects. Conventional monolayer cell cultures systems cannot provide the necessary three-dimensional structural and
mechanical information to support complex tissue development, and this limitation has prompted scientists to look for
other insights.
Initially, researchers combined the stepwise differentiation strategy with three-dimensional culture systems to treat
ESC/iPSC-derived differentiated progenitors. Spence and
colleagues (2011) applied this strategy to generate threedimensional intestinal organoids consisting of a polarized, columnar, epithelium. The tissue was patterned into
villus-like structures and crypt-like proliferative zones that
expressed intestinal stem cell markers (Spence et al. 2011).
A similar strategy was applied to achieve differentiation of
human iPSCs into brain cell types. The resulting cerebral
organoids could develop structures characteristic of various
brain regions in a dish. Further examination revealed a cerebral cortex containing organized progenitor populations that
produced mature cortical neuron subtypes, recapitulating
features of human cortical development and regionalization
(Lancaster et al. 2013). Takebe and colleagues (2013) further
applied this strategy by taking advantage of the self-assembly
between hepatocytes and supporting cells. They put human
iPSC-derived hepatic cells together with endothelial and
mesenchymal cells and found these cells self-organized into
liver-bud-like structures. More important, functional human
vasculatures quickly developed after the structures were
transplanted into mice and this stimulated the maturation
of hepatic functions comparable to those of the adult liver
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(Takebe et al. 2013). These pioneering studies collectively
show the significant advantages of organoid culture over
monolayer culture. This indicates the promise of providing
higher dimensional structures for regenerative medicine.
Tissue engineering in cell differentiation
Tissue engineering is “an interdisciplinary field that applies
the principles of engineering and life sciences toward the
development of biological substitutes that restore, maintain,
or improve tissue function or a whole organ,” as defined by
Langer and Vacanti (1993). The field has advanced greatly
with the accumulating knowledge of stem cell biology in
recent years (figure 2).
Every cell type has its unique shape and structure. Kilian
and colleagues (2010) demonstrated that cell shape, independently of soluble growth factors, has a strong influence
on the differentiation of human mesenchymal stem cells.
Geometric features that increased actomyosin contractility
could promote osteogenesis, and cytoskeleton-disrupting
pharmacological agents modulated shape-based trends in
lineage commitment. Together, these findings indicate that
geometric shape cues can play a decisive role in orchestrating the mechanochemical signals directing cell fates (Kilian
et al. 2010).
How can we control the shape that cells form? One simple
idea is by patterning the cells within a fixed shape using a
matrix with differential cell affinity. One successful example
used human liver cells. These cells are prone to dedifferentiate and quickly lose their hepatocyte functions during
conventional culture in a dish. Khetani and Bhatia (2008)
reported a type of optimized microscale architecture for
human liver cells. This culture system patterns liver cells
in small cell clusters and improves their survival as well as
maintains hepatic phenotypic functions, as was assessed
by gene expression profiles and physiological metabolism
(Khetani and Bhatia 2008). A further study (Stevens et al.
2013) achieved the microscale organization of many cell
types, within a variety of synthetic and natural extracellular
matrices. It demonstrated that compartment microstructure and cellular composition modulate hepatic functions,
further suggesting the importance of architectural optimization. Meanwhile, a very interesting report suggests that
physical effects influence cell lineage differentiation (Engler
et al. 2006). The authors used naive mesenchymal stem cells
as the starting material and grew them in different matrices
with variable elasticity. Surprisingly, they found that cell
lineage specification was extremely sensitive to tissue level
elasticity: Soft matrices that mimic brain are neurogenic,
stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone are
osteogenic (Engler et al. 2006). Inspired by this pioneering
study, another group established a model to comprehensively investigate the effect of matrix elasticity on myocytes’
contractility. Their finding indicated that matrix elasticity
dominates over intracellular elasticity and that myocytes
with lower aspect ratios have a functional advantage when
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kidney (Song et al. 2013) have been created. Additional data strongly suggested
Cell contact
that the decellularized matrix could support the survival and function of termiCell support
nally differentiated cells.
2D Cell pattern
The success of decellularized organs
and tissues as a scaffold for generation of
artificial organs has sparked a search for
new scaffold materials. Ideal candidates
should be biodegradable, demonstrate
3D
support
cell compatibility, be nontoxic, and be
Decellularization
Purification
available at low cost. For the scaffolds,
Function rebuild pore distribution, exposed surface area,
and porosity play a major role (O’Brien
Differentiated
Self-assembly
et al. 2005). Polymers both natural and
cells in mixture
synthetic are widely used in tissue engiNiche effect
Polymer-based 3D
population
neering. Collagen, an important comSignal transit
ponent of skin, bone, cartilage, and
blood-vessel walls, has been designed
as a scaffold product that takes the
form of a sheet used in the treatment of
Hydrogel-based 3D
burns (Delatte et al. 2001) and as a tube
used in blood vessels (Flanagan et al.
2006) and peripheral nerve regeneration
Figure 2. Various methods to engineer tissues. Purified desired cell types
(Gu et al. 2011). Silk fibroin exhibits good
derived from stem cells can be further engineered into a tissue or organ. ­
biocompatibility and is able to support
Two-dimensional (2D) cell patterning can provide the cell–cell interaction
the growth of human differentiated cells
and support. Three-dimensional (3D) engineering including decellularization,
polymer-based or hydrogel-based scaffold can provide the structure that further (Nunes et al. 2013). Synthetic degradable
polymers are also widely used as scaffold
functions as tissue or organ.
material in tissue engineering because of
their high versatility and because they
can be obtained in a consistent quality, which leads to
the elasticity of the extracellular matrix decreases because
reproducibility of results (Oh and Lee 2013). Compared
of conditions such as fibrosis (McCain et al. 2014). Taken
with natural polymers, synthetic polymers are always easier
together, these results indicate that artificially patterning
to process but generally are less biocompatible. By using
stem cells by mimicking the appropriate elasticity matrix or
microfabrication techniques, the polymers can be made into
natural cell shape will facilitate functional maturation.
unique three-dimensional structures, which usually mimic
Patterning cells in monolayers is apparently not enough,
the natural tissue morphology and can help the seeding cells
as the monolayer does not accurately reflect the anatomic
improve their biological function (Engelmayr et al. 2008).
interaction in a tissue. How do we correctly position cells
Hydrogels are another appealing scaffold material because
in a three-dimensional environment? The most straightforthey are structurally similar to the extracellular matrix of
ward strategy is using decellularized primary tissue, from
many tissues, can often be processed under relatively mild
which all the live cells have been removed while keeping the
conditions, and may be delivered in a minimally invasive
underlying structure intact. In the past decade, a variety of
manner such as injection. The hydrogel will help to maintain
decellularized methods, including tissue- or organ-specific
and facilitate the self-assembly of cells into natural tissue-like
physical, chemical, and enzymatic methods, have been sucstructure (Drury and Mooney 2003). One typical application
cessfully developed for different organs or tissues (Gilbert
of hydrogel is in cardiac tissue engineering. Zimmerman and
et al. 2006). Using an acellular nature matrix and reseeding
colleagues (2002) first reported an engineered heart tissue by
with proper endothelial or other types of cells, an artificial
mixing neonatal cardiac myocytes with collagen and matrix
organ can be generated. The first proof-of-concept trial was
factors, casting them in circular molds, and subjecting them
reported in 2008 by Ott and colleagues (2008): They decelto phasic mechanical stretch. These cells displayed imporlularized a rat heart and then reseeded the matrix with pritant hallmarks of differentiated myocardium (Zimmermann
mary cardiac and endothelial cells. They further found that
et al. 2002). After improving the technique, Zhang and colthese bioartificial hearts could maintain a pumping function
leagues (2002) developed a hydrogel patch that allowed prowhen cultured in a bioreactor for up to four weeks (Ott et
liferation and building up into a muscle network of human
al. 2008). Using similar strategies, bioartificial rodent organs
ESC-derived cardiomyocytes. This engineered patch greatly
such as liver (Uygun et al. 2010), lung (Ott et al. 2010), and
472 BioScience • May 2015 / Vol. 65 No. 5
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Ge
Di
ffe
re nom
nt
e
iat
ion ed
it
/P
ur ing
ific
at
ion
Treat patients
Purification
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Differentiation
by using gene editing technology, they
further demonstrated that correction of
the TAZ mutation can rescue the disReprogramming
Collect fibroblast
ease phenotype. Finally, linoleic acid was
tested for its ability to rescue the phenotype, which pointed to a new treatment
strategy for BTHS. Notably, the researchers took advantage of the combination of
BTHS patients
a monolayer culture with a two-dimensional cell pattern (to visualize sarcomere
3D Tissue
organization) and a three-dimensional
engineered “heart on a chip” (to measure
contractile stress generation by cultured
cardiomyocytes) to fully mimic the disease phenotype (Wang et al. 2014). This
excellent example of engineering strategy
combined with the iPSC technique to
model disease and test potential drug
Drug test
Function test
therapies initiates a new era (­figure 3).
The maturity of three-dimensional tissue
culture not only promises drug discovery
at the functional tissue level, but also
Figure 3. A summary of the remarkable work about disease modeling, which
facilitates regenerative therapy if comcombined induced pluripotent stem cells (iPSCs) and the heart-on-a-chip tissue bined with cutting-edge genome editing
engineering technique (Wang et al. 2014). In this study, patient-specific iPSCs
techniques.
were generated from two Barth syndrome (BTHS) individuals who were caused
Safety is always a concern in the clinic,
by TAZ mutation. They first differentiated the patient-specific iPSCs into
because of the ability of pluripotent stem
cardiomyocytes (CM) and modeled the BTHS in cell level; they assayed some
cells to self-renew. Recent studies have,
known chemicals to test the rescue effect with the hope to search for new drugs
however, provided promising insights.
to treat BTHS. Moreover, they used the cutting-edge gene editing technique and Metabolic activity is different in undiffound the disease phenotype can be rescued by correcting the TAZ mutation,
ferentiated cells and terminally differenwhich provided the hope that autologous iPSC-derived cardiomyocytes with
tiated cells, which prompts scientists to
corrected mutation could be engineered and transplantable. Abbreviations: 2D, search for chemicals that specifically kill
two dimensional; 3D, three dimensional.
undifferentiated stem cells. For instance,
Hirata and colleagues (2014) screened
fluorescent chemical libraries with human iPSCs and identiimproves the electrophysiological function of cardiomyofied a fluorescent compound, KP-1, which could selectively
cytes (Zhang et al. 2013).
label human pluripotent stem cells. They further reported
that this selectivity was mainly due to a distinct expression
Conclusions and challenges
pattern of transporter genes and the transporter selectivTissue engineering and regenerative medicine have grown
ity of KP-1 (Hirata et al. 2014). Similarly, another group
quickly and will reinvent humanity’s future. Engineered tisdemonstrated that cytotoxic small molecules with approprisues could be used for drug screening instead of the rare and
ate selectivity for ATP-binding cassette transporters could
expensive primary human tissues. Most important, because
specifically kill human pluripotent stem cells (Kuo et al.
patient-specific iPSCs can be robustly generated from the
2014). Another recent paper revealed the unique requiresomatic cells, disease modeling and personalized drug
ment of proliferative ESCs for histone deacetylase 1 and 2
screening should be feasible. A recent study successfully
(HDAC1/2) activity. The researchers found that deleting
applied the combination of stem cell techniques and tissue
HDAC1/2 could make ESCs lose their viability, which sugengineering to model disease (Wang et al. 2014). In this
gests that an HDAC1/2 inhibitor might be able to selectively
study, patient-specific iPSCs were generated from two indikill proliferative ESCs and progenitor cells (Jamaladdin et
vidual Barth syndrome (BTHS) patients. The disease, which
al. 2014). A variety of such methods could be combined to
leads to abnormalities of mitochondrial function that affect
eliminate the risk of undesired cell proliferation.
many tissues, especially heart tissue, is caused by a single
Functionality of the generated organs or tissues is another
mutation in gene TAZ which encodes the phospholipidmajor concern in applications. Organ is composed of mullysophospholipid transacylase Tafazzin. The researchers
tiple cell types with well-organized structure, and the hetfirst proved that the mitochondrial abnormality was preserogeneity of cells that make up a particular organ presents
ent in patient-specific iPSC-derived cardiomyocytes; then,
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Overview Articles
an additional challenge. For instance, scientists have recently
made the breakthrough that generating insulin-secreting
pancreatic beta cells in vitro from human pluripotent stem
cells is successfully achieved (Pagliuca et al. 2014, Rezania
et al. 2014), but it meets a huge challenge to make a functional pancreas or even a much smaller islet unit. Great
progresses have been achieved in the past years: Pluripotent
stem cell derived organoids could recapture hallmarks of
organogenesis to a certain extent (Lancaster and Knoblich
2014); the technology of decellularization and recellularization to engineer entire organs is under development
(Badylak et al. 2011). Notably, Kamao and colleagues (2014)
reported the robust generation of retinal pigment epithelium cell sheets from human iPSCs and recently initiated a
clinical trial to treat the severe visual impairment caused by
­age-related macular degeneration. Despite those advancements, the challenge of how to generate a functional tissue
or organ with relatively higher complexity still exists and
requires collaborative multidisciplinary expertise.
Other issues, such as production of large amount of
clinically relevant and functionally mature cells with high
purity at a reasonable cost (Chen et al. 2014), the elimination of immunogenicity, and tumorigenecity due to in vitro
manipulation (Lee et al. 2013), should be intensively studied
before clinical application. However, the now well-developed reprogramming technique, combined with advanced
tissue engineering strategies, is initiating a new era in cell
therapy and disease modeling, as well as in drug discovery.
Moreover, the rapid progression of other technologies, such
as genome editing (Wang et al. 2014) should greatly facilitate the development of stem cell and tissue engineering;
meanwhile, achievements in stem cell and tissue engineering can also provide invaluable research platforms and
insights to help the study of human development and disease
pathogenesis.
Acknowledgments
We thank Katie Kester for critical reading. We apologize
for papers that are not directly cited because of space limitations. WJ is supported by a Juvenile Diabetes Research
Foundation postdoctoral fellowship. The authors declare no
competing financial interests.
References cited
Badylak SF, Taylor D, Uygun K. 2011. Whole-organ tissue engineering: Decellularization and recellularization of three-dimensional
matrix scaffolds. Annual Review of Biomedical Engineering 13:
27–53.
Chen KG, Mallon BS, McKay RD, Robey PG. 2014. Human pluripotent
stem cell culture: Considerations for maintenance, expansion, and
therapeutics. Cell Stem Cell 14: 13–26.
Cheng X, et al. 2012. Self-renewing endodermal progenitor lines generated
from human pluripotent stem cells. Cell Stem Cell 10: 371–384.
Chong JJ, et al. 2014. Human embryonic-stem-cell-derived cardiomyocytes
regenerate non-human primate hearts. Nature 510: 273–277.
Chung YG, et al. 2014. Human somatic cell nuclear transfer using adult
cells. Cell Stem Cell 14: 777–780.
474 BioScience • May 2015 / Vol. 65 No. 5
Delatte SJ, Evans J, Hebra A, Adamson W, Othersen HB, Tagge EP. 2001.
Effectiveness of beta-glucan collagen for treatment of partial-thickness
burns in children. Journal of Pediatric Surgery 36: 113–118.
Drury JL, Mooney DJ. 2003. Hydrogels for tissue engineering: scaffold
design variables and applications. Biomaterials 24: 4337–4351.
Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG,
Gramolini A, Keller G. 2011. SIRPA is a specific cell-surface marker for
isolating cardiomyocytes derived from human pluripotent stem cells.
Nature Biotechnology 29: 1011–1018.
Engelmayr GC Jr, Cheng M, Bettinger CJ, Borenstein JT, Langer R, Freed
LE. 2008. Accordion-like honeycombs for tissue engineering of cardiac
anisotropy. Nature Materials 7: 1003–1010.
Engler AJ, Sen S, Sweeney HL, Discher DE. 2006. Matrix elasticity directs
stem cell lineage specification. Cell 126: 677–689.
Flanagan TC, Wilkins B, Black A, Jockenhoevel S, Smith TJ, Pandit AS.
2006. A collagen-glycosaminoglycan co-culture model for heart valve
tissue engineering applications. Biomaterials 27: 2233–2246.
Gilbert TW, Sellaro TL, Badylak SF. 2006. Decellularization of tissues and
organs. Biomaterials 27: 3675–3683.
Gonzalez F, Boue S, Izpisua Belmonte JC. 2011. Methods for making
induced pluripotent stem cells: Reprogramming a la carte. Nature
Reviews Genetics 12: 231–242.
Graf T. 2011. Historical origins of transdifferentiation and reprogramming.
Cell Stem Cell 9: 504–516.
Grskovic M, Javaherian A, Strulovici B, Daley GQ. 2011. Induced pluripotent stem cells: Opportunities for disease modelling and drug discovery.
Nature Reviews Drug Discovery 10: 915–929.
Gu X, Ding F, Yang Y, Liu J. 2011. Construction of tissue engineered nerve
grafts and their application in peripheral nerve regeneration. Progress
in Neurobiology 93: 204–230.
Guha P, Morgan JW, Mostoslavsky G, Rodrigues NP, Boyd AS. 2013. Lack of
immune response to differentiated cells derived from syngeneic induced
pluripotent stem cells. Cell Stem Cell 12: 407–412.
Han W, Zhao Y, Fu X. 2010. Induced pluripotent stem cells: The dragon
awakens. BioScience 60: 278–285.
Hirata N, et al. 2014. A chemical probe that labels human pluripotent stem
cells. Cell Reports 6: 1165–1174.
Hou P, et al. 2013. Pluripotent stem cells induced from mouse somatic cells
by small-molecule compounds. Science 341: 651–654.
Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M,
Soreq H, Benvenisty N. 2000. Differentiation of human embryonic stem
cells into embryoid bodies compromising the three embryonic germ
layers. Molecular Medicine 6: 88–95.
Jamaladdin S, Kelly RD, O’Regan L, Dovey OM, Hodson GE, Millard
CJ, Portolano N, Fry AM, Schwabe JW, Cowley SM. 2014. Histone
deacetylase (HDAC) 1 and 2 are essential for accurate cell division and
the pluripotency of embryonic stem cells. Proceedings of the National
Academy of Sciences 111: 9840–9845.
Jiang W, Sui X, Zhang D, Liu M, Ding M, Shi Y, Deng H. 2011. CD24: a
novel surface marker for PDX1-positive pancreatic progenitors derived
from human embryonic stem cells. Stem Cells 29: 609–617.
Jiang W, Zhang D, Bursac N, Zhang Y. 2013. WNT3 is a biomarker capable
of predicting the definitive endoderm differentiation potential of
hESCs. Stem Cell Reports 1: 46–52.
Johannesson B, et al. 2014. Comparable frequencies of coding mutations
and loss of imprinting in human pluripotent cells derived by nuclear
transfer and defined factors. Cell Stem Cell 15: 634–642.
Kamao H, Mandai M, Okamoto S, Sakai N, Suga A, Sugita S, Kiryu J,
Takahashi M. 2014. Characterization of human induced pluripotent
stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports 2: 205–218.
Kelly OG, et al. 2011. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nature
Biotechnology 29: 750–756.
Khetani SR, Bhatia SN. 2008. Microscale culture of human liver cells for
drug development. Nature Biotechnology 26: 120–126.
http://bioscience.oxfordjournals.org
Overview Articles
Kilian KA, Bugarija B, Lahn BT, Mrksich M. 2010. Geometric cues for
directing the differentiation of mesenchymal stem cells. Proceedings of
the National Academy of Sciences 107: 4872–4877.
Kim H, Lee G, Ganat Y, Papapetrou EP, Lipchina I, Socci ND, Sadelain M,
Studer L. 2011. miR-371-3 expression predicts neural differentiation
propensity in human pluripotent stem cells. Cell Stem Cell 8: 695–706.
Kim JB, Greber B, Arauzo-Bravo MJ, Meyer J, Park KI, Zaehres H, Scholer
HR. 2009. Direct reprogramming of human neural stem cells by OCT4.
Nature 461: 649–643.
Kuo TF, et al. 2014. Selective elimination of human pluripotent stem cells by
a marine natural product derivative. Journal of the American Chemical
Society 136: 9798–9801.
Lancaster MA, Knoblich JA. 2014. Organogenesis in a dish: Modeling
development and disease using organoid technologies. Science 345:
1247125.
Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME,
Homfray T, Penninger JM, Jackson AP, Knoblich JA. 2013. Cerebral
organoids model human brain development and microcephaly. Nature
501: 373–379.
Langer R, Vacanti JP. 1993. Tissue engineering. Science 260: 920–926.
Lee AS, Tang C, Rao MS, Weissman IL, Wu JC. 2013. Tumorigenicity as a
clinical hurdle for pluripotent stem cell therapies. Nature Medicine 19:
998–1004.
Liu H, et al. 2008. Generation of induced pluripotent stem cells from adult
rhesus monkey fibroblasts. Cell Stem Cell 3: 587–590.
Ma H, et al. 2014. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511: 177–183.
McCain ML, Yuan H, Pasqualini FS, Campbell PH, Parker KK. 2014. Matrix
elasticity regulates the optimal cardiac myocyte shape for contractility.
American Journal of Physiology Heart and Circulatory Physiology 306:
H1525–1539.
Mummery C, et al. 2003. Differentiation of human embryonic stem cells
to cardiomyocytes: Role of coculture with visceral endoderm-like cells.
Circulation 107: 2733–2740.
Murry CE, Keller G. 2008. Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell
132: 661–680.
Nunes SS, et al. 2013. Biowire: A platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nature Methods 10: 781–787.
O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. 2005. The effect of pore size
on cell adhesion in collagen-GAG scaffolds. Biomaterials 26: 433–441.
Oh SH, Lee JH. 2013. Hydrophilization of synthetic biodegradable polymer
scaffolds for improved cell/tissue compatibility. Biomedical Materials 8
(art. 014101).
Osafune K, Caron L, Borowiak M, Martinez RJ, Fitz-Gerald CS, Sato Y,
Cowan CA, Chien KR, Melton DA. 2008. Marked differences in differentiation propensity among human embryonic stem cell lines. Nature
Biotechnology 26: 313–315.
Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, Taylor DA.
2008. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nature Medicine 14: 213–221.
Ott HC, Clippinger B, Conrad C, Schuetz C, Pomerantseva I, Ikonomou L,
Kotton D, Vacanti JP. 2010. Regeneration and orthotopic transplantation of a bioartificial lung. Nature Medicine 16: 927–933.
Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH,
Peterson QP, Greiner D, Melton DA. 2014. Generation of functional
human pancreatic beta cells in vitro. Cell 159: 428–439.
Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch
MW, Cowan C, Hochedlinger K, Daley GQ. 2008. Disease-specific
induced pluripotent stem cells. Cell 134: 877–886.
http://bioscience.oxfordjournals.org
Rais Y, et al. 2013. Deterministic direct reprogramming of somatic cells to
pluripotency. Nature 502: 65–70.
Rezania A, et al. 2014. Reversal of diabetes with insulin-producing
cells derived in vitro from human pluripotent stem cells. Nature
Biotechnology 32: 1121–1133.
Schulz TC, et al. 2012. A scalable system for production of functional
pancreatic progenitors from human embryonic stem cells. PLOS ONE
7 (art. e37004).
Sneddon JB, Borowiak M, Melton DA. 2012. Self-renewal of embryonicstem-cell-derived progenitors by organ-matched mesenchyme. Nature
491: 765–768.
Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. 2013.
Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nature Medicine 19: 646–651.
Spence JR, et al. 2011. Directed differentiation of human pluripotent stem
cells into intestinal tissue in vitro. Nature 470: 105–109.
Stevens KR, et al. 2013. InVERT molding for scalable control of tissue
microarchitecture. Nature Communications 4: 1847.
Tachibana M, et al. 2013. Human embryonic stem cells derived by somatic
cell nuclear transfer. Cell 153: 1228–1238.
Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell
126: 663–676.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K,
Yamanaka S. 2007. Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131: 861–872.
Takebe T, et al. 2013. Vascularized and functional human liver from an
iPSC-derived organ bud transplant. Nature 499: 481–484.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,
Marshall VS, Jones JM. 1998. Embryonic stem cell lines derived from
human blastocysts. Science 282: 1145–1147.
Uygun BE, et al. 2010. Organ reengineering through development of a
transplantable recellularized liver graft using decellularized liver matrix.
Nature Medicine 16: 814–820.
Wang G, et al. 2014. Modeling the mitochondrial cardiomyopathy of Barth
syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nature Medicine 20: 616–623.
Warren L, et al. 2010. Highly efficient reprogramming to pluripotency and
directed differentiation of human cells with synthetic modified mRNA.
Cell Stem Cell 7: 618–630.
Williams LA, Davis-Dusenbery BN, Eggan KC. 2012. SnapShot: Directed
differentiation of pluripotent stem cells. Cell 149: 1174–1174 e1171.
Yamada M, et al. 2014. Human oocytes reprogram adult somatic nuclei
of a type 1 diabetic to diploid pluripotent stem cells. Nature 510:
533–536.
Zhang D, Shadrin IY, Lam J, Xian HQ, Snodgrass HR, Bursac N. 2013.
Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials 34:
5813–5820.
Zhao T, Zhang ZN, Rong Z, Xu Y. 2011. Immunogenicity of induced pluripotent stem cells. Nature 474: 212–215.
Zimmermann WH, Schneiderbanger K, Schubert P, Didie M, Munzel F,
Heubach JF, Kostin S, Neuhuber WL, Eschenhagen T. 2002. Tissue
engineering of a differentiated cardiac muscle construct. Circulation
Research 90: 223–230.
Donghui Zhang ([email protected]) is affiliated with the
Department of Cardiology and Wei Jiang ([email protected])
is affiliated with the Program in Cellular and Molecular Medicine, at Boston
Children’s Hospital, Boston, Massachusetts.
May 2015 / Vol. 65 No. 5 • BioScience 475