FOCUS ON STEM CELLS

REVIEW

Harnessing the potential of induced pluripotent stem

cells for regenerative medicine
Sean M. Wu and Konrad Hochedlinger

The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small
combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment
of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here
we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and
biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the
molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in
regenerative medicine.

Medical advances within the past century, such as the discovery of of iPSC technology in disease modelling and drug screening, and could
antibiotics and the development of vaccines, have led to remarkable enable autologous cell transplantation in clinical therapy in the future. As
breakthroughs in our ability to treat and even cure some of the most our understanding of the inherent similarities and differences between
challenging ailments. The recent finding that pluripotency can be ESCs and iPSCs improve3, we will be better equipped to tackle challenges
induced in somatic cells may represent yet another key discovery in the that have hampered the use of ESCs in clinical and translational
area of drug discovery and cell-based therapy. applications thus far (see Box 1).
The search for a method to induce developmental reprogramming of a Here, we explore the growing interest in using disease-specific iPSCs
somatic cell into an embryonic state stems from seminal frog studies that as an in vitro platform for drug screening and disease pathway discovery.
demonstrated that differentiated cell nuclei introduced into enucleated Given the potential for iPSCs to serve as a source of cell replacement
oocytes support the development of genetically identical animals or in degenerative diseases, we will also discuss recent preclinical animal
clones1–3. Cloned animals were also later produced in mammalian studies using iPSC derivatives in cell-based therapy and outline the
species4–9. However, the identity of the cocktail of factors from the challenges to be overcome before the full potential of iPSC technology
oocyte cytoplasm that was reverting the differentiated nucleus to its can be realized in pharmaceutical and clinical applications.
primitive state remained elusive. By systematically examining the effect
of pluripotency-specific transcription factors on fibroblasts, Takahashi iPSC-based disease modelling
and Yamanaka discovered in 2006 that retroviral expression of a set of The ability to generate pluripotent cell lines from patients afflicted
four genes (Oct4, Sox2, Klf4 and c-Myc) converted somatic cells into a with diseases of known and suspected aetiologies should allows us to
pluripotent state, albeit at an extremely low efficiency10. These iPSCs obtain, in theory, genetically matched cell types from all major organs
exhibited transcriptional and epigenetic features that were highly of interest in unlimited quantity. Indeed, recent studies have described
similar to those of embryonic stem cells (ESCs)11–13. Different groups the generation of iPSC lines from patients with a full range of genetically
subsequently repeated these findings with human cells14–16. inherited as well as sporadic diseases (Table 1). In most cases, in vitro
Remarkable progress made in reprogramming technology over the differentiation of iPSCs to the cell type relevant to the disorder has been
past few years has facilitated the generation of virus-free and/or vector- reported, and there are now many studies that suggest that patient-
free iPSCs, eliminating the potential risk of virally-induced tumour specific iPSCs exhibit certain disease features. For example, a progressive
formation17–23. iPSCs have been derived at increased efficiencies loss of motor neurons was observed during in vitro differentiation of
from several easily accessible human cell types, including blood cells, iPSCs derived from spinal muscular atrophy (SMA) patients, which
keratinocytes and dermal fibroblasts23–27. These and other advances now may reflect the developmental loss of motor neurons seen during this
allow basic and translational scientists to develop strategies for the use disease28. Similarly, cardiomyocytes derived from iPSCs from patients

Sean M. Wu is at the Cardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Boston 02114, Massachusetts, USA and the Harvard
Stem Cell Institute, Cambridge, Massachusetts 02138, USA. Konrad Hochedlinger is at the Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA;
the Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, USA; the Department of Stem Cell
and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA and the Howard Hughes Medical Institute, Department of Stem Cell and
Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA
e-mail: khochedlinger@helix.mgh.harvard.edu or smwu@partners.org

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associated with in vitro differentiation of iPSCs derived from SMA

BOX 1 Challenges in pluripotent stem cell clinical trials
patients was ameliorated by treatment with small-molecule candidates
The recent trial by Geron using human ESC-derived oligodendrocytes that reverse disease features in other neuronal culture assays28. Likewise,
for patients with spinal cord injury provides a salient example of the sensory neuron defect of familial-dysautonomia-iPSCs and the
the challenges facing pluripotent stem cell therapy105. During the synapse defect of RETT-iPSCs were partially restored on exposure
initial stage of FDA filing, graft-derived microscopic cysts were of cells to previously reported candidate drugs32,33. Thus, iPSCs from
found in mice transplanted with cell preparations enriched for human patients suffering from various diseases can be used to bridge the
human ESC-derived oligodendrocytes. Subsequently, extensive gap between small animal models, which may not always reflect the true
analyses were performed to evaluate the lot-to-lot differences in human disease phenotype, and clinical testing, which is expensive and
cyst formation and to assess whether an in vitro assay could predict time consuming. However, it would be premature to assume that small
this potential. Furthermore, safety data from a 9-month follow-up of molecules active in iPSC-based assays in vitro would be immediately
these cyst-containing grafts was demanded by the FDA. Geron has ready for clinical studies in humans. Further evaluation of their ADME
recently demonstrated that the microscopic cysts do not represent (absorption, distribution, metabolism and excretion) properties and of
a manifestation of teratoma and are not harmful. Consequently, the their toxicity and efficacy will need to be done in animal models if they
clinical advisory panel of the FDA granted permission to Geron to have not been tested previously in human patients.
resume enrolment of patients for the trial. It is worth noting that there have been no published studies that
It will be interesting to see whether other human ESC-based use diseased iPSCs in a high-throughput screening platform to
clinical studies will encounter similar issues. For example, Advanced discover novel small molecules that can potentially reverse a disease
Cell Technology has recently received approval by the FDA to phenotype. However, given the converging interests of chemical
conduct a Phase I/II clinical trial using human ESC-derived retinal and stem cell biology, successful applications of such therapeutic
pigment epithelial cells to treat Stargardt disease and age-related screens using diseased iPSCs are expected in the near future. Besides
macular regeneration. Likewise, Novocell will perform trials using providing a powerful tool for drug discovery, iPSC technology may
human ESC-derived pancreatic progenitor cells for the treatment allow researchers to model pre-symptomatic abnormalities in patient-
of type 1 diabetes. Although immunorejection of allogeneic grafts derived cells that could yield valuable insights into disease mechanisms
remains a serious challenge in hESC-based trials, a recent study and may lead to the development of diagnostic tools and drugs for
found that short-term suppression of leukocyte co-stimulatory early intervention.
molecules could significantly improve engraftment efficiencies of
both hESC- and hiPSC-derived cells in mice106. iPSCs in cell therapy
One of the most exciting aspects of iPSC technology is the possibility
with LEOPARD syndrome were found to be enlarged, possibly reflecting of generating autologous cells for cell-replacement therapy (Fig. 1).
the hypertrophic cardiomyopathy associated with this disease29. Patients The somatic origin of iPSCs has minimized but not eliminated some
suffering from Long QT and Timothy syndrome exhibit increased QT of the challenges that have hampered the development of human
intervals on electrocardiography, and differentiated cardiomyocytes ESC-based therapies. As cell transplantation for tissue repair outside
produced from iPSCs from such patients had prolongation of action of the haematopoietic and skin systems is a relatively nascent area of
potentials in single-cell electrophysiological assays30,31. iPSCs derived investigation, the regulatory requirements for the approval of pluripotent
from methyl CpG binding protein 2 (MeCP2)-deficient female patients stem cell-derived cells in clinical studies remain extremely high. A recent
with RETT syndrome give rise to glutamatergic neurons with fewer trial by the biopharmaceutical company Geron provides insights into the
synapses and decreased calcium transients when compared with challenges that lie ahead (see Box 1).
controls, as is usually seen in RETT patients32. Familial dysautonomia- Ultimately, our ability to bring pluripotent stem cell biology into
derived iPSCs exhibit decreased neurogenic differentiation and cell-based therapy will depend on the efficiency of cell-lineage-
migration behaviours, compared with control iPSCs33. Two recent studies specific differentiation, efficiency of cell purification to eliminate
investigated the disease phenotypes of iPSCs derived from Hutchinson- the risk of teratoma, and development of novel cell delivery methods
Gilford progeria patients and found that the differentiated smooth to introduce cells of interest into relevant organs (Fig. 1). Despite a
muscle cells had premature senescence, demonstrating that vascular decade of research on the mechanisms driving human pluripotent
defects seen in patients could also be observed in vitro34,35. Because some stem cell differentiation, it remains challenging to reliably generate
of these studies were performed with rather low numbers of iPSC lines large quantities of well-differentiated and functional cells from human
or used ESC lines instead of iPSC lines from unaffected individuals as ESCs or iPSCs.
controls (Table 1), it remains an open question how reproducible the With regards to transplantation of iPSCs for therapeutic regeneration,
observed phenotypes are when larger sets of genetically matched patient the most compelling study so far showed that haematopoietic cells
and control cell lines are being compared with each other. Nevertheless, derived from iPSCs can reduce the blood cell phenotype in a humanized
these in vitro studies provide the first proof-of-principle that disease mouse model of sickle cell anemia36. iPSCs were derived from a transgenic
modelling using iPSC technology may indeed be feasible. mouse carrying a mutation in the human haemoglobin sequence and
Although production of disease phenotypes from differentiated then genetically corrected through homologous recombination. In vitro
iPSCs in vitro is the necessary first step towards disease modelling, the differentiation of the ‘corrected’ iPSCs into haematopoietic progenitors
identification of novel pathways or drugs that could affect the disease and subsequent transplantation into the original transgenic mice
process is the ultimate goal of this approach (Fig. 1). The loss of neurons resulted in restoration of normal haemoglobin levels and an improved

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Table 1 Summary of published human iPSC disease models
Genetic Number Control Drug
Disease type Disease name Cell type Phenotype Reference
cause of lines line test
Dopaminergic
Neurological Parkinson’s disease Polygenic 23 hiPSC No obvious defect ND 20
neurons
Polygenic
Dopaminergic Neuronal death with
(with LRRK2 4 hiPSC Yes 51
neurons chemicals
mutation)
Amyotrophic lateral
Polygenic 3 Motor neurons hESC ND ND 107
sclerosis
Loss of neuron
Spinal muscular
Monogenic 2 Motor neurons hiPSC formation, loss of SMN Yes 28
atrophy
gene expression
Familial hiPSC,
Monogenic 2 Neural crest cells Loss of neural crest cells Yes 33
dysautonomia hESC
Loss of synapses, reduced
RETT syndrome Monogenic 4 Neurons hiPSC spine density, smaller Yes 32
soma size
hiPSC,
Huntington’s disease Monogenic 2 ND ND ND 108
hESC

Friedreich ataxia Monogenic 6+ ND hESC Changes GAA-TTC repeat ND 109

hiPSC, Corrected loss of FANCA

Blood Fanconi anaemia Monogenic 19 Blood cell ND 110
hESC function
hiPSC,
Fragile X syndrome Monogenic 11 ND Loss of FMR1 expression ND 111
hESC
Cardiac and Increased cardiomyocyte
Long QT 1 syndrome Monogenic 6 Cardiomyocytes hiPSC Yes 30
vascular depolarization
Not Increased cardiomyocyte
Long QT 2 syndrome Monogenic Cardiomyocytes hiPSC Yes 112
reported depolarization
Increased cardiomyocyte
hiPSC,
LEOPARD syndrome Monogenic 6 Cardiomyocytes size, decreased MAPK ND 29
hESC
signalling
Increased cardiomyocyte
Timothy syndrome Monogenic 16 cardiomyocytes hiPSC Yes 31
depolarization
Smooth muscle
Hutchinson Gilford hiPSC, Smooth muscle and mes-
Monogenic 4 cells, mesenchymal ND 34
Progeria hESC enchymal cell apoptosis
stem cells
Smooth muscle cell
Smooth muscle
Monogenic 6 hiPSC nuclear morphology and ND 35
cells
ageing phenotype
Duchenne muscular hiPSC,
Monogenic 2 ND ND ND 108
dystrophy hESC
Insulin- and glu-
Pancreatic Type 1 diabetes Polygenic 4 cagon- hESC ND ND 113
producing cells
A1-antitrypsin Loss of A1-antitrypsin
Hepatic Monogenic 19 Hepatocytes hiPSC Yes 114
deficiency expression
hiPSC,
Others Prader-Willi syndrome Monogenic 4 Neurons Imprint disorder ND 115
hESC
Angelman and Prader- hiPSC, Loss of paternal UBE3A
Monogenic 13 Neurons ND 116
Willi syndrome hESC expression
hiPSC,
Down syndrome Monogenic 2 ND ND ND 108
hESC
ND – not determined

phenotype. Although this rescue is remarkable, the fact that retroviral Similar transplantation-based approaches have been reported for other
HoxB4-transduced haematopoietic progenitor cells were used is a caveat, organs. For example, partially-purified dopaminergic neurons derived
and it remains to be seen whether similar rescue effects can be obtained from mouse iPSCs improved the clinical symptoms of a rat model of
using non-HoxB4 transduced cells. More importantly, a bona fide Parkinson’s disease37. Likewise, transplantation of human iPSC-derived
haematopoietic stem cell with the capacity for long-term multilineage cells into experimentally injured rodent heart showed some degree of
reconstitution has yet to be generated from human iPSCs. Thus, the short-term functional improvement in cardiac contractile function38,39.
translational potential of this strategy for sickle cell anaemia patients These examples of successful transplantation of iPSC-derived mesodermal
remains unclear. and ectodermal cells into animals involved the cell lineages that are more

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- Reproduction of disease-
specific phenotype
- Scalability of assay
for drug screens - In vivo efficacy
Drug screening - Toxicity in large
(’Disease modelling’) population

Treatment of
multiple patients

+
Patient Patient-specific Specialized cells Cell therapy Single
iPSCs treated patient
- Detection of - Availability of efficient - Successful engraftment - In vivo efficacy
high-quality differentiation protocols - Teratoma risk - Costs associated
iPSCs - Proper preclinical - Maturation level and with patient-specific
animal models functionality of cells therapy

Figure 1 Schematic representation of the potential utility of iPSC technology in cultured iPSC-derived cells as it occurs in the patient. Such a phenotype
in regenerative medicine. Introduction of reprogramming factors, such as could be employed to model this disease for mechanistic studies as well
Oct4, Sox2, Klf4 and c-Myc, into somatic cells of patients (for example, as for large-scale drug screening efforts to identify compounds that could
skin cells, keratinocytes or blood cells) gives rise to iPSCs. These patient- be used to treat any patient suffering from the same disease. The idea
specific iPSCs can then be differentiated into a variety of specialized cell behind cell therapy is to generate autologous specialized cells from iPSCs
types for a potential use in disease modelling (top) or cell therapy (bottom). for transplantation into individual patients. Shown in purple are the current
The concept behind disease modelling is to reproduce a cellular phenotype limitations in using iPSC technology in regenerative medicine.

easily produced from pluripotent stem cells (for example, neurons, blood (Fig. 1). The most critical issues are whether the relevant disease
and cardiomyocytes)40. The generation of endodermal lineage cells with phenotypes can be faithfully reproduced in vitro and, if so, whether
bona fide differentiated characteristics has been more difficult. However, they can accurately predict disease behaviour in vivo. Despite promising
researchers have recently succeeded in generating insulin-producing cells41, studies suggesting that certain features of familial dysautonomia, SMA
hepatocytes42, anterior foregut endoderm43 and intestinal cells44 from and RETT syndrome can be generated using iPSC-derived neural
human pluripotent stem cells. As previously shown for the derivation of cells, other neurological disorders such as Parkinson’s disease seem
motor neurons from mouse ESCs45, the exposure of pluripotent cell lines more difficult to model so far20. Three main factors may influence the
to growth factors encountered by embryonic cells from that tissue during amenability of diseases to in vitro modelling: the onset of disease in
normal development improves differentiation towards the lineage of choice. patients, the cell-autonomous nature of the disorder and the complexity
Generating chimaeric animals could also allow the production of of the underlying genetic defects. For example, evidence from animal
endodermal tissue. In a recent study, wild-type iPSCs were injected models and clinical data indicate that familial dysautonomia, SMA
into the blastocysts of mice unable to form a normal pancreas (Pdx1 and RETT syndrome manifest early in life, may have a strong cell-
mutants)46 resulting in chimaeric mice that harboured pancreases autonomous component and are caused by mutations in single genes,
composed entirely of the introduced iPSCs. Mature β cells from whereas Parkinson’s disease generally occurs later in life and is caused by
these mice were then harvested and transplanted into syngeneic mice environmental and complex genetic factors. However, it is still unclear
treated with streptozocin to induce diabetes, leading to recovery of which of these three elements most strongly influences our ability to
their glucose regulatory capacity46. Wild-type rat β cells generated generate the relevant disease phenotype in vitro. It is possible that a
by injection of Pdx1–/– mouse blastocysts with rat iPSCs also rescued disease such as autism, which involves complex genetics but manifests
the glucose dysregulation in streptozocin-treated rats following their early in life, could still be modelled with an appropriate iPSC-derived cell
transplantation (Fig. 2). Although it is unclear if such interspecies type. Many diseases with the greatest societal impact are polygenic and
chimeras would ever be ethically or technically feasible with human highly influenced by environment (for example, congestive heart failure,
iPSCs and whether the resulting cells would ever be safe for human Alzheimer’s disease, diabetes, sudden cardiac death, emphysema and
application given the potential for reactivation of endogenous Parkinson’s disease). It remains to be seen whether their key phenotypes
host-derived viruses47, the use of such assays to generate mature, can be reproduced in vitro using iPSCs.
differentiated and functional derivatives from iPSCs may open a new If the aetiology for disease development is known or suspected, there
approach for disease modelling in vivo. may be ways to introduce the causal agent into purified iPSC-derived
cells to induce or accelerate the manifestation of disease phenotypes
Challenges to iPSC-based disease modelling and drug discovery (see Table 2). For example, in amyotrophic lateral sclerosis (ALS),
Several challenges must be overcome before successful implementation superoxide dismutase (SOD) mutations affect the function of glial
of iPSC-based drug screening and pathway discovery can be achieved cells surrounding motor neurons. Studies have shown that co-culture

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types of neurons52,53, cardiomyocytes54–57, blood58–61 and pancreatic

cells41,62, none of these protocols generates the cell types of interest
with > 95% purity. Sorting of these cells from the heterogeneous iPSC
Rat mixture to reproduce the disease phenotypes for high-throughput
small molecule screening remains a challenge. Improvements in
+ Oct4, Sox2, cell-purification strategies (for example, fluorescence activated cell
Klf4, c-Myc
sorting, drug selection, gradient centrifugation and functional marker
isolation) may eventually allow us to overcome this barrier. The use
Rat iPSCs of small-molecule screens to identify compounds that can enrich for
a cell type of interest has also proved valuable51,63–67. Despite these
challenges, some companies already offer human iPSC (hiPSC)-derived
cardiomyocytes in quantities that are suitable for drug discovery and
Injection into
Pdx1–/– mouse toxicology testing.
blastocysts The heterogeneity of the maturation stage of the differentiated iPSCs is
also a potential limitation. A high-throughput screen aimed at identifying
small molecules that improve cardiomyocyte contractility may have a
high rate of false-positive and -negative hits if there are well-to-well
Rat-mouse differences in differentiation state, as mature cardiomyocytes exhibit
chimaera
greater contractility than their immature counterparts. Similar issues
may apply to small-molecule screening using hepatocytes or pancreatic
Pancreas β cells if the end-point of analysis is the secretion of specific enzymes or
entirely derived hormones, which strictly depend on the cells’ maturation stages.
from rat iPSCs
Once these barriers to the development of robust in vitro disease
models using iPSCs are overcome and small molecules that can reverse
Figure 2 Xenogeneic rat-mouse chimaera to produce entirely iPSC-derived the disease phenotype in vitro have been identified, an appropriate
rat pancreas. The introduction of wild type rat iPSCs into Pdx1-deficient animal model will be needed to validate the in vitro screen ‘hits’ in
blastocyst-stage mouse embryos resulted in the generation of a chimaeric rat-
vivo. For candidates that are Food and Drug Administration (FDA)-
mouse that harbours a rat iPSC-derived pancreas. This pancreas is expected
to be composed entirely of rat iPSC-derived cells as the loss of Pdx1 in mouse approved drugs, with known pharmacokinetic and toxicity profiles, no
embryos results in the complete absence of a developing pancreas. additional animal studies might be needed. For small molecules that
have not been previously tested for their pharmacokinetic, toxicity and
of human ESC-derived motor neurons with glial cells carrying the efficacy profiles, a standard pre-clinical evaluation of these molecules
mutation induces neuronal death48,49. An in vitro disease model could in vivo will still be required. It is likely that a number of small-molecule
therefore potentially be generated that uses glial cells and motor candidates identified from such screens might show efficacy only in
neurons derived from iPSCs from an ALS patient in a similar co-culture the artificial conditions of an in vitro assay. Thus, large-animal models
system. Another example is Duchenne muscular dystrophy (DMD); the of disease would be essential to help eliminate these candidates with
skeletal muscle phenotype of this disease is thought to be due to both the insufficient biological efficacy or enhanced toxicity in vivo. Investment
presence of dystrophin mutations and cumulative mechanical stretch of research resources to create reliable animal disease models should thus
injury from muscle use50. Thus, mechanical stress (or catecholamine be a significant priority if we are to realize the full potential of therapeutic
stimulation) may need to be applied to iPSC-derived skeletal muscle to drug screening efforts using disease-specific human iPSCs.
appropriately model this disease in vitro. For other complex diseases,
exposure of relevant chemical agents or toxins to iPSC-derived cells Challenges to iPSC-based therapy
may reveal phenotypes that would otherwise remain undetectable. For Major hurdles remain before iPSC-derived cells can be safely introduced
example, in one study that generated dopaminergic neurons from iPSCs into human patients. First, as for any pluripotent stem-cell-based
derived from patients with sporadic cases of Parkinson’s disease, no therapy, the risk of teratoma formation can be substantial. As most pre-
obvious abnormalities could be detected20. However, in a subsequent clinical human ESC/iPSC-derived cell transplantation studies have been
study, dopaminergic neurons derived from iPSCs obtained from a single performed in immunosuppressed animals, it is unclear whether the risk
Parkinson’s disease patient harbouring a mutation in the leucine-rich for teratoma formation will be similar or greater with patient-matched
repeat kinase 2 gene (LRRK2) were exposed to oxidative stress and iPSCs than that observed in immunosuppressed animals. The frequency
demonstrated increased susceptibility to cell death51. of teratoma formation following human ESC transplantation into
Assuming that disease features can be reproduced in vitro, it is still animal hosts is directly related to the degree of immunosuppression68,
unclear whether the phenotypes can be used for high throughput so transplantation of genetically matched iPSC derivatives into
small-molecule screening. A major limitation is the lack of robust patients, which are expected to elicit no immune reactivity against
lineage-specific differentiation protocols that enable researchers to the transplanted cells, may result in an even greater rate of teratoma
generate sufficient quantities of purified cells of a specific type for formation than the rate observed in animal studies. Alternatively,
large-scale screening applications. Although significant advances have incomplete reprogramming or genetic aberrations accrued during
been made to direct the differentiation of ESCs or iPSCs into certain the iPSC derivation process (see below) may render even genetically

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Table 2 Potential human iPSC models of complex diseases involving environmental factors

Possible pertubations to induce iPSC

disease phenotype in vitro

Disease Name Toxin Genes Chemical Physical Cells

Amyotrophic lateral
Selenium SOD, TDP-43, FUS/TLS Reactive oxygen – Astrocytes
sclerosis
Duchenne muscular Dystrophin/delta-sarcoglycan
Cardiotoxin Catecholamine Mechanical stretch –
dystrophy gene knockdown
Hypertrophic or dilated Introduce sarcomeric gene Mechanical stretch
Ethanol Doxorubicin, herceptin –
cardiomyopathy mutations or electrical pacing
Vinyl chloride,
Hepatitis B and C virus,
Hepatocellular carcinoma Aflatoxin B1, ethanol, arsenic Polycyclic aromatic – –
p53, K-ras
hydrocarbon
Chronic obstructive Nicotine, reactive
Tobacco Elastase, α1 anti-trypsin Mechanical stretch Neutrophils
pulmonary disease oxygen

matched iPSC lines immunogenic. So far, no study has evaluated the mature β-globin expression, and this could affect their functionality.
immunogenicity of genetically matched iPSCs on transplantation into Whether transplanted cells can undergo further maturation over time
syngeneic hosts. within their site of engraftment remains to be determined. Thus, future
At present, it is unclear whether any of the currently available developments within this area should aim at enhancing the maturation
strategies to generate differentiated cells from iPSCs and to separate of pluripotent stem-cell-derived cells in vitro before their therapeutic
them from residual pluripotent cells is able to eliminate the risk of application78–80. Notably, a recent study suggested that direct conversion
teratoma formation. Although it is encouraging that lineage selective of human fibroblasts into haematopoietic cells entails activation of adult
survival or engraftment has been observed on transplantation of human globin genes rather than fetal globins as is generally seen following ESC/
ESC-derived cardiomyocytes into immunosuppressed rodents 55,56 or iPSC differentiation, suggesting a possible alternative to producing
murine iPSC-derived neurons into Parkinsonian rats37, it is unknown certain mature cell types69.
whether such effects would persist when autologous human iPSC- A final consideration for the successful application of iPSC-derived
derived cells are transplanted. The transdifferentiation of one adult cells in regenerative medicine is their ability to integrate with existing
cell type into another cell type would circumvent the teratoma risk cells in the tissue. Most solid organs harbour an intrinsic architecture
associated with pluripotent cells and may provide an alternative that shows an appropriate balance between the number of each cell
approach to produce clinically relevant autologous cell types. Indeed, type and their geometrical arrangement to reflect their developmental
recent data suggest that the introduction of either pluripotency genes relationships. It remains to be seen whether transplantation of isolated
or lineage-specific transcription factors into fibroblasts can give rise single cells in suspension (mostly of one cell type) could auto-regulate
to cells resembling haematopoietic progenitors69, cardiomyocytes70,71, the number of each of the cell types to produce the endogenous tissue
myogenic cells72 and neurons73. It remains to be seen, however, if architecture. Furthermore, the engrafted cells will need to function in
transdifferentiated cell types are as functional as ESC-/iPSC-derived concert with the existing cells. This is particularly important for organs
cells and whether lineage switching into other cell types can be achieved such as the heart, lung, kidney and liver where individual functional
with this strategy. A serious disadvantage of transdifferentiation over units (for example, ventricular muscles, nephrons, alveolar sacs and the
directed differentiation from pluripotent cells is that somatic cells hepatobiliary network) are interconnected with other functional units
in general have a limited lifespan and are therefore not expandable, and with the vasculature. Although the successes of haematopoietic
whereas ESCs/iPSCs have limitless growth and can hence be repeatedly stem cell transplantation therapy over the past four decades have
coaxed into the desired cell types. spurred the interest and development of cell transplantation strategies
Beyond the issue of teratoma formation, there is now a growing in solid organs, it should be cautioned that observations made in the
recognition that differentiated cells derived from ESCs/iPSCs are haematopoietic system may not necessarily apply to solid organs. The
mostly immature. These cells mimic embryonic development and recent disappointing clinical data from the transplantation of skeletal
adopt phenotypes that resemble fetal or neonatal cells74,75. Whether this myoblasts81 and bone marrow mononuclear cells82,83 for the treatment of
immaturity will influence their clinical applicability may vary between myocardial injury should remind us that the route to a durable clinical
diseases and cell types. For the treatment of degenerative diseases, such therapy using stem cells remains largely obscure and much greater
as Parkinson’s disease, Alzheimer’s disease or congestive heart failure, the understanding in cell lineage specification, differentiation and function
transplanted cells would need to be sufficiently mature to replace the lost will be needed to advance this field.
cells of similar type (for example, dopaminergic neurons and ventricular Recent progress in tissue engineering raises the possibility that some
cardiomyocytes) to ensure proper function. Likewise, cell maturity may of the structural limitations associated with cell transplantation may
also be critical for diseases that require the transplanted cells to correct in fact be surmountable. Several reports have provided exciting proof-
lost secretory function or cell number (for example, pancreatic islet β of-principle evidence that the seeding of decellularized tissue scaffolds
cells, hepatocytes or haematopoietic cells). For instance, human ESC with endothelial and epithelial cells grown in bioreactors can produce
(hESC)-derived erythroid progenitors express mostly embryonic and bioartificial lungs84,85, livers86 and hearts87 that engraft in animals and
fetal haemoglobin but have none76 or only limited77 ability to activate exhibit normal tissue function for up to several days.

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Are hiPSCs and hESCs equivalent? respectively, to predict hiPSC differentiation potentials, which may be
To what extent pluripotent cell lines exhibit biological variability among time- and cost-inefficient. In the future, however, it may become possible
one another and whether hiPSCs have the same properties as hESCs are to rapidly pre-screen newly derived hiPSCs and hESCs for their potential
two additional important questions in the field. These issues may affect to differentiate into desired cell lineages of all three germ layers using a
the functionality and safety of hiPSC-derived mature cells and answers smaller set of markers.
to these questions may determine how many cell lines need to be derived In conclusion, although there is evidence for subtle differences
to observe the desired phenotypes in a reliable fashion. These two topics between ESCs and iPSCs at the transcriptional, epigenetic, genetic and
are actively debated and we will attempt to summarize recent findings functional levels, it remains unclear which of these are solely the result of
that address these issues. biological variation or handling of the cells and which are a consequence
Although initial studies concluded that hiPSCs are highly similar or of the reprogramming process itself. Further work is needed to examine
even indistinguishable from hESCs15,16,88, a number of laboratories have this issue and to determine if the observed aberrations have any
subsequently documented substantial differences in gene expression89, functional impact on their potential therapeutic utility.
DNA methylation90,91, in vitro differentiation potential92,93 and teratoma-
forming propensity94. However, it remains unclear which of these Future perspectives
differences are due to inherent differences between hESCs and hiPSCs Although significant progress still needs to be made in understanding the
rather than to differences associated with the generation of hiPSCs3. molecular mechanisms of cellular pluripotency and reprogramming, the
Indeed, recent studies showed that genetic background95, the use of viral possibility that novel pathways and drugs may be discovered through the
integration20, lab-to-lab variation96 and passage number89,97 can have use of iPSC technology should sustain the great enthusiasm that basic and
profound effects on gene expression and function in pluripotent cells. clinical/translational scientists have bestowed on this area of research. The
In addition, three independent studies found that hiPSCs carry idea that we have the knowledge and means to generate ‘spare parts’ for
copy number variations (deletions and duplications)98–100 and point every failing organ may belong to the realm of science fiction for now.
mutations101, as assessed by SNP (single nucleotide polymorphism) However, it is worth noting that the rat-mouse interspecies chimaerism
arrays and exon sequencing, respectively. Some of these alterations seem study by Kobayashi et al. suggests one possible route to generate human
to be the result of culturing, as has been seen before for hESCs, whereas tissues (if not organs) using iPSCs46.
other mutations pre-existed in the somatic donor cells. Some may have The remarkable discovery of iPSCs by Takahashi and Yamanaka may
arisen de novo during the reprogramming process. A limitation of these be the molecular equivalent of the discovery of antibiotics or vaccines in
reports is that hESC lines, which probably originate from multiple the last century. Time will tell whether the efforts of stem cell biologists
embryonic founder cells, were compared with hiPSCs that are, per and translational scientists in this area today will be discussed in
definition, clonal cell lines derived from a single fibroblast of unknown the same way. Our endeavour to overcome the barriers that prevent
genomic integrity. It should, therefore, be informative to include clonal successful translation of stem cell biology into clinical therapy should
fibroblast and early-passage subcloned hESC lines in these analyses to help to improve our knowledge regarding disease pathogenesis itself and
evaluate the exact contributions of cell of origin, subcloning procedure ways to prevent their onset or progression. In the end, this may prove to
and passage number on the mutational profile. be the most important contribution of iPSC technology.
In any case, these studies clearly indicate that many hESCs and hiPSCs
ACKNOWLEDGMENTS
harbour subtle or severe chromosomal abnormalities, and thus may We thank J. Wu and G. Mostoslavsky for their insightful comments and critical
warrant careful examination before their potential use in therapy or reading of the manuscript and J. Gold for detailed discussion regarding the human
disease modelling. Although large genomic amplifications, especially ESC-derived oligodendrocyte clinical trial at Geron. This work was supported by
grants from the NIH (OD003266 and HD058013 to K.H.; OD004411, HL081086,
of areas comprising cancer-associated genes, would certainly be a reason HL100408 to S.M.W.), the Harvard Stem Cell Institute (K.H. and S.M.W.), and the
for excluding such hiPSC lines in therapeutic applications, future work Howard Hughes Medical Institute (to K.H.). We apologize to colleagues whose
is needed to evaluate whether the observed heterozygous small deletions work we could not cite in this brief review article.

and point mutations result in functional consequences and thus pose a COMPETING FINANCIAL INTERESTS
risk in a potential therapeutic setting. K.H. is an advisor to iPierian, Inc.
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