Stem cells and their niche
Stem cells, in contrast to progenitor cells, harbor the
unique ability to divide and generate additional stem cells
(self-renew) and to produce progeny that diff erentiate
into tissue-specifi c cells with defi ned physiological func-
tions. Th ese properties make embryonic stem (ES) cells,
induced pluripotent stem (iPS) cells [1,2] and tissue-
specifi c adult stem cells (aSCs) well suited for regenera-
tive medicine applications. Nevertheless, the clinical use
of ES cells, iPS cells, and aSCs for cell-based therapies is
hindered by a number of critical hurdles. In addition to
the ethical considerations associated with the generation
of ES cells, cell populations derived from totipotent ES
and iPS cells have the potential to generate teratomas
upon transplantation if the fi delity and effi ciency of
diff erentiation and enrichment protocols are not ideal.
aSCs are intrinsically wired to diff erentiate effi ciently into
cells from their tissue of origin. However, their relative
infrequency in tissues and our limited under standing of
the parameters regulating their diff erentiation and self-
renewal currently precludes most aSC-based clinical
applications. However, the medical potential of stem
cells, specifi cally aSCs, can be realized by placing un-
precedented emphasis on elucidating the mechanisms
governing their behavior and fate.
aSC regulation is largely attributed to dynamic bidirec-
tional interactions made with the tissue environment in
the immediate vicinity of the cell, termed the ‘niche’
(Figure 1). First formally described in the fruit fl y, Droso-
phila [3,4], the stem cell niche, or microenvironment, is
composed of both biochemical (growth factors, cyto-
kines, receptor ligands, and so on) and biophysical
(matrix stiff ness, topography/architecture, fl uidity, and
so on) factors that act singly and in concert to continu-
ously modulate cell fate. Despite widespread recognition
of its importance, our understanding of niche elements
and their cell and molecular infl uence on aSCs is limiting.
We can remedy this by adopting creative research
approaches that allow systematic analysis of candidate
niche factors and are amenable to screens to identify
presently unrecognized niche elements. By advancing our
understanding of stem cell niche regulation we can begin
to envision regenerative medicine applications built on
principles derived from fundamental niche biology.
Naturally derived (that is, collagen, fi brin, MatrigelTM)
and synthetic (that is, polyethylene glycol, polyacryla mide,
nanofi bers) biomaterials can be designed and patterned
down to minute detail, off ering the possibility to engineer
stem cell niches and test eff ects of putative biochemical
and biophysical features on stem cell fate in culture. Using
biomaterials as a design framework, our under standing of
niche composition and how components regulate stem
cells is limited only by the imagination. In this review we
will discuss two- and three-dimensional biomaterial
approaches to deconvolve the niche and its regulatory
eff ects, and we will provide several examples of clinical
applications that may benefi t from biomaterials research.
Engineering two-dimensional stem cell
Th e native aSC niche is a three-dimensional entity, and
ultimately the most representative culture model of any
In the body, tissue homeostasis is established and
maintained by resident tissue-specifi c adult stem
cells (aSCs). Through preservation of bidirectional
communications with the surrounding niche and
integration of biophysical and biochemical cues,
aSCs actively direct the regeneration of aged, injured
and diseased tissues. Currently, the ability to guide
the behavior and fate of aSCs in the body or in
culture after prospective isolation is hindered by our
poor comprehension of niche composition and the
regulation it imposes. Two- and three-dimensional
biomaterials approaches permit systematic analysis
of putative niche elements as well as screening
approaches to identify novel regulatory mechanisms
governing stem cell fate. The marriage of stem cell
biology with creative bioengineering technology
has the potential to expand our basic understanding
of stem cell regulation imposed by the niche and to
develop novel regenerative medicine applications.
© 2010 BioMed Central Ltd
Engineering a stem cell house into a home
Penny M Gilbert* and Helen M Blau*
*Correspondence: email@example.com; firstname.lastname@example.org
Baxter Laboratory in Stem Cell Biology, Department of Microbiology and
Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University School of Medicine, Stanford, CA 94305, USA
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
© 2011 BioMed Central Ltd
tissue must refl ect this detail. However, the eff ect of
dimensionality on cells is complex to study and a means to
do this has yet to be fully realized, making two-dimensional
biomaterials approaches to deconstruct and study indi-
vidual niche components particularly attrac tive. Extrinsic
regulation of aSCs by niche elements - including cell-cell
contact mediators, secreted signaling factors, extracellular
matrix (ECM), substrate stiff ness and topography,
nutritional para meters (O2, nutrients), pH, temperature,
fl uid fl ow, mecha nical stress (that is, cyclic strain) and even
gravity - can all be probed in two-dimensions to generate a
modular toolbox of stem cell regulation that can be used in
future three-dimensional niche reconstruction . While
our focus here is extrinsic stem cell regulation, it should be
noted that intrinsic regulation is fundamentally important
and typically both intrinsic and extrinsic regulation act in
concert to modu late cell behavior . In this section we
will discuss several niche parameters and the approaches
used to probe them in two dimensions using examples
from the literature.
Exploring cell-cell interactions
Tissue regeneration requires resident aSCs to survey the
status of the microenvironment and respond appro-
priately when alterations resulting from aging, injury or
disease are detected. In addition to changes incurred by
the surrounding ECM or the infl ux of circulating factors
from the vasculature, aSC behavior is guided through
direct and indirect interactions with cells in close juxta-
position. Employing a biomaterials-based approach allows
for fundamental insight into the spatial and temporal
nature of aSC interactions with the surrounding support
cells in the resting microenvironment and dis covery of
how those relationships change upon tissue insult.
Typically, co-culture of two or more cell types in a
culture dish is used to study cell-cell interactions, though
it is notoriously diffi cult to draw defi nitive conclusions
about mechanism due to the complexity of the system.
Rather than studying a heterogeneous mix of two cell
types, clever biomaterials-based strategies were deve loped
to generate isolated cell ‘pairs’. Microfl uidics technology
 combined with patterning on polydimethylsiloxane
(PDMS; a silicone polymer that can harden to a rubber-
like material) to create an array of cell ‘traps’ and a three-
step loading protocol, was used to create a grid contain-
ing hundreds of ‘co-culture’ replicates . Spatially
segregating the cell pairs enables the user to evaluate cell
fate changes over time at the pair level. Physical isolation
of two cell types can also be achieved using synthetic,
polyethylene glycol (PEG) hydrogels or PDMS patterned
with microwells [9-15]. Tunable PEG hydrogel provides
the additional fl exibility to interrogate cell pairs while
altering additional microenvironmental parameters such
as matrix rigidity and ECM/ligand identity, density or
mode of presentation (that is, tethered or soluble).
Impor tantly, these approaches are all amenable to high-
throughput screening and time-lapse microscopy to
assess co-culture eff ects on stem cell behavior and fate
changes over time, such as division resulting in sym-
metric or asymmetric self-renewal, diff erentiation and
changes in viability.
To investigate whether observed co-culture behaviors
are contingent on the direct interaction of two cells or
due to indirect paracrine eff ects, a co-culture approach
utilizing two interlocking combs was developed . In
this paradigm each cell type is cultured on an individual
silicon comb and cell behavior and fate are assessed while
combs are interlocked or when separated at known
micro meter scale distances. Th is biomaterials strategy
can spatially resolve the distance of relevant cell-cell
commu nications, but unlike the cell trap and microwell
technology it is diffi cult to reliably study cell-cell
interactions at the pair level and the approach is limited
to adherent cell types.
Elucidating cell-extracellular matrix communications
In addition to cell-cell interactions, aSC fate is modifi ed
by interactions with the ECM. Upon injury and aging or
during disease progression the matrix composition is
Figure 1. The satellite cell niche. Adult stem cells, such as skeletal
muscle satellite cells, engage in bidirectional communication with
the surrounding niche to maintain tissue homeostasis. Pax7 (green)
expressing satellite cells receive direct biophysical and biochemical
cues from the multinucleated (blue) skeletal muscle fi bers (black) they
sit on top of and the laminin (red) containing basement membrane
with associated growth factors and cytokines surrounding each fi ber
and encasing the stem cell. This confocal image of a muscle cross-
section further illustrates the architecture of the resting niche, which
poses an additional level of regulation on stem cells.
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 2 of 9
dramatically altered, cryptic binding sites are exposed
and aSCs can gain direct exposure to ECM ligands they
were previously sheltered from. Identifi cation of putative
ECM ligands present in resting and activated tissue and
their impact on aSC behavior and fate is enabled by
recent advances utilizing robotic spotting to print single
and combinations of ECM ligands as arrays and subse-
quently culture and follow the fate of exposed cells
[17,18]. Using this type of unbiased throughput approach
can greatly advance our basic understanding of cell
regulation by the matrix in the niche as well as to provide
a catalogue of matrix-mediated cellular outputs that can
be used to direct stem cell fate.
Standard tissue culture protocols typically supplement
growth factors and cytokines in the soluble media milieu,
while in tissues these secreted morphogens are most
commonly presented to cells tethered to the ECM .
Covalent attachment of secreted growth factors to
biomaterial surfaces demonstrated improved stability of
labile proteins and persistent signaling resulting in long-
term maintenance of signaling without the requirement to
supply additional protein [20-23]. In addition to protein
stabilization, mode of ligand presentation (soluble versus
tethered) was shown to have profoundly divergent eff ects
on cell fate underlying the relevance of this distinction [20-
22,24,25]. Studies investigating ligand presentation and
assessing how the mode of presentation infl uences cell fate
promise not only to advance our basic under standing of
aSC regulation, but also to aid researchers in the smart
design of culture conditions to promote a desired fate.
As described above, the ECM can directly modulate
aSC behavior in the niche through direct receptor-ligand
interactions. In addition, the density, fi ber alignment and
porosity of the ECM can impart spatial infl uence over
cells to dictate cell shape, an aspect which is progressively
gaining needed attention . For example, cells cultured
on micropatterned ECM islands with the same ligand
density but with diff erent surface area generate distinct
spreading phenotypes resulting in marked cell shapes
(rounded versus spread), which impose impressive
infl uence over cell viability . More recently, the
molecu lar mechanisms and signaling pathways driving
cell shape-mediated eff ects on stem cell populations have
been described [28,29]. Importantly, during wound
healing and disease progression, tissues undergo pro-
found alterations in the identity and organization of the
ECM, whose cellular and molecular eff ects are a topic of
intense investigation. Niche architectural eff ects confer a
unique dimension of aSC regulation by the ECM and
warrant greater focus by stem cell researchers.
Investigating cell-matrix interplay
Imagine pulling a string to turn on or off a lamp. Typically
the string is attached to something stationary and stiff
allowing you to generate resistance and activate the
switch. Imagine instead that the string is attached to
something soft like putty; the more you pull the string,
the more the soft putty will stretch preventing force
generation or activation of the light bulb. Adherent cells
are constantly assessing their microenvironment by mak-
ing contact with and pulling at the ECM. Cells pulling on
adhesion ligands attached to a stiff as opposed to a soft
matrix experience cytoskeletal reorganization resulting
in distinct intracellular signaling that can profoundly
alter cell fate [30-32]. Th us, the mechanical properties of
the niche, a biophysical cue, add yet another level of
regulation imposed by the ECM.
First demonstrated using immortalized cell lines ,
the ability of matrix stiff ness to regulate cell fate is now
widely accepted. In a groundbreaking study exploring the
impact of substrate rigidity on stem cell fate, mesen-
chymal stem cells were shown to diff erentiate into bone,
muscle or brain when cultured on polyacrylamide sub-
strates mimicking the mechanical properties of each
tissue . Since then, a similar biomimetic approach to
tune the culture substrate to the stiff ness of the endoge-
nous tissue has been used to encourage lineage-specifi c
diff erentiation to additional multipotent stem cells, such
as neural progenitors, and to culture ES and iPS cell
colonies long term without loss of stemness in the
absence of the fi broblast feeder layer [35-37]. Notably,
soluble factors present in culture media typically act
together with the culture matrix to regulate cell fate and
these interactions should be considered when drawing
conclusions. Also, in contrast to standard tissue culture
plastic, porous matrices (polyacrylamide, PEG) permit
diff usion of soluble molecules to both the apical and
basal cell surfaces, and decoupling the eff ects of substrate
stiff ness from bidirectional diff usion is still a challenge.
Unlike ES and iPS cells, prospectively isolated aSCs, such
as skeletal muscle satellite cells, are notoriously diffi cult to
expand in culture due to their natural inclina tion to
diff erentiate upon exposure to rigid tissue culture plastic
. Satellite cells were fi rst identifi ed by electron
microscopy according to their anatomic location and des-
cribed as a mononucleated cell that resides atop multi-
nucleated postmitotic skeletal fi bers and beneath a thin
basement membrane (Figure 1) . Despite the current
knowledge that satellite cells are responsible for the
remarkable ability of postnatal skeletal muscle tissue to
regenerate in response to injury, aging and disease [38,40-
46], surprisingly little is known about the compo nents of
the niche or the extrinsic regulation imposed by the niche
on satellite cell fate. However, recently developed strategies
to prospectively isolate satellite cells to relatively high
purity [38,41-46] in con junction with robust in vivo
functional assays of muscle stem cell fate [9,46] render the
satellite cell ready for interro gation in culture.
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 3 of 9
To investigate the role of matrix rigidity on satellite cell
fate, freshly isolated and FACS (fl uorescence activated
cell sorting) enriched muscle stem cells (MuSCs) were
cultured on PEG hydrogels with diff ering mechanical
proper ties but constant ligand density . Timelapse
videos of MuSC clonal division within microwells were
automatically analyzed using the Baxter algorithm and
revealed improved survival when MuSCs were cultured
on substrates that mimic the mechanical properties of
skeletal muscle tissue. Noninvasive bioluminescence
imaging of luciferase-expressing MuSCs transplanted
intra muscularly into mice after culture on hydrogels of
varied stiff ness demonstrated that culture on a muscle
biomimetic substrate provides the optimal condition to
maintain ‘stemness’ long term (Figure 2). Further, an in
vivo functional assay showed defi nitively that MuSCs
cultured on pliant hydrogel could self-renew in culture
while those propagated on plastic lost self-renewal
potential in as few as 2 days. Critical to the conclusions
drawn in these studies is the use of freshly isolated aSCs
in combination with functional assays in mice to validate
all culture observations; an experimental paradigm that
sets the bar for future applications of biomaterial
approaches to study stem cell fate.
In conclusion, two-dimensional biomaterial approaches
are exceptionally well suited to study the cellular and
molecular mechanisms governing stem cell fate regula-
tion by the immediately opposing niche as well as the
greater surrounding microenvironment. Tunable synthetic
polymer platforms off er the fl exibility to study stem cell
fate in response to simple or complex combinations of
putative niche parameters. In addition, these systems are
highly amenable to time-lapse microscopy analysis and
with recently developed strategies to automatically
analyze cell behavior and lineage relationships, it is now
feasible to evaluate the vast amounts of data generated by
such studies [9,11,47,48]. Th e success of two-dimensional
biomaterials approaches to study stem cell regulation in
culture is contingent on the availability of markers and/or
behaviors that accurately predict stem cell fate in vivo
. Transgenic reporter animals used for prospective
isolation of aSC populations can be used to dynamically
assay stem cell fate in real time and are particularly
advan tageous. Without robust, simple readouts it is diffi -
cult to perform high-throughput analysis of aSC popu-
lations to screen for novel biochemical and biophysical
features that regulate stem cell fate and further refi ne the
resting, aged, injured and diseased niches. Nevertheless,
by implementing two-dimensional biomaterials-based
approaches to study aSC regulation, we are likely to
expand our current diagnostic capa bilities, enable in vivo
modulation of aSC populations, and develop strategies to
expand aSCs in culture for use in cell-based therapies.
Engineering three-dimensional stem cell
In contrast to two-dimensional tissue culture approaches,
many aSCs are embedded within a complex, instructive
three-dimensional matrix, often in intimate contact with
additional cell types and in proximity to nutrient and
oxygen-delivering vasculature. While two-dimensional
approaches enable well controlled interrogation of single
putative niche elements on cell fate, the focus of three-
dimensional tissue engineering is to reconstruct the
complex architecture of stem cells within a three-
dimensional matrix to achieve a physiologically relevant
Figure 2. Substrate rigidity regulates muscle stem cell self-renewal in culture. A biomimetic biomaterials approach in conjunction with
functional assays in mice demonstrated that muscle stem cell (MuSC) self-renewal can be maintained in culture if cells are propagated upon a
substrate that recapitulates the mechanical properties of the native skeletal muscle tissue, a physical property of the stem cell niche . Pliant
culture substrates enabled propagation of additional Pax7 (green) expressing MuSCs and improved survival (middle), while culture on softer (left) or
stiff er (right) matrices decreased cell survival (gray) and promoted diff erentiation. Image is courtesy of Stephane Corbel, Blau Laboratory.
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 4 of 9
structure. Of course, this goal is highly complicated, but
by comparing to and extending the design principles
estab lished in two-dimensional
dimensional material biology has the greatest potential to
impact our understanding of in vivo tissue function. As
there are several excellent reviews describing the current
technical advances in the relatively nascent fi eld of three-
dimensional tissue model generation [50-54], here we
will focus on the challenges and potential of three-
dimensional matrix biology.
Challenges of three-dimensional culture models
Th ree-dimensional biomaterials to encapsulate stem cells
and investigate niche-mediated eff ects come with a
number of design challenges absent in two-dimensional
culture that must be overcome prior to use of the
materials by the biological community. A fi rst design
concern is the nutrient and oxygen requirements of fully
encapsulated stem cells [55,56]. Hydrogel systems with
the fl exibility to optimize matrix porosity can easily meet
this design challenge and provide adequate energy
requirements to maintain viability. A second criterion to
consider is the mechanism of polymer polymerization.
While natural matrices and some synthetic polymer
systems spontaneously interact over time to form a three-
dimensional network, other synthetic hydrogel matrices
rely on chemical or photo-initiators to achieve polymer
crosslinking and have potentially toxic eff ects on
encapsulated cells. An additional challenge inherent to
synthetic three-dimensional scaff olds is the need to
design strategies permitting cell migration after encap su-
lation. Th is has been successfully achieved through
incorporation of matrix metalloproteinase or other
proteolytic cleavage sequences into the polymer sequence
. An added benefi t of polymer design is the ability to
design scaff olds that permit migration of specifi c cell
types based on whether or not they secrete certain
enzymes. A fi nal design challenge is development of
independent tuning of biophysical and biochemical
parameters allowing three-dimensional culture optimiza-
tion on a cell type basis. Extending this to permit matrix
tunability over time in a spatial and temporal manner has
the potential to enable exquisite study of stem cell fate
changes as they may occur during disease progression
. Th rough the careful design and thoughtful
characterization of the parameters described above it is
now possible to produce biomaterials that promote long-
term survival, prolifera tion and diff erentiation of stem
cells in three dimensions.
matrices that permit
Establishing the eff ects of dimensionality
One of the most exciting research areas enabled by three-
dimensional biomaterials technology is the ability to
determine the behavioral and molecular eff ects of dimen-
sionality. While standard two-dimensional approaches
essentially defi ne the apical and basal surface of the
cultured cells, three-dimensional culture provides a
situation wherein the cell actively directs its own polarity.
By comparing cell behavior in three dimensions to that in
two dimensions it is feasible to probe the infl uence of
dimensionality on cultured cells. However, it is critically
important to consider the limitations of the system
employed, asan observed eff ect could be due to a
constraint in the culture system and not dimensionality
per se. For example, a diff erence in cell behaviour or
function may be confounded by a lack of appropriate
growth factor and nutrient diff usion through three-
dimensional biomaterials. Culture systems designed to
overcome this common diff usion barrier in the three-
dimensional culture setting are needed to draw meaning-
ful conclusions about the eff ects of dimensionality on cell
Recent studies exploring the eff ect of dimensionality on
cell behavior and fate have revealed several surprising
fi ndings. For example, a comparison of breast tumor cells
lacking or re-expressing HOXA9, a novel breast tumor
suppressor gene, exhibited no diff erence in cell growth
when assayed in two dimensions, but when the cells were
embedded within a three-dimensional reconstituted
basement membrane (mimicking the in vivo micro-
environ ment) distinct diff erences in proliferation were
observed . Th ese studies underscore the importance
of studying cells in the context of a three-dimensional
tissue-like structure in order to fully realize the eff ects of
a genetic (intrinsic) alteration. Further, when reconstruct-
ing a three-dimensional stem cell microenvironment it
should not be assumed that observations made in two
dimensions will necessarily translate into a similar eff ect
in three dimensions. Often additional tweaking of bio-
physical and biochemical parameters in three dimensions
is necessary to optimize a desired stem cell behavior
[19,36,61]. Arguably, one of the most interesting
dimensionality-related discrepancies arose from studies
on cell migration. Until now, models of cell migration
were derived from two-dimensional studies of cell
motility and led to an understanding that migration is
intimately linked to the formation of distinct sites of cell
attachment containing paxillin, vinculin, actin, focal
adhesion kinase as well as other structural and signaling
molecules necessary for focal adhesion formation and
force generation. However, in three dimensions it was
noted that migration occurs in the absence of distinct
focal adhesion formation and the characteristic molecules
observed in focal adhesion aggregates in two dimensions
(paxillin, vinculin, and so on) were found diff usely
localized throughout the cell during three-dimensional
movement . Similar comparisons of two-dimensional
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 5 of 9
behaviors in three-dimensional culture systems may
reveal similar discrepancies and contribute to our under-
standing of how dimensionality regulates stem cells.
Potential of three-dimensional matrix biology
Th ree-dimensional biomaterials enable reconstruction of
physiological models of tissue matrix scaff olds and their
accompanying cell types in both homeostatic and disease
states . Not only can they be used to expand our basic
knowledge of stem cell regulation by the microenviron-
ment, but these models can also facilitate identifi cation
of therapeutics targeting the stem cell niche to treat aged,
injured and diseased tissues. While it is unreasonable to
expect three-dimensional models to mimic the native
tissue down to molecular detail, by recapitulating certain
fundamental physiological functions, such models can be
used to study how perturbations to systems such as the
human airway wall, the lung or liver eff ect specifi c
functional outcomes to investigate the effi cacy and mode
of action of novel and currently prescribed medications
[63-65]. In addition, these models can be used to test the
toxicity of drugs intended for use in patients. Finally,
three-dimensional biomaterials can be expected to play a
substantial role in directing tissue regeneration or even
act as replacement tissues as described in the following
Clinical translation of engineered
Th e integration of bioengineering approaches with stem
cell biology has the potential to substantially change the
practice of medicine as we know it today. While
hematopoietic cell transplantation therapies have been
used in the clinic for more than a decade to resolve blood
malignancies, most solid tissues are precluded from
treatment with cell-based therapies to regenerate defects
and restore function. Several complicated factors lend to
this discrepancy, but the lack of suitable strategies to
expand isolated aSCs or to robustly diff erentiate ES or
iPS cells into a single tissue-specifi c lineage is a major
limitation to the progress of cell-based therapies. Using
two-dimensional or three-dimensional biomaterials
approaches, it is realistic to imagine that in the near
future we will identify simple strategies based on smart
design principles to expand aSCs and direct ES and iPS
cell fate, enabling cell-based regenerative therapeutics.
After injury, or as result of aging or disease, the
homeostatic microenvironment can undergo substantial
remodeling and reconstruction and, consequently, render
the environment ill-instructive for resident tissue-specifi c
aSCs. For example, it is hypothesized that extrinsic
changes to the satellite cell microenvironment prevent
eff ective skeletal muscle regeneration rather than in-
trinsic changes to the satellite cell itself during aging .
As an alternative to cell based therapies, studies suggest
that simply providing an instructive cell-free scaff old to
artifi cially modify the microenvironment and direct the
aSCs residing in tissue could prove useful to regenerate
damaged tissue . Th is approach was fi rst developed
and utilized in the repair of critical sized defects in bone
through the use of allogeneic demineralized bone matrix,
a US Food and Drug Administration approved product,
and has now been extended to many other tissue types
[68,69]. For example, cell-free scaff old-based strategies
are already used in the clinic to repair open skin wounds
on war victims . By focusing on biochemical and
biophysical parameters governing stem cell fate decisions
(that is, directed migration, proliferation, diff erentiation,
and so on), materials impregnated with signaling mole-
cules designed for release in a temporally and spatially
regulated manner are a viable option to modulate cell fate
and promote repair over time within the intact patient
Regenerative medicine using cell-free scaff olds relies
on the patient’s own cells to migrate into and repopulate
the acellular scaff old (Figure 3). To overcome this poten-
tial challenge, strategies combining synthetic or natural
matrices repopulated with cell types required for long-
term function of the replacement tissue are being
developed. For example, large cartilage defects resulting
from injury or aging are notoriously diffi cult to repair.
Use of a nanofi brous scaff old seeded with human mesen-
chymal stem cells (which evade the immune response)
demonstrated the ability of a bioengineering approach to
repair large cartilage defects in swine while restoring
smooth cartilage at the surface and withstanding use-
associated compression force . Similarly, corneal
function was restored in patients affl icted by debilitating
burns using autologous limbal stem cells embedded in
fi brin gels .
A major challenge in the clinic is the availability of
donor tissue for transplantation into patients with critical
organ failure. A tissue-engineering approach based upon
the principle of designing stem cell microenvironments
that incorporate the cell types, signaling cues and
structure required for long-term physiological function
and incor poration in a living patient has the potential to
sub stantially reduce the current reliance on organ donors
to provide tissues to patients in critical need. Th ough
generation of functional three-dimensional organs is an
extraordinary challenge, several research groups are
actively pursuing this goal and the literature is already
repleat with successes. To overcome the challenge of lost
bladder function in young patients affl icted with disease
rendering malfunction, researchers utilized a bioengi-
neer ing approach to construct collagen scaff olds in the
likeness of the human bladder. To ensure proper long-
term function and to reduce the possibility of tissue
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 6 of 9
rejection, the engineered bladders were seeded with
urothelial and muscle cells isolated from the patient prior
to transplantation. Follow-up studies 2 years following
transplantation concluded that the bioengineered bladders
had not only maintained architecture, but were also still
fully functional in the patient recipients . Organ trans-
plantation is typically accompanied by use of immune
suppression treatment to reduce the incidence of immune
rejection. To improve transplantation success, several
researchers are adopting a bioengineering approach that
entails decellularizing donor tissue (to remove the major
histocompatibility complex (MHC) component) with a
gentle, multistep detergent treatment that leaves the
matrix scaff old intact and permits recolonization with
patient derived cells. Th is approach has been used
successfully to treat a patient suff ering from broncho-
malacia (loss of airway function). Trans plant of a
decellularized donor trachea repopulated with epithelial
cells and chondrocytes from patient-derived mesenchymal
stem cells led to successful long-term repair of the airway
defect and restoration of mechanical properties .
Finally, a recent study demonstrated the possibility of
using a bioengineering approach to construct corporal
tissue to facilitate penile reconstruc tion. In a multistep,
dynamic process the three-dimensional corporal tissue
was engineered from a naturally derived collagen matrix
reseeded with autologous cells and trans planted into
rabbits with excised corpora. Amazingly, the bio engi-
neered phallus was structurally similar to the native tissue
and function was demonstrated by successful impreg-
nation of female rabbits with the engineered tissue .
Together these examples exemplify the potential impact
that material science will have on the treatment of human
disease in the not so distant future.
Both two-dimensional and three-dimensional biomaterials
approaches are changing the way scientists think about
the stem cell microenvironment and are providing
strategies to regulate the fate of prospectively isolated
stem cells in culture and of stem cells residing in intact
tissues. More importantly, current biomaterials tech nolo-
gies and the inevitable future technological advances in
the fi eld provide a novel toolbox for stem cell biologists
to investigate the impact of niche biochemical and
biophysical properties in unprecedented ways. Th ese
engineering approaches can be extended to all pros-
pectively isolated stem cell populations for the purpose of
elucidating the mechanisms governing their regulation.
To accelerate the impact of biomaterials towards the
treatment of human disease, it is essential to incorporate
in vivo functional assays as a standard practice to validate
observations made in culture. Furthermore, by placing
more emphasis on human stem cells and their niche
regulation, we can advance the translation of material-
based therapeutics from the bench to the bedside.
Bioengineering approaches to study the stem cell micro-
environment have the potential to revolutionize regener a-
tive medicine by providing physicians with tools to
regulate resident aSC behavior (that is, self-renewal,
diff er en tiation, migration) in patients, cells for cell-based
therapies, and perhaps even bioengineered organs to
replace defective tissues. Ultimately, the active colla-
boration of engineers, biologists, physicians, chemists,
computational scientists and physicists towards the goal
of understanding the niche, how it regulates stem cell fate
and how it changes with aging, injury and disease will
allow us to harness this knowledge and generate novel
regenerative medicine therapeutics.
Figure 3. Alternative approaches to functional organ replacement. Organ transplant is plagued by lack of available tissue, the short window
of tissue viability prior to transplant and graft rejection after transplant. A new bioengineering approach promises to overcome many of these
challenges in the near future. Donor tissue, such as liver (A), is decellularized (B) through a multistep process that leaves the extracellular matrix
scaff old intact. The matrix is then repopulated with tissue-specifi c cells that are compatible to the patient - for example, by diff erentiating patient-
derived iPS cells into hepatocytes. Resultant tissues can either be studied in culture to gain insight into tissue function (C) or used for transplant in
the clinic (D). Since this approach capitalizes on the remaining matrix scaff old and removes donor cells, tissues that would normally be discarded
due to viability issues can be salvaged. Further, scaff olds repopulated with cells derived from the patient are less likely to be rejected.
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 7 of 9
aSC, adult stem cell; ECM, extracellular matrix; ES, embryonic stem;
iPS, induced pluripotent stem; MuSC, muscle stem cell; PDMS,
polydimethylsiloxane; PEG, polyethylene glycol.
The authors declare that they have no competing interests.
PMG and HMB drafted, read and approved the fi nal manuscript.
This work was supported by the following funding agencies: CIRM TG2-01159
and PHS CA09151 to PMG and NIH grants HL096113, AG009521, AG020961,
U01-HL100397, JDRF 34-2008-623, MDA Grant 4320, LLS Grant TR6025-09,
CIRM Grant RT1-01001, Stanford BioX Award IIP3-34, and the Baxter
Foundation in Stem Cell Biology to HMB.
Published: 31 January 2011
1. Yamanaka S, Blau HM: Nuclear reprogramming to a pluripotent state by
three approaches. Nature 2010, 465:704-712.
2. Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse
embryonic and adult fi broblast cultures by defi ned factors. Cell 2006,
3. Scadden DT: The stem-cell niche as an entity of action. Nature 2006,
4. Morrison SJ, Spradling AC: Stem cells and niches: mechanisms that
promote stem cell maintenance throughout life. Cell 2008, 132:598-611.
5. Yamada KM, Cukierman E: Modeling tissue morphogenesis and cancer in
3D. Cell 2007, 130:601-610.
6. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K,
Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM: Matrix
crosslinking forces tumor progression by enhancing integrin signaling.
Cell 2009, 139:891-906.
7. Whitesides GM: The origins and the future of microfl uidics. Nature 2006,
8. Skelley AM, Kirak O, Suh H, Jaenisch R, Voldman J: Microfl uidic control of cell
pairing and fusion. Nat Methods 2009, 6:147-152.
9. Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P,
Nguyen NK, Thrun S, Lutolf MP, Blau HM: Substrate elasticity regulates
skeletal muscle stem cell self-renewal in culture. Science, 329:1078-1081.
10. Chin VI, Taupin P, Sanga S, Scheel J, Gage FH, Bhatia SN: Microfabricated
platform for studying stem cell fates. Biotechnol Bioeng 2004, 88:399-415.
11. Dykstra B, Ramunas J, Kent D, McCaff rey L, Szumsky E, Kelly L, Farn K, Blaylock
A, Eaves C, Jervis E: High-resolution video monitoring of hematopoietic
stem cells cultured in single-cell arrays identifi es new features of self-
renewal. Proc Natl Acad Sci U S A 2006, 103:8185-8190.
12. Khademhosseini A, Ferreira L, Blumling J 3rd, Yeh J, Karp JM, Fukuda J, Langer
R: Co-culture of human embryonic stem cells with murine embryonic
fi broblasts on microwell-patterned substrates. Biomaterials 2006,
13. Karp JM, Yeh J, Eng G, Fukuda J, Blumling J, Suh KY, Cheng J, Mahdavi A,
Borenstein J, Langer R, Khademhosseini A: Controlling size, shape and
homogeneity of embryoid bodies using poly(ethylene glycol) microwells.
Lab Chip 2007, 7:786-794.
14. Lutolf MP, Doyonnas R, Havenstrite K, Koleckar K, Blau HM: Perturbation of
single hematopoietic stem cell fates in artifi cial niches. Integr Biol (Camb)
15. Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW: Reproducible, ultra
high-throughput formation of multicellular organization from single cell
suspension-derived human embryonic stem cell aggregates. PLoS One
16. Hui EE, Bhatia SN: Micromechanical control of cell-cell interactions. Proc
Natl Acad Sci U S A 2007, 104:5722-5726.
17. Anderson DG, Levenberg S, Langer R: Nanoliter-scale synthesis of arrayed
biomaterials and application to human embryonic stem cells. Nat
Biotechnol 2004, 22:863-866.
18. LaBarge MA, Nelson CM, Villadsen R, Fridriksdottir A, Ruth JR, Stampfer MR,
Petersen OW, Bissell MJ: Human mammary progenitor cell fate decisions
are products of interactions with combinatorial microenvironments. Integr
Biol (Camb) 2009, 1:70-79.
19. Griffi th LG, Swartz MA: Capturing complex 3D tissue physiology in vitro. Nat
Rev Mol Cell Biol 2006, 7:211-224.
20. Alberti K, Davey RE, Onishi K, George S, Salchert K, Seib FP, Bornhäuser M,
Pompe T, Nagy A, Werner C, Zandstra PW: Functional immobilization of
signaling proteins enables control of stem cell fate. Nat Methods 2008,
21. Nur EKA, Ahmed I, Kamal J, Babu AN, Schindler M, Meiners S: Covalently
attached FGF-2 to three-dimensional polyamide nanofi brillar surfaces
demonstrates enhanced biological stability and activity. Mol Cell Biochem
22. Fan VH, Tamama K, Au A, Littrell R, Richardson LB, Wright JW, Wells A, Griffi th
LG: Tethered epidermal growth factor provides a survival advantage to
mesenchymal stem cells. Stem Cells 2007, 25:1241-1251.
23. Mehta G, Williams CM, Alvarez L, Lesniewski M, Kamm RD, Griffi th LG:
Synergistic eff ects of tethered growth factors and adhesion ligands on
DNA synthesis and function of primary hepatocytes cultured on soft
synthetic hydrogels. Biomaterials 2010, 31:4657-4671.
24. Beckstead BL, Santosa DM, Giachelli CM: Mimicking cell-cell interactions at
the biomaterial-cell interface for control of stem cell diff erentiation.
J Biomed Mater Res A 2006, 79:94-103.
25. Suzuki T, Yokoyama Y, Kumano K, Takanashi M, Kozuma S, Takato T, Nakahata T,
Nishikawa M, Sakano S, Kurokawa M, Ogawa S, Chiba S: Highly effi cient ex
vivo expansion of human hematopoietic stem cells using Delta1-Fc
chimeric protein. Stem Cells 2006, 24:2456-2465.
26. Folkman J, Moscona A: Role of cell shape in growth control. Nature 1978,
27. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE: Geometric control
of cell life and death. Science 1997, 276:1425-1428.
28. Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A, Kumacheva E,
Zandstra PW: Niche-mediated control of human embryonic stem cell
self-renewal and diff erentiation. EMBO J 2007, 26:4744-4755.
29. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS: Cell shape,
cytoskeletal tension, and RhoA regulate stem cell lineage commitment.
Dev Cell 2004, 6:483-495.
30. Mammoto T, Ingber DE: Mechanical control of tissue and organ
development. Development, 137:1407-1420.
31. Discher DE, Mooney DJ, Zandstra PW: Growth factors, matrices, and forces
combine and control stem cells. Science 2009, 324:1673-1677.
32. Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS: Control of
stem cell fate by physical interactions with the extracellular matrix. Cell
Stem Cell 2009, 5:17-26.
33. Pelham RJ Jr, Wang Y: Cell locomotion and focal adhesions are regulated by
substrate fl exibility. Proc Natl Acad Sci U S A 1997, 94:13661-13665.
34. Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs stem cell
lineage specifi cation. Cell 2006, 126:677-689.
35. Georges PC, Miller WJ, Meaney DF, Sawyer ES, Janmey PA: Matrices with
compliance comparable to that of brain tissue select neuronal over glial
growth in mixed cortical cultures. Biophys J 2006, 90:3012-3018.
36. Saha K, Keung AJ, Irwin EF, Li Y, Little L, Schaff er DV, Healy KE: Substrate
modulus directs neural stem cell behavior. Biophys J 2008, 95:4426-4438.
37. Mei Y, Saha K, Bogatyrev SR, Yang J, Hook AL, Kalcioglu ZI, Cho SW, Mitalipova
M, Pyzocha N, Rojas F, Van Vliet KJ, Davies MC, Alexander MR, Langer R,
Jaenisch R, Anderson DG: Combinatorial development of biomaterials for
clonal growth of human pluripotent stem cells. Nat Mater, 9:768-778.
38. Montarras D, Morgan J, Collins C, Relaix F, Zaff ran S, Cumano A, Partridge T,
Buckingham M: Direct isolation of satellite cells for skeletal muscle
regeneration. Science 2005, 309:2064-2067.
39. Mauro A: Satellite cell of skeletal muscle fi bers. J Biophys Biochem Cytol 1961,
40. Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB: Syndecan-3
and syndecan-4 specifi cally mark skeletal muscle satellite cells and are
implicated in satellite cell maintenance and muscle regeneration. Dev Biol
41. Fukada S, Higuchi S, Segawa M, Koda K, Yamamoto Y, Tsujikawa K, Kohama Y,
Uezumi A, Imamura M, Miyagoe-Suzuki Y, Takeda S, Yamamoto H: Purifi cation
and cell-surface marker characterization of quiescent satellite cells from
murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res 2004,
42. Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL,
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 8 of 9
Wagers AJ: Isolation of adult mouse myogenic progenitors: functional Download full-text
heterogeneity of cells within and engrafting skeletal muscle. Cell 2004,
43. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE:
Stem cell function, self-renewal, and behavioral heterogeneity of cells
from the adult muscle satellite cell niche. Cell 2005, 122:289-301.
44. Kuang S, Kuroda K, Le Grand F, Rudnicki MA: Asymmetric self-renewal and
commitment of satellite stem cells in muscle. Cell 2007, 129:999-1010.
45. Cerletti M, Jurga S, Witczak CA, Hirshman MF, Shadrach JL, Goodyear LJ,
Wagers AJ: Highly effi cient, functional engraftment of skeletal muscle
stem cells in dystrophic muscles. Cell 2008, 134:37-47.
46. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM: Self-renewal and
expansion of single transplanted muscle stem cells. Nature 2008,
47. Ravin R, Hoeppner DJ, Munno DM, Carmel L, Sullivan J, Levitt DL, Miller JL,
Athaide C, Panchision DM, McKay RD: Potency and fate specifi cation in CNS
stem cell populations in vitro. Cell Stem Cell 2008, 3:670-680.
48. Eilken HM, Nishikawa S, Schroeder T: Continuous single-cell imaging of
blood generation from haemogenic endothelium. Nature 2009,
49. Fu J, Wang YK, Yang MT, Desai RA, Yu X, Liu Z, Chen CS: Mechanical
regulation of cell function with geometrically modulated elastomeric
substrates. Nat Methods 2010, 7:733-736.
50. Annabi N, Nichol JW, Zhong X, Ji C, Koshy S, Khademhosseini A, Dehghani F:
Controlling the porosity and microarchitecture of hydrogels for tissue
engineering. Tissue Eng Part B Rev 2010, 16:371-383.
51. Hennink WE, van Nostrum CF: Novel crosslinking methods to design
hydrogels. Adv Drug Deliv Rev 2002, 54:13-36.
52. Lutolf MP, Hubbell JA: Synthetic biomaterials as instructive extracellular
microenvironments for morphogenesis in tissue engineering. Nat
Biotechnol 2005, 23:47-55.
53. Lutolf MP, Gilbert PM, Blau HM: Designing materials to direct stem-cell fate.
Nature 2009, 462:433-441.
54. Kopecek J: Hydrogel biomaterials: a smart future? Biomaterials 2007,
55. Derda R, Laromaine A, Mammoto A, Tang SK, Mammoto T, Ingber DE,
Whitesides GM: Paper-supported 3D cell culture for tissue-based
bioassays. Proc Natl Acad Sci U S A 2009, 106:18457-18462.
56. Valentin JE, Freytes DO, Grasman JM, Pesyna C, Freund J, Gilbert TW, Badylak
SF: Oxygen diff usivity of biologic and synthetic scaff old materials for
tissue engineering. J Biomed Mater Res A 2009, 91:1010-1017.
57. Lin CC, Anseth KS: PEG hydrogels for the controlled release of
biomolecules in regenerative medicine. Pharm Res 2009, 26:631-643.
58. Kloxin AM, Kasko AM, Salinas CN, Anseth KS: Photodegradable hydrogels for
dynamic tuning of physical and chemical properties. Science 2009,
59. Raghavan S, Shen CJ, Desai RA, Sniadecki NJ, Nelson CM, Chen CS:
Decoupling diff usional from dimensional control of signaling in 3D
culture reveals a role for myosin in tubulogenesis. J Cell Sci 2010,
60. Gilbert PM, Mouw JK, Unger MA, Lakins JN, Gbegnon MK, Clemmer VB,
Benezra M, Licht JD, Boudreau NJ, Tsai KK, Welm AL, Feldman MD, Weber BL,
Weaver VM: HOXA9 regulates BRCA1 expression to modulate human
breast tumor phenotype. J Clin Invest 2010, 120:1535-1550.
61. Little L, Healy KE, Schaff er D: Engineering biomaterials for synthetic neural
stem cell microenvironments. Chem Rev 2008, 108:1787-1796.
62. Fraley SI, Feng Y, Krishnamurthy R, Kim DH, Celedon A, Longmore GD, Wirtz D:
A distinctive role for focal adhesion proteins in three-dimensional cell
motility. Nat Cell Biol 2010, 12:598-604.
63. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE:
Reconstituting organ-level lung functions on a chip. Science 2010,
64. Choe MM, Tomei AA, Swartz MA: Physiological 3D tissue model of the
airway wall and mucosa. Nat Protoc 2006, 1:357-362.
65. Khetani SR, Bhatia SN: Microscale culture of human liver cells for drug
development. Nat Biotechnol 2008, 26:120-126.
66. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA:
Rejuvenation of aged progenitor cells by exposure to a young systemic
environment. Nature 2005, 433:760-764.
67. Badylak SF, Freytes DO, Gilbert TW: Extracellular matrix as a biological
scaff old material: Structure and function. Acta Biomater 2009, 5:1-13.
68. Urist MR: Bone: formation by autoinduction. Science 1965, 150:893-899.
69. Reddi AH, Huggins C: Biochemical sequences in the transformation of
normal fi broblasts in adolescent rats. Proc Natl Acad Sci U S A 1972,
70. Cornwell KG, Landsman A, James KS: Extracellular matrix biomaterials for
soft tissue repair. Clin Podiatr Med Surg 2009, 26:507-523.
71. Borselli C, Storrie H, Benesch-Lee F, Shvartsman D, Cezar C, Lichtman JW,
Vandenburgh HH, Mooney DJ: Functional muscle regeneration with
combined delivery of angiogenesis and myogenesis factors. Proc Natl Acad
Sci U S A, 107:3287-3292.
72. Li WJ, Chiang H, Kuo TF, Lee HS, Jiang CC, Tuan RS: Evaluation of articular
cartilage repair using biodegradable nanofi brous scaff olds in a swine
model: a pilot study. J Tissue Eng Regen Med 2009, 3:1-10.
73. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G: Limbal
stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010,
74. Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB: Tissue-engineered autologous
bladders for patients needing cystoplasty. Lancet 2006, 367:1241-1246.
75. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, Dodson A,
Martorell J, Bellini S, Parnigotto PP, Dickinson SC, Hollander AP, Mantero S,
Conconi MT, Birchall MA: Clinical transplantation of a tissue-engineered
airway. Lancet 2008, 372:2023-2030.
76. Chen KL, Eberli D, Yoo JJ, Atala A: Bioengineered corporal tissue for
structural and functional restoration of the penis. Proc Natl Acad Sci U S A
Cite this article as: Gilbert PM, Blau HM: Engineering a stem cell house into
a home. Stem Cell Research & Therapy 2011, 2:3.
Gilbert and Blau Stem Cell Research & Therapy 2011, 2:3
Page 9 of 9