TRANSPLANTATION SURGERY AND RESEARCH
The Seed and the Soil
Optimizing Stem Cells and Their Environment for Tissue Regeneration
Jeong S. Hyun, MD, Daniel T. Montoro, BS, David D. Lo, MD, Ryan A. Flynn, BS, Victor Wong, MD,
Michael Thomas Chung, BS, Michael T. Longaker, MD, MBA, and Derrick C. Wan, MD
Abstract: The potential for stem cells to serve as cellular building blocks for
reconstruction of complex defects has prompted significant enthusiasm in
the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly
hinder their tissue forming capacity. To circumvent this obstacle, novel
approaches have been developed to manipulate both the stem cell itself and
its surrounding environmental niche. By understanding this paradigm of
seed and soil optimization, innovative strategies may thus be developed to
harness the true promise of stem cells for tissue regeneration.
Key Words: stem cells, cytokines, scaffolds, niche, tissue regeneration
(Ann Plast Surg 2013;70: 235Y239)
T
reatment of tissue loss and organ failure in an aging population
represents a significant biomedical burden, with present modalities such as artificial implants or organ transplantation offering less
than ideal solutions. Regenerative medicine, however, holds significant promise as a novel approach to address insufficient, diseased,
or damaged tissues throughout the body. By introducing cellular elements adept at participating in the process of repair, the potential
exists to create de novo tissue capable of dynamic response, thus providing clinicians with the ability to treat tissue and organ loss in a
functional and lasting manner.
Despite tremendous advances in the field of regenerative medicine, however, several complex issues still remain. Although both
pluripotent and multipotent stem cells have been shown to undergo
in vitro differentiation along a wide variety of lineages, the actual
in vivo implantation of these cells continues to present a challenging
dilemma. The clinical application of stem cells often involves placement into a wound environment that is hypoxic, acidic, and with dramatically up-regulated inflammatory mediators. Within this hostile
environment, cellular survival is uncertain, and the utility of
implanted cells can be dramatically compromised (Fig. 1). In light
of this, there is now a growing recognition that simple placement of
stem cells into a wound is not sufficient to ensure maximum utility
for tissue regeneration.
Recent investigations have begun to evaluate the use of novel
approaches including gene therapy to augment the regenerative capacity of progenitor cells. Alternatively, a variety of biomimetic scaffolds, mechanical stimuli, and exogenous cytokines have also been
studied as a means to both deliver and guide in vivo differentiation
of stem cells. Collectively, these findings highlight the ‘‘seed and
Received July 12, 2012, and accepted for publication, after revision, July 16, 2012.
From the Hagey Laboratory for Pediatric Regenerative Medicine, Department of
Surgery, Plastic and Reconstructive Surgery Division, Stanford University
School of Medicine, Stanford, CA.
Conflicts of interest and sources of funding: none declared.
Reprints: Derrick C. Wan, MD, Department of Surgery, Stanford University Medical Center, 257 Campus Drive West, Stanford, CA 94305. E-mail: dwan@
stanford.edu.
Copyright * 2013 by Lippincott Williams & Wilkins
ISSN: 0148-7043/13/7002-0235
DOI: 10.1097/SAP.0b013e31826a18fb
Annals of Plastic Surgery
& Volume 70, Number 2, February 2013
soil’’ concept, as we can either manipulate the progenitor cell itself
(seed) to ensure survival, or we can augment the surrounding environmental niche (soil) to maximize the ability of implanted cells to contribute toward tissue-specific regeneration. By understanding this
paradigm, innovative strategies may be developed allowing clinicians
to take maximal advantage of stem cell potential.
MANIPULATING THE SEED
To maximize cellular viability and promote tissue-specific differentiation of stem cells, there has been dramatic progress made over
recent years with manipulation of the seed. Specifically, regulation of
the genome has been found to facilitate expression or inhibition of
certain key cellular processes. An important component of promoting
early in vivo cellular survival is the development of a vascular network to support the implanted cells. This is especially critical in
ischemic wound environments where reconstruction is often
attempted. Several studies have thus evaluated ways the implanted
cell itself can serve as a means to enhance local angiogenesis. Yang
and colleagues1 used nonviral, biodegradable polymeric nanoparticles to increase vascular endothelial growth factor (VEGF) expression in human mesenchymal stem cells (MSCs) and embryonic
stem cells (ESCs). Implantation of these modified cells resulted in enhanced cellular viability and engraftment into target tissues when
compared to untreated cells. Similarly, Das et al2 overexpressed
VEGF and platelet-derived growth factor in MSCs before placing
them into a rat cardiac infarct model. Using these manipulated stem
cells, they were able to demonstrate increased cardiac function and
cardiac tissue angiogenesis. Furthermore, VEGF has been transduced
in concert with bone morphogenetic protein (BMP)-2Vin effect,
‘‘priming’’ the seedVand this has been shown to promote in vivo bone
regeneration by MSCs in rabbits.3 Like VEGF, hypoxia-inducible
factor-1> has been found to play a critical role in stimulating angiogenesis in hypoxic conditions. Zou et al4 overexpressed this gene in
bone-marrow-derived MSCs and demonstrated that an increase in
hypoxia-inducible factor-1> expression augmented in vivo bone regeneration in calvarial defects. Considering the hypoxic nature of
many wounds, strategies to improve angiogenesis through stem cell
manipulation could thus have important clinical implications for
cell-based approaches to tissue regeneration.
Aside from local ischemia, additional barriers that may also
hinder stem cell efficacy and tissue regeneration include inflammatory
mediators present in all wound environments. Inflammation has been
shown to have wide ranging deleterious effects, and some of this
may be attributed to activation of Toll-like receptor-4 signaling. Using
MSCs from TLR4 knock-out mice, however, Wang and colleagues5
showed enhanced cardioprotection in a rat model of myocardial
ischemia-reperfusion injury. This was also associated with increased
proliferation and differentiation of implanted cells. Similarly, Lee et al6
demonstrated that injected MSCs produce the anti-inflammatory mediator TNF-> induced protein 6 (TSG-6), and increased expression
of TSG-6 after intravenous injection of MSCs in mice subsequently
decreased the size of infarcts and improved cardiac function. In addition, the aggregation of MSCs into 3-dimensional spheroids was
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FIGURE 1. Schematic of hostile wound environments. Infection, low pH, hypoxia, ischemia, and inflammation may all contribute to
impaired tissue regeneration by implanted stem cells.
found to enhance expression of TSG-6 and stanniocalcin-1, a protein with both anti-inflammatory and antiapoptotic properties.7
Although there remains no consensus over the use of systemic antiinflammatory agents to augment local wound regeneration, there is
nonetheless strong evidence that targeted manipulation of specific
inflammatory mediators in stem cells may increase their effectiveness after implantation for tissue repair.
Another strategy to increase stem cell viability after implantation is to enhance prosurvival pathways within the cell itself. Wei
et al8 overexpressed bcl-2, an antiapoptotic protein, in retinoic acid
treated ESCs and found increased survival as well as neuronal differentiation after transplantation into postinfarct brain cavities in rats.
Over 8 weeks, lesion cavities were found to be filled with ESCderived neurons, astrocytes, oligodendrocytes, and endothelial cells,
and enhanced functional recovery on neurological and behavioral
tests was noted. Laflamme and colleagues9 also identified a ‘‘prosurvival cocktail’’ including cyclosporine A, insulin-like growth factor-1,
and Bcl-xL which was found to augment ESC survival and engraftment
in regions of infarcted rat myocardium. Researchers have likewise
evaluated the inhibition of cellular apoptosis through down-regulation
of caspase activity. As caspases reside at the convergence of major apoptotic pathways, Imai et al10 used the caspase inhibitor ZVAD-fmk
to enhance engraftment and survival of donor hematopoietic stem
cells (HSCs) for bone marrow transplantation in mice. In light of
these reports, a combination of these strategies may potentially increase the ability of stem cells to survive and differentiate after
implantation for reconstructive tissue engineering purposes.
CULTIVATING THE SOIL
The stem cell itself does not exist on an island. The surrounding environment, or niche, plays a critical role in the overall regulation of cellular behavior and development. This notion takes
root from early studies examining HSCs. In 1978, Schofield11 was
one of the first to note that ‘‘the stem cell is seen in association with
other cells which determine its behavior.’’ More recent studies with
Drosophila and Caenorhabditis elegans have paralleled this observation, showing that cells adjacent to the germline stem cells were
critical for their maintenance and viability.12,13 This concept of a
stem cell niche has also been described in muscle and bone.14
A stem cell niche can be roughly defined as the microenvironment surrounding the stem cell that maintains its viability and regulates its function. The niche can be comprised of the 3-dimensional
extracellular macrostructure, from neighboring cells that may secrete
specific cytokines and growth factors, or by a combination of the
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2.13,14 The bone marrow environment surrounding HSCs represents
the classic mammalian niche.11 In-depth understanding of this arrangement has resulted in significant implications for regenerative
medicine. Under steady-state conditions, HSCs reside within the
bone marrow space in contact with a variety of cells contributing to
their physiologic regulation.15 Early studies with bone marrow transplantation have led to definition of the HSC niche as the site where
purified progenitor fractions localize at early time points after injection.15,16 More stringent criterion have recently been proposed,
however, defining the niche as the location where cells may be functionally regulated and contribute to hematopoiesis.16
Investigations with long-term culture of HSCs in vitro have
elucidated some of the critical elements, collectively termed stroma,
which positively regulate and sustain these cells. Osteoblasts have
been shown to support HSCs, as parathyroid hormone induced osteoblastic expansion has concomitantly led to increased numbers of
HSCs.16,17 Conversely, a reduction of osteoblastic cells has been associated with negative effects on HSC homeostasis and their retention
in the marrow space.18 Recent work by Chan and colleagues19 has
suggested the process of endochondral ossification, whereby bone
formation proceeds through a cartilaginous intermediate, to be necessary for development of the HSC niche. Like osteoblasts, adipocytes
arise from a common shared progenitor with osteoblasts, may also
contribute to the support of HSCs. In states of stress, Walkley
et al20 described the ability for adipocytes to support certain aspects
of hematopoiesis. Adipocytes have also been found to secrete various cytokines capable of positively regulating HSC differentiation.21
Other studies, however, have suggested a negative correlation between fat and hematopoiesis. With age, human long bones become
filled with increasing numbers of adipocytes which contribute to
the HSC microenvironment, and this has been suggested to result in
senile down-regulation of HSC function.22
Aside from these mesenchymal-derived components of the
HSC niche, the endothelial compartment has become recognized as
another key contributor to the bone marrow supportive stroma. Kiel
and colleagues23 have shown highly purified HSCs to colocalize with
the vasculature at a greater frequency than osteoblasts on the endosteal surface. Furthermore, stimulation of endothelial cells has been
found to positively regulate HSCs.24,25 By Akt activation of endothelial cells, release of angiogenic paracrine growth factors has been described resulting in an increase in the number of colony forming units
for HSCs and ultimate hematopoietic recovery.25 These data therefore
establish the importance of a vascular component for the HSC niche
which is capable of balancing the rate of proliferation and lineagespecific differentiation of HSCs.15
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How these components of the bone marrow niche interact to
control HSC survival and differentiation has yet to be determined.
There is undoubtedly a tight coupling between osteogenic, adipogenic, and vasculogenic elements at both the cellular and a larger
macrostructural level. It has even become apparent that multiple divergent specialized niches may exist within the bone marrow microenvironment designed to support and direct HSCs along particular
blood cell lineages.15,26,27 Despite these complexities, however, as
our understanding of the HSC niche continues to evolve, important
implications have arisen for tissue engineering. Given the ability for
the niche to regulate stem cell function, by manipulation of this microenvironment, the potential exists to precisely control stem cell survival, proliferation, and lineage-specific differentiation. This may be
accomplished through modification of the surrounding 3-dimensional
structure, neighboring niche cells, and through the use of various
growth factors and cytokines (Fig. 2).
Crafting the Structural Niche
The ability to fabricate a 3-dimensional structure with synthetic
biopolymers around which stem cells may be implanted allows for
potential modulation of cellular activity in vivo. Not only should it
be a means for delivery, but it should also serve to both enhance stem
cell survival and proliferation. Specific biomaterials have been
designed which can interact with stem cells directly or indirectly
through modification of the mechanical environment. Phipps and colleagues28 demonstrated that a tricomponent scaffold consisting of
polycaprolactone, collagen I, and hydroxyapatite nanoparticles electrospun into a 3-dimensional lattice was capable of inducing MSCs
to adhere, spread, and proliferate. This has also been reported by
many with apatite-coated and noncoated polylactic-co-glycolic acid
scaffolds.29Y32 Similar effects on stem cells have recently been shown
The Seed and the Soil
using hydrogel scaffolds, with Rustad et al33 describing a pullulancollagen construct to be effective as a biomaterial-based niche
for delivery of MSCs into wounds. Within such an environment,
MSCs were found to up-regulate expression of transcription factors
associated with maintenance of pluripotency and self-renewal, secrete
VEGF, and accelerate wound healing in mice. Importantly, cells delivered within the hydrogel were found to remain viable longer and
demonstrated enhanced engraftment when compared to cells injected
alone.33 These data highlight the ability for a carefully constructed
3-dimensional environment to significantly impact stem cell biology.
As a functional niche, these structures are thus capable of augmenting
both stem cell viability and their regenerative potential.
Modifying the Cellular Niche
As neighboring cells within the microenvironmental niche
have been shown to play an instructive role for HSCs, researchers
have also sought to manipulate cell-cell interactions to dictate stem
cell activity for tissue engineering purposes. Mesenchymal stem cells
are known to strongly interact with endothelial cells within the intima
and adventitia of blood vessels. In light of this, investigators have
established that MSCs seeded within a 3-dimensional niche composed of collagens I and III in addition to endothelial cells are capable
of forming vessel-like tubes.34 In contrast, constructs without endothelial cells were unable to effect similar results.
The ability for cell-cell interactions to also direct chondrogenic
stem cell differentiation has been evaluated using primary chondrocytes to alter MSC biology. By culturing either bone marrow-derived
MSCs or adipose-derived stromal cells (ASCs) in concert with primary human chondrocytes, increased glycosaminoglycan content could
be detected along with enhanced levels of type II collagen.35 Such
chondroinduction was found to be secondary to both MSC-induced
chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis, highlighting the potential complex 2-way interactions between stem cells and their surrounding niche.
Finally, to promote osteogenic differentiation of stem cells,
several studies have evaluated the capacity for multiple types of
neighboring cells to enhance bone formation. Interestingly, human
umbilical vein endothelial cells within a 3-dimensional spheroid construct have been found to inhibit adipogenic differentiation of MSCs
and instead activate expression of both endogenous Wnts and BMPs
to promote osteogenic differentiation.36 Alternatively, MSCs have
also been found to interact with primary osteoblasts or periosteal
derived cells, resulting in enhanced levels of collagen type I and
Runx-2 transcripts.37 Collectively, these findings underscore the importance of heterotypic cell-cell interactions in the regulation of
stem cell behavior, as previously described with the HSC niche. In
a 3-dimensional environment, both human umbilical vein endothelial cells and primary osteoblasts may thus augment the osteogenic
potential of MSCs, providing potential strategy to enhance bone
tissue engineering.
Optimizing the Cytokine Niche
FIGURE 2. The microenvironmental niche can regulate stem
cell behavior and development. This can be accomplished
through biomaterial composition and regulation of mechanical
stimuli, incorporation of protein mimetics, small molecules,
or vehicles for gene therapy, or by facilitating heterotypic
cell-cell interactions.
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As demonstrated by HSCs, the cytokine milieu created by the
niche can have profound effects on stem cell activity. With this in
mind, researchers have thus sought to manipulate local concentrations
of various morphogens to effect changes in cellular differentiation capacity. In particular, the ability to fabricate biocompatible scaffolds
has allowed for the delivery of cytokines in concert with stem cells.
Whang and colleagues38 demonstrated the capacity for BMP adsorbed onto calcium coated polyethylene glycol scaffolds to be capable of inducing significantly greater ectopic bone formation in vivo
than scaffolds without BMP. Several reports have since demonstrated
similar findings, with direct incorporation of BMP-2 into a variety of
scaffolds for bone tissue engineering.39Y42 Such a means for cytokine
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Annals of Plastic Surgery
Hyun et al
delivery has been shown to dramatically enhance the osteoinductive
properties of the niche and result in accelerated bone regeneration
in multiple animal models.39Y42 From a clinical perspective, the incorporation of rhBMP-2 into collagen sponges have found use in singlelevel anterior lumbar interbody fusion and to promote bone formation
in some cases of tibial fractures.43 Although use in such situations
continues to evolve, they nonetheless highlight the potential to locally
manipulate cytokine levels to construct a highly specialized niche
directing cellular activity.
In similar fashion, investigators have also looked to fabricate
proangiogenic microenvironments through the delivery of various
growth factors.44,45 Heparan binding modification of various biosynthetic scaffolds has allowed researchers to enhance VEGF, fibroblast
growth factor-2, and insulin-like growth factor-1 delivery, each of
which have been found to promote vessel growth by vascular progenitors.46 Vascular endothelial growth factor has also been delivered
through coprecipitation onto calcium phosphates, allowing for both
angiogenesis and enhanced bone formation in cranial defects.47 As
previously elaborated, adequate vascularization to support viability,
growth, and function remains a challenge to cellular-based strategies
for wound repair. Such findings, however, underscore the potential
for modification of the cytokine niche to stimulate both angiogenesis and lineage-specific differentiation by stem cells.
INTEGRATING THE SEED AND THE SOIL
Although significant progress has been made in stem cell regulation at both the cellular and microenvironmental niche level, ultimate translation for regenerative medicine will likely require a
synthesis of both the ‘‘seed and the soil.’’ This means integrating a
modified, primed stem cell with a carefully constructed niche designed to maximize cellular survival and guide differentiation. Bone
regeneration represents one particular area where such an approach
may prove clinically valuable given the suboptimal nature of present
modalities. Work from our own laboratory has thus sought to develop
a novel strategy to maximize bone tissue engineering through manipulation of both the stem cell and niche.
Adipose-derived stromal cells represent a readily available osteogenic cellular building block for tissue engineering purposes, and
multiple reports have shown their capacity for in vivo bone regeneration.48Y50 As exogenous BMP signaling has been demonstrated to
promote osteogenic differentiation in these cells, augmentation of this
pathway at the cellular level may therefore serve to enhance ASCmediated bone formation. Specifically, down-regulation of the BMP
antagonist Noggin in ASCs resulted in greater endogenous BMP
signaling as well as augmented ability for these cells to promote
calvarial regeneration.40 These findings thus highlight the potential
to optimize the seed for the purposes of bone tissue engineering.
Studies from our laboratory have similarly evaluated the
effects of manipulating the microenvironmental niche to maximize
bone formation. After implantation of ASCs into critical-sized calvarial defects, subcutaneous injection of BMP-2 into the region of injury
has been found to significantly enhance bone formation by engrafted
cells.49 This confirmed the ability to craft an early pro-osteogenic
niche designed to guide osteogenic differentiation of ASCs. A more
elegant approach, however, has recently been developed with a biomimetic scaffold designed to allow for slow release of BMP-2. Delivery of ASCs on such a scaffold has been found to dramatically
augment calvarial healing.40
To harness the true potential of stem cells, however, primed cellular building blocks and specially designed niches must be orchestrated in
such a manner as to maximize formation of desired tissues. This has
been specifically accomplished through the incorporation of Noggin
shRNA lentiviral particles onto apatite-coated PLGA scaffolds previously loaded with BMP-2 (Fig. 3).40 Using this delivery vehicle for
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& Volume 70, Number 2, February 2013
FIGURE 3. Diagram of highly osteogenic niche with PLGA
core surrounded by hydroxyapatite, BMP-2, and DNA/RNA
constructs to enhance bone differentiation by stem cells. The
potential also exists for incorporation of additional components
which may likewise promote robust bone formation by
stem cells.
ASCs, simultaneous stem cell manipulation and creation of a highly
osteogenic cytokine microenvironmental niche could be achieved, facilitating the most robust healing of calvarial defects observed.40 By
synthesizing an approach using the concept of the ‘‘seed’’ and the
‘‘soil,’’ an optimal strategy may therefore be developed for skeletal
regeneration.
CONCLUSIONS
Stem cells represent a powerful tool for regenerative medicine
given their potential to restore disparate tissue types. With the complexity of wound environments, however, the application of primed
stem cells alone to restore specific deficits will likely yield suboptimal results. Through a better comprehension of how the microenvironment may influence cellular biology, novel modalities may thus
be developed to manipulate the niche and enhance both cellular survival and differentiation. In the future, integration of these 2 strategies
using stem cell modification and a carefully constructed specific tissue promoting microenvironment may prove to be the most effective
therapy for complex tissue reconstruction.
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