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First published online 6 June 2007
doi: 10.1242/dev.003707
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Meeting Review |
1 Vascular Biology Program, Departments of Pathology and Surgery, Children's
Hospital and Harvard Medical School, Boston, MA 02115, USA.
2 Center for Regenerative and Developmental Biology, Forsyth Institute, and
Developmental Biology Department, Harvard School of Dental Medicine, Boston,
MA 02115, USA.
* Author for correspondence (e-mail: donald.ingber{at}childrens.harvard.edu)
SUMMARY
At a recent Keystone Symposium on `Developmental Biology and Tissue Engineering', new findings in areas ranging from stem cell differentiation, embryonic pattern formation and organ regeneration to engineered cell microenvironments, synthetic biomaterials and artificial tissue fabrication were described. Although these new advances were exciting, this symposium clarified that biologists and engineers often view the challenge of tissue formation from different, and sometimes conflicting, perspectives. These dichotomies raise questions regarding the definition of regenerative medicine, but offer the promise of exciting new interdisciplinary approaches to tissue and organ regeneration, if effective alliances can be established.
Introduction
Developmental biologists strive to understand how the cells of our tissues and organs come to be specified and placed in their correct positions. Tissue engineers seek to create artificial materials to repair tissues when they are lost due to injury or disease. Both strive to identify crucial cues that trigger these processes, and to control the cells that execute these programs. That tissue engineering might gain from developmental biology, and vice versa, seems obvious; however, investigators in each of these fields generally attend their own meetings and publish in their own journals. But the recent Keystone Symposium on `Developmental Biology and Tissue Engineering' (organized by Gordana V. Vunjak-Novakovic, Randall T. Moon and David Kaplan) in Snowbird, Utah, suggests that this paradigm is shifting. Here, we describe key themes from this symposium, and consider the crucial challenges that must be overcome to establish the key principles of regenerative medicine and translate them into powerful therapeutic strategies.
What is regenerative medicine?
Regenerative medicine is a burgeoning new field that promises to improve
health and quality of life by repairing or regenerating cells, tissues or
organs. This must be accomplished under a diverse set of circumstances,
including acute injury, surgical resection, inflammation, pathological
remodeling, ageing and progressive degeneration. Some biologists view the goal
as the discovery of master switches and stem cells that drive embryonic organ
formation, or the inductive organizers that induce a blastema to regenerate a
limb, and to use this knowledge to reform damaged organs in humans. Bob Nerem
(Georgia Institute of Technology, GA, USA) opened the symposium noting that
many, including NIH, consider tissue engineering to be the replacement of
tissues by fabricating substitutes ex vivo for implantation. However, this
might be an incorrect assumption. Fred Schoen (Brigham and Women's Hospital
and Harvard Medical School, MA, USA) described how artificial heart valves
composed of resorbable synthetic polymer scaffolds containing cultured bone
marrow-derived cells produce functional valves even though the implanted cells
are likely to be replaced during remodeling in vivo. Apparently, the
mechanical microenvironment of the leaflet induces ingrowth and
differentiation of the tissue, and results in regeneration of normal valve
architecture (Mendelson and Schoen,
2006
). Arnold Caplan (Case Western Reserve University, OH, USA)
showed results from human clinical trials, as reported by Osiris Therapeutics,
in which adult mesenchymal stem cells (MSCs) improved health in patients with
myocardial infarct, graft-versus-host disease and Crohn's disease after
intravenous injection, not by differentiating into various cell types, but by
suppressing immune responses and permitting natural healing. Meanwhile, David
Mooney (Harvard University, MA, USA) described injectable polymer systems that
control the spatiotemporal dynamics of morphogens and in situ programming of
stem cells at injury sites (Hill et al.,
2006). So rigid definitions of regenerative medicine are not
constructive while the principles that define the field are still being
delineated.
Won't stem cells solve the problem?
Stem cells are at the center of expectations of regenerative medicine, and
many exciting results relating to stem cell therapies were presented at the
Snowbird meeting. Robert Lanza (Advanced Cell Technologies, MA, USA) described
how somatic cell nuclear transplantation produces immune-compatible cells that
can repopulate the bone marrow without requiring myelosuppression, as well as
how human embryonic stem cell (hESC) lines can be generated without destroying
embryos (Klimanskaya et al.,
2006). This is accomplished by extracting a single cell from
eight-cell stage embryos, or by creating haploid embryos through
parthenogenesis.
Alan Colman (Singapore Institute of Medical Biology and ESC International,
Singapore) focused on identifying the crucial growth factors and cytokines
necessary to induce hESCs to differentiate into cardiomyocytes. By using
cell-free and serum-free culture conditions, combined with genetic selection,
they have been able to obtain a 99.9%-pure cardiomyocyte population. However,
when GFP-labeled hESCs are injected into ischemic hearts of NOD/SCID mice,
most of the implanted cells die. Clinical trials involving injection of stem
cells into the heart have been similarly ineffective at maintaining viable
cells at the injection site in humans
(Hofmann et al., 2005).
Nevertheless, the field is still young, and it seems likely that cellular
therapies using stem cells will be effective for certain conditions,
especially those in which only dysfunctional cells need to be replaced and
tissue structure remains intact (e.g. diabetes, Parkinson's disease).
Aren't tissue engineers already building tissues and putting them into people?
If the press seems to be overselling stem cells now, one only needs to go
back a few years to find that they had done the same for tissue engineering.
Yet only a few engineered tissues (e.g. Apligraf and Integra artificial skin
products) are now in clinical use. At Snowbird, Shulamit Levenberg (Technion
University, Haifa, Israel), who presented work on co-culturing vascular
endothelial cells with other cell types as an approach to improve the
vascularity and functionality of engineered tissues, noted that many
developmental biologists ask tissue engineers: "It's so complex, how are
you going to try to mimic it?". Farshid Guilak (Duke University, NC,
USA) reaffirmed the difficulty of this challenge and explained that some of
the frustration in the field might have been based on oversimplified
assumptions, such as what works in animals will also work in patients, and
that cost is no object. He also described how existing polymer scaffold-cell
composites often do not have the appropriate material properties to bear
physiological mechanical loads. Guilak is approaching this challenge in a new
way by applying a three-dimensional (3D) weaving technology to create porous
fabrics that exhibit different moduli (stiffness and elasticity) in different
directions and that are composed of interwoven fibers of
poly(
-caprolactone) (Moutos et al.,
2007); these scaffolds can exhibit mechanical properties similar
to those of native articular cartilage.
However, the challenge of integrating this or any other type of artificial scaffold into a diseased tissue filled with inflammatory cytokines remains a tough one. Fred Schoen emphasized that clinical testing and validation of engineered materials will be complicated by the heterogeneity of tissue responses among patients, as well as differences in response in old versus young. Although exciting and effective in certain applications, tissue engineering approaches, like stem cell therapies, need to be greatly improved to match the promises made in the press.
What can we learn from developmental biology?
We should be able to develop new, more powerful regenerative therapeutic
strategies if we could decipher the control mechanisms responsible for normal
developmental patterning in the embryo and the adult. This promise is seen in
powerful regenerative model systems such as Planaria, which perfectly
regenerate after the excision of nearly every part of their body, and stop as
soon as the pre-existing structures are rebuilt
(Sanchez Alvarado, 2003).
Human liver can similarly regenerate its original functional mass after
removal of more than 50% of the organ, and it shuts off this program when its
normal size is restored. However, demonstrating that this type of regenerative
capacity can be reactivated in other human organs remains a fundamental
challenge in the regenerative medicine field.
Randall Moon (HHMI, University of Washington School of Medicine, WA, USA)
showed that Wnt signaling through ß-catenin is required for epithelial
and mesenchymal cell proliferation, and cell fate specification, during tail
regeneration in zebrafish. Conversely, a gain-of-function Wnt8 accelerates the
regeneration process without altering final tail size
(Stoick-Cooper et al., 2007).
Moon also reported that ß-catenin-mediated gene expression increases near
sites of injury in mouse liver and fish heart, and showed that Wnt5a enhances
bone marrow engraftment of hematopoietic progenitor cells when they are
injected intravenously in mice (Trowbridge
et al., 2006). This is an example of where the dissection of a
developmental signaling mechanism has led to tangible results that might have
a significant impact on tissue engineering and stem cell therapies. However,
the question of morphogen specificity remains: how can a molecule, such as
Wnt, that produces diverse effects in many tissues at different times and in
different spatial contexts be used as part of a therapeutic strategy in humans
where specificity and lack of toxicity are crucial? Also, as Wnts can
contribute to tumor formation (Kikuchi,
2003), how can this potential complication be prevented?
Another major approach pursued by developmental biologists centers on
defining inductive factors that guide stem cells to differentiate into various
specialized cell types. Didier Stainier (University of California San
Francisco, CA, USA) described how Wnt2b, bone morphogenetic protein (BMP),
fibroblast growth factor (FGF), and retinoic acid (RA) are required for
endoderm to differentiate into liver, whereas FGF and RA promote exocrine
pancreas formation, and hedgehog signaling and RA stimulate pancreatic
ß-cell differentiation (Ober et al.,
2006). Thomas Reh (University of Washington, WA, USA) showed that
NOTCH maintains hESC progenitors in a self-renewing state, and that neural
differentiation can be synchronized by inhibiting NOTCH. Moreover, in other
experiments with human ES cells, he reported that insulin-like growth factor,
DKK1 and noggin direct the cells to generate human retinal progenitors, which
can be induced to differentiate into retinal photoreceptors by simultaneously
blocking NOTCH and adding RA (Lamba et
al., 2006).
But generating the right kinds of cell types is not the same as
regenerating tissues and organs. The correct cues must also be provided to
position these cells appropriately, to induce them to deposit extracellular
matrices (ECMs), and to organize these elements spatially across several
levels of scale as components of larger tissue and organ structures
(Fig. 1). For example, in mouse
Ds (disorganization) mutants, normal cell and tissue types are
produced, but their overall placement with respect to each other is disturbed
at the organ/appendage level (Robin and
Nadeau, 2001). So induction of cell differentiation alone is not
sufficient to meet the regenerative medicine challenge
(Fig. 1). Interestingly, the
spatial organization of the microenvironment itself can dictate the final
output of multicellular and multimolecular developmental programs. For
example, Stainier found that although Smoothened (Smo) is required for
pancreatic ß-cell differentiation in zebrafish, cells from smo
knockouts differentiate into ß cells when placed in wild-type embryos. In
this case, spatial context overrides gene expression. Reh also showed that
when GFP-labeled hESCs are injected into the vitreous of newborn mice, the
cells invade all of the retinal layers and differentiate into appropriate
neural cell types (Lamba et al.,
2006). Thus, stem cells can sense local environmental cues, place
themselves in relatively normal positions, and differentiate appropriately
based on their location.
|
). Thus, we need to learn more about normal tissue
construction, and not only how to chemically induce stem cells to form various
specialized cell types (Fig.
1).
Classic epithelium-mesenchyme recombination experiments demonstrated that
whereas the epithelium specifies the function of its cells
(cytodifferentiation), the mesenchyme often governs the 3D form of the
epithelium (histodifferentiation) and the overall morphology of the organ
(Sakakura et al., 1976). The
mesenchyme influences pattern formation by producing morphogens, and by
secreting ECM components and matrix-modifying enzymes. At Snowbird, Donald
Ingber (Children's Hospital and Harvard Medical School, MA, USA) extended this
view by describing how cytoskeletal contractile forces that cells exert on ECM
scaffolds and on each other contribute to morphogenetic control during mouse
embryonic lung development. When he altered physical interactions between
cells and ECM by modulating cytoskeletal tension generation by manipulating
Rho GTPase or Rho-associated kinase (ROCK), he was able to selectively speed
up or slow down epithelial budding morphogenesis and angiogenesis
(Moore et al., 2005). Other
groups have found that physical forces and cell distortion regulate axis
formation (Farge, 2003),
tissue remodeling during gastrulation
(Beloussov et al., 1990) and
whole organ size in animals (van Rooij et
al., 2007).
Jeff Axelrod (Stanford University, CA, USA) and Suzanne Eaton (Max Planck Institute, Dresden, Germany) explored the importance of cell-cell adhesions and mechanical forces during the establishment of epithelial planar cell polarity (PCP) in Drosophila. PCP requires the coordination of local and long-range signaling pathways to orient cells consistently in an epithelial sheet with respect to the polarity of the whole tissue. Axelrod showed that in a domino-like cascade, the interaction between Fz and Vang is mediated by the atypical cadherin Flamingo (Starry night - Flybase) that is present on both sides of the cell-cell adhesion complex and transmits a directional signal between adjacent cells. Eaton examined the mechanism of PCP in the Drosophila wing epithelium. She finds that wing cells become hexagonally packed just before initiating bristle formation in a time frame that exactly correlates with polarization of PCP proteins. By using physical modeling approaches, she showed that different cell packing and cell shape configurations are governed by the level of mechanical tension at the junctional region, and demonstrated the presence of tension directly using laser ablation. Thus, cell packing geometry in Drosophila wings is governed by a balance of mechanical forces, much like what Ingber observed during the control of mouse lung development.
Michael Levin (Forsyth Institute and Harvard School of Dental Medicine, MA,
USA) demonstrated that another physical cue - bioelectricity - drives
regeneration in lower organisms (Adams et
al., 2007; Levin,
2007). He found that induction of spinal cord and muscle
regeneration in the Xenopus tail requires the expression of a
cell-surface H+ pump in the wound epithelium that changes
transmembrane potential in the regeneration bud, and creates a long-range
electric field that appears to promote nerve ingrowth. Moreover, misexpression
of a heterologous (yeast) H+ pump is sufficient to induce the whole
regeneration cascade in the Xenopus tail at non-regenerative stages.
Most intriguing was the finding that ectopic expression of a K+
channel in Xenopus can induce the formation of complex eyes that
contain both expressor and non-expressor cells. Together, these findings
provide another example of how a physical cue, such as an electric potential,
can be as important as chemicals and genes are for developmental control. They
also clarify that organ regeneration is a unique developmental program
(distinct from wound repair) that may be reinitiated by a relatively simple
signal (e.g. the activity of specific ion transporters), which reboots adult
cells and reprograms their development, if presented in a receptive tissue
microenvironment.
Does combining engineering and biological approaches advance the field?
Numerous presentations described examples of how developmental biologists
and engineers are beginning to work together and incorporate each other's
tools and approaches. These experiments often fundamentally change the way in
which we view the problem. Ingber, Christopher Chen (University of
Pennsylvania, PA, USA) and Dennis Discher (University of Pennsylvania) all
presented results using engineered substrates that show that mechanical
interactions between cells and ECM (and related changes of cell shape) control
cell fate switching in vitro. Ingber described how endothelial cells, liver
cells and smooth muscle cells can be switched between growth, differentiation
and apoptosis in the presence of a constant amount of soluble growth factors
by varying cell spreading (Singhvi et al.,
1994; Chen et al.,
1997). This was accomplished by culturing cells on
micrometer-sized ECM islands created with a microfabrication technique that
holds one cell on each island. Chen used a similar approach to show that when
larger ECM islands are created that support the adhesion of multicellular
monolayers, growth patterns are not homogeneous; rather, increased DNA
synthesis is observed in regions where tensional forces are concentrated owing
to the geometry of the island (McBeath et
al., 2004). He also discovered that cell shape distortion governs
stem cell lineage switching (McBeath et
al., 2004). hMSCs differentiate into bone cells with high
efficiency when cultured on large ECM islands that promote spreading, whereas
the same MSCs in the same medium switch on adipose cell differentiation when
plated on small islands. Discher fabricated ECM-coated polyacrylamide gels of
different stiffnesses to match the elasticity of soft tissues that include
brain, muscle and the bone matrix called `osteoid'. The hMSCs sense these
differences in elasticity and respond by differentiating into neurons, muscle
cells and osteoblasts, respectively
(Engler et al., 2006). As in
Chen's studies with shape distortion, ECM mechanics proved as potent as
induction cocktails. Matthias Chiquet (Friedrich Miescher Institute, Basel,
Switzerland) showed that altering the mechanical forces balanced across
integrin receptors induces expression of the tenascin-C-encoding gene in
embryonic fibroblasts (Sarasa-Renedo et
al., 2006). Interestingly, this mechanical signaling pathway is
mediated by integrin-linked kinase, as well as Rho and ROCK, which are also
crucial for establishment of localized growth differentials in vitro
(Nelson et al., 2005) and in
vivo (Moore et al., 2005).
These results provide the first examples of quantitative design criteria that tissue engineers might use to help design artificial tissue scaffolds. In fact, Jeremy Mao (Columbia University, NY, USA), David Kaplan (Tufts University, MA, USA), Gordana Vunjak-Novakovic (Columbia University), and others are already incorporating mechanical loading in their tissue engineering design strategies. Equally important, however, these results suggest that developmental biologists might want to focus more on the role of mechanical forces and material elasticity in their studies on `stem cell niches' that are responsible for controlling stem cell behavior in situ.
Biologists tend to consider one cytokine (or gene) at a time, and to
determine whether it is present (on) or absent (off) when analyzing its role
in cell and tissue regulation. Mooney described how the use of synthetic
polymer-based drug delivery systems to deliver two angiogenic factors - VEGFA
and PDGFBB - with different dynamics (VEGFA fast; PDGFBB slow) in a defined
spatial gradient in the same tissue produces much more robust vascular
development than either alone, or when both are added simultaneously
(Hao et al., 2007). Moreover,
he showed that controlling the spatiotemporal dynamics of growth factor
delivery can help regenerate the vasculature and save whole limbs in a mouse
leg ischemia model. These findings are likely to resonate with developmental
biologists because they know that tissue formation is regulated by multiple
soluble factors that exhibit varying concentrations over time and space. But
it is often difficult, if not impossible, to measure or control these
parameters; so this type of engineering approach might open entirely new
avenues of research in embryology.
Peter Zandstra (University of Toronto, Canada) described related studies
using microfabricated substrates and automated microfluidic systems in which
he found that mouse progenitor cells and ESCs differ in their ability to sense
signal strength and spatial cues, and that they exhibit different time windows
of sensitivity to the same factors. Zandstra's computational models also
revealed that ESCs exhibit robust autocrine signaling that has a buffering
effect on differentiation, that they exhibit feed-forward regulatory loops,
and that loss of signal responsiveness is an early reversible step in ESC
commitment prior to differentiation (Davey
and Zandstra, 2006). Furthermore, when he cultured ESCs on
microfabricated ECM islands of different sizes, he observed that endogenous
signaling gradients can be regulated in a spatially controlled manner to
control ESC fate. These engineering approaches might prove extremely useful
for ESC production for regenerative medicine applications, as well as for
developmental biologists interested in stem cell niche function.
A common theme that emerged in this meeting was the need to understand how
robust behaviors emerge from collective interactions. Anand Asthagiri
(California Institute of Technology, CA, USA) created quantitative integrative
models of vulva cell type specification in C. elegans. He deduced a
phase diagram of multicellular patterns in a parameter-unbiased approach, and
identified parameters that optimally shift a wild-type phenotype into a
mutant. This revealed that the phenotypic capacity of the molecular circuit he
studied is constrained (i.e. not all possible variations can be explored), and
that this approach enables control over large-scale patterns (e.g.
transitioning from reflectional to translational symmetry in the linear order
of vulval cell types) (Giurumescu et al.,
2006). These types of systems-based approaches may be augmented by
automated text-mining/curating systems, such as the one described by Andrey
Rzhetsky (Columbia University) (Rzhetsky
et al., 2004).
Cells cannot explore all possible phenotypic states because regulatory
interactions within their gene and signaling networks make certain states
impossible. Regulatory constraints also exist at the level of cell-cell and
tissue-tissue networks that exhibit similar complex interdependent
interactions during embryological development. As Ingber discussed, this
phenomenon of dynamic network complexity has been analyzed using physics-based
mathematical approaches to explore the implications of much larger networks,
on the scale of whole genomes (Kauffman,
2004). This work revealed that the complex web of gene
interactions funnels down to only a limited number of stable states over time
called `attractors' (in the same way that water droplets falling on a hilltop
eventually roll down to a common low point in one valley or another). In this
framework, a `master' gene or inductive signal effectively acts by lowering
the hilltops in the attractor landscape (e.g. by simultaneously altering the
activation state of multiple downstream network elements). Once the system
(gene, cell or tissue network) passes over the lowered peak, it will fall into
another stable attractor and hence generate only distinct types of cells,
tissues or organs, because the fixed landscape is determined by the
architecture and system-wide dynamics of the underlying regulatory networks.
Ingber described gene microarray studies that experimentally confirmed the
existence of attractors in the gene regulatory network of human HL60
promyelocytic precursor cells that were induced to differentiate into
neutrophils by two different factors, one specific (RA) and one highly
non-specific (the solvent DMSO) (Huang et
al., 2005). The existence of attractors might explain how
generalized stimuli, such as cell shape distortion, changes in ECM mechanics,
or a short-circuit electric current flowing through breaks in epithelia, can
control cell fate switching and produce identical responses to those induced
by cytokines. This physics-based view of cellular signaling appears to
conflict with current paradigms in the biology; however, it might greatly
simplify approaches to regenerative medicine because it suggests that we may
not have to recreate every step in a signaling cascade to produce a stable
functional tissue.
Presentations by engineers described many tools and approaches that might
also be of value to developmental biologists. Mooney and Tabata described
biodegradable polymers that provide the controlled delivery of proteins,
plasmid DNA and siRNAs to selected sites
(Silva and Mooney, 2007).
Levin described techniques to guide cell migration and differentiation using
voltage gradients (Adams et al.,
2007), and Vunjak-Novakovic showed how cultured cardiomyocytes
exhibit regular cardiac beating rhythms in engineered microsystem bioreactors
when electrical potentials are applied
(Gerecht-Nir et al., 2006).
These bioreactors, which culture cells and tissues at high density within
micro-channelled scaffolds perfused with culture medium containing oxygen
carriers, might also prove useful for embryological studies.
Another area where biologists and engineers could learn from each other is
the materials science of living tissues. Gabor Forgacs (University of
Missouri, MI, USA) described an automated 3D printer that produces functional
organoid-like structures of any shape by depositing liquid-like multicellular
spheroids as ink particles drop-by-drop. Buddy Ratner (University of
Washington) summarized results showing that biocompatibility can be controlled
by selectively engineering the surface properties of materials to control
protein orientation. Peter Lelkes (Drexel University, PA, USA) has used FGF
proteins and tenascin C to stimulate concomitant epithelial and endothelial
branching morphogenesis in an in vitro model of lung assembly
(Mondrinos et al., 2006).
Kaplan demonstrated the ability to combine the strength and biocomptability of
natural silk with the specific adhesivity of cell-binding sequences from ECM
molecules within chimeric proteins that mimic natural hierarchical
self-assembly properties (Wong Po Foo et
al., 2006).
These various engineering approaches could potentially revolutionize
developmental biology. But the Symposium made it clear that tissue engineers
can learn a great deal from developmental biologists as well. Caplan
highlighted properties of MSCs, such as homing and secretion of trophic or
inhibitory factors, that might prove helpful to those desiring to create
materials that can recruit endogenous MSCs or modify their function when
injected in vivo (Caplan and Dennis,
2006). Charles Murry (University of Washington) is engineering a
3D scaffold-free patch of human cardiac tissue using hESC-derived cardiac
myocytes (McDevitt et al.,
2003). Levenberg finds that co-culturing similar hESC-derived
cardiomyocytes with hESC-derived endothelial cells and embryonic fibroblasts
results in increased cardiac cell proliferation and enhanced vascularization
(Caspi et al., 2007). Dan Gazit
(Hebrew University and Cedars Sinai Medical Center, CA, USA) is genetically
engineering MSCs to express osteogenic BMP or brachyury transcription factor
to regenerate bone and nucleus pulposus within intervertebral spinal discs
(Aslan et al., 2006). He has
also developed numerous sophisticated in vivo imaging techniques, including
micro-CT analysis and fiber optic-based confocal fluorescence imaging, that
might help developmental biologists and tissue engineers alike.
A new area of biology that might impact tissue engineers, as well as
biologists, is that of micro-RNAs (miRNAs) in developmental control. Alexander
Schier (Harvard University) showed that a specific miRNA (miR430) regulates
the maternal-zygotic transition in zebrafish, not by inducing the switch, but
by erasing the previous state (Giraldez et
al., 2006). He speculated that tissue engineers might potentially
use miRNAs in the future to hold cells in a particular developmental state.
Eric Olson (University Texas Southwestern Medical Center, TX, USA) used
microarrays to identify miRNAs that increase their expression in response to
structural perturbations that produce cardiac muscle hypertrophy and heart
dilation in mice (van Rooij et al.,
2007). He found that a particular miRNA (miR208) in the intron of
the myosin heavy chain gene plays a central role in the control of heart form
and function: strikingly, hearts of miR208-knockout mice do not
undergo hypertrophy or fibrosis in response to thoracic artery banding. Tissue
engineers and biologists may find miRNAs to be extremely useful gene-silencing
tools.
Why don't more tissue engineers and developmental biologists work together?
When it comes to clinical translation of fundamental research, tissue engineering should be to developmental biology what drug development is to molecular biology. Yet many biologists find it difficult to see how they can gain from engineers who mix cells together with polymers, inject them into animals, and expect to find that they can regenerate lost tissues without understanding basic developmental principles. Tissue engineers cannot always appreciate why biologists are so fascinated by individual molecules or genes, or even by entire signaling pathways, because they concentrate more on the physical properties of the microenvironment. Developmental biologists also work with cell and molecular biologists in basic academic departments, whereas tissue engineers often collaborate with clinical champions in a hospital setting or interact with industry. Thus, it is not surprising that there has been significant tension between tissue engineers and developmental biologists, or at least a lack of camaraderie, in the past. However, the symposium clarified that this clash of perspectives has not restricted some investigators from bridging this gap. Moreover, a positive feed-back loop has been created, as those scientists and engineers who effectively span this interface are now able to act as `match-makers' between other biologists and engineers who seek to form new alliances.
So what is the best regeneration strategy?
More questions were generated than answered at the symposium. Do we develop optimal culture conditions for growing and differentiating stem cells outside our bodies, or do we fabricate injectable biomaterials that target to injury sites and recruit endogenous stem cells in vivo? Do we build fully functional adult organ replacements, or do we construct microenvironments that mimic embryonic organs, healing wounds or developmentally active inducer tissues? Should we develop cells and biomaterials that enhance existing tissue repair processes, or create reprogramming protocols that can induce adult human tissues to form blastemas and regenerate whole organs, as newts can? Do different kinds of injury (acute amputation, slow degeneration, hypoxic death, crush, etc.) exhibit the same propensity for regeneration, and how might we determine or control this behavior? Much of the discussion centered on exactly how much micromanaging of cellular behavior will be necessary to promote regeneration, regardless of the approach. Must we provide detailed instructions, such as precise time-varying changes of concentration and spatial gradients for every cytokine and morphogen used during normal development, or will we be able to rely on self-organizing properties of cell collectives and endogenous cues provided by the host environment that harness natural attractor-switching mechanisms?
Each investigator must follow their own vision of what form regenerative
therapies will take in the future. However, the possibility of identifying
`master regulators' - signals that activate numerous downstream events in a
coordinated fashion to generate complex structures - is an attractive one
(Fig. 1). This approach is
plausible given the demonstrated master regulator functions of molecular
signaling molecules, such as Wnt and ß-catenin, which can induce a new
primary axis during embryogenesis
(Funayama et al., 1995), or of
the Apc and Runx2 genes whose knockout results in
supernumerary teeth (Aberg et al.,
2004). However, this meeting clarified that there are also
biophysical master regulators, such as ion fluxes that alter electrical
potential and trigger regeneration of whole organs in Xenopus (see
above).
The activation of such high-level morphogenetic programs has the benefit of inducing a coherent response that stops when the structure is rebuilt. This is important because little is known about the precise signals that tell organs when to stop growing. However, triggering embryonic cascades is not always desirable. For example, Olson showed that stress-induced heart remodeling triggers activation of embryonic gene programs (expression of fetal forms of myosin heavy chain) that cause pathological changes in adult cardiac myocytes; reactivation of embryonic programs is also one of the hallmarks of cancer. Thus, a key question is whether we can fully reactivate embryonic programs in a regenerative context. If not, it will be necessary to develop more-focused strategies that selectively trigger limited aspects of the regenerative process, or artificial approaches that are `inspired' by normal developmental programs, rather than attempting to recreate them step-by-step.
It is also essential to identify the best target for a regeneration
strategy. Should we implant ESCs, control the activities of adult MSCs,
stimulate tissue renewal by somatic cells, dedifferentiate mature terminal
cells, or a combination of the above? All of these may be of value, and
although the current emphasis seems to focus on stem cells, data suggest that
exploring the neglected possibility of triggering growth of terminally
differentiated somatic cells (e.g. central nervous system neurons) might also
pay off (Cone and Cone, 1976;
Stillwell et al., 1973).
Likewise, biophysical approaches (e.g. using fluorescent voltage- or
pH-reporter dyes, measuring micromechanical properties) might make it feasible
to identify stem cell niches in vivo and to isolate and grow cells from adult
tissues with important self-renewal and differentiation properties. Discovery
of unique chemical or biophysical properties of these cells or of the local
tissue microenvironment might also lead to novel ways to preferentially target
drug payloads or novel self-assembling nanomaterials to these regions, and
thereby provide new approaches to modulate stem cell function and
developmental programs in situ.
Yet another problem that must be overcome is that developmental signaling can be very context-dependent. The same biochemical (e.g. Wnt) or physical signal (e.g. cell distortion) might be interpreted differently at different times and places, and thus give rise to entirely different developmental responses. The importance of a cell's provenance through the embryo for its final functional specialization emphasizes the crucial role that spatial and temporal context play in the conversion of a signal into a response in living systems. Appropriately crafting the regenerative microenvironment is likely to require combining biological and engineering strategies, as well as computational modeling approaches.
Conclusion
The emergence of regenerative medicine has created a new and larger tent, within which tissue engineers and developmental biologists find themselves to be only smaller acts. But at present, it appears unlikely that a stem cell, morphogen or artificial biomaterial will solve the problem of organ regeneration on its own. In the embryo, developmental responses result from the cumulative life experiences of cells as they pass through different spatial contexts, each with its own special regulatory milieu. Tissue engineers could not do what they do without the knowledge of the specific molecular regulators and ECM components that guide these developmental processes, which were uncovered by biologists. Biologists, likewise, are beginning to seek out engineers and physical scientists to address questions that cannot be effectively answered using their existing biological tools, such as how physical forces, electric potentials, spatiotemporal gradients and system-level dynamics influence morphogenetic control. Thus, alliances between researchers in these different fields are already self-organizing. However, complex biological behaviors emerge from collective interactions among numerous components, whether at the molecular, cellular, tissue or organ levels. Thus, to create therapeutics that repair injuries by promoting tissue and organ reconstruction rather than by scarring, we must recreate the correct microenvironment containing the right combination of physical, as well as chemical cues, and ensure that they are acting in the appropriate spatial and temporal context. To meet this challenge, we will need to combine our expertise in biology and engineering, and appropriate tools and approaches from many other disciplines as well. Only then will it be possible to develop effective therapeutics that can reprogram damaged tissues so that they are able to regenerate themselves.
ACKNOWLEDGMENTS
We thank G. Vunjak-Novakovic, R. T. Moon and D. Kaplan for organizing the meeting, P. G. Chao and N. Tandon for sharing their presentation notes, and Kristin Johnson for artwork. We apologize to those whose work could not be discussed because of space constraints. D.E.I. and M.L. gratefully acknowledge support from NIH, NSF, AHA and NHTSA.
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