Regrowing Limbs: Can People Regenerate Body Parts?
C. Ben Mitchell
Regrowing Limbs: Can People Regenerate Body Parts?
Progress on the road to regenerating major body parts,
salamander-style, could transform the treatment of amputations and major
wounds
By Ken Muneoka, Manjong Han and David M. Gardiner
A salamander*s limbs are smaller and a bit slimier than those of
most people, but otherwise they are not that different from their human
counterparts. The salamander limb is encased in skin, and inside it is
composed of a bony skeleton, muscles, ligaments, tendons, nerves and
blood vessels. A loose arrangement of cells called fibroblasts holds all
these internal tissues together and gives the limb its shape.
Yet a salamander*s limb is unique in the world of vertebrates in that
it can regrow from a stump after an amputation. An adult salamander can
regenerate a lost arm or leg this way over and over again, regardless of
how many times the part is amputated. Frogs can rebuild a limb during
tadpole stages when their limbs are first growing out, but they lose
this ability in adulthood. Even mammalian embryos have some ability to
replace developing limb buds, but that capacity also disappears well
before birth. Indeed, this trend toward declining regenerative capacity
over the course of an organism*s development is mirrored in the
evolution of higher animal forms, leaving the lowly salamander as the
only vertebrate still able to regrow complex body parts throughout its
lifetime.
Humans have long wondered how the salamander pulls off this feat. How
does the regrowing part of the limb *know* how much limb is missing
and needs to be replaced? Why doesn*t the skin at the stump form a
scar to seal off the wound as it would in humans? How can adult
salamander tissue retain the embryonic potential to build an entire limb
from scratch multiple times? Biologists are closing in on the answers to
those questions. And if we can understand how the regeneration process
works in nature, we hope to be able to trigger it in people to
regenerate amputated limbs, for example, and transform the healing of
other major wounds.
The human body*s initial responses to such a serious injury are not
that different from those of a salamander, but soon afterward the human
and amphibian wound-healing strategies diverge. Ours results in a scar
and amounts to a failed regeneration response, but several signs
indicate that humans do have the potential to rebuild complex parts. The
key to making that happen will be tapping into our latent abilities so
that our own wound healing becomes more salamanderlike. For this reason,
our research first focused on the experts to learn how it is done.
Lessons from the Salamander
When the tiny salamander limb is amputated, blood vessels in the
remaining stump contract quickly, so bleeding is limited, and a layer of
skin cells rapidly covers the surface of the amputation site. During the
first few days after injury, this so-called wound epidermis transforms
into a layer of signaling cells called the apical epithelial cap (AEC),
which is indispensable for successful regeneration. In the meantime,
fibroblasts break free from the connective tissue meshwork and migrate
across the amputation surface to meet at the center of the wound. There
they proliferate to form a blastema*an aggregation of stemlike cells
that will serve as progenitors for the new limb.
Many years ago studies in the laboratory of our colleague Susan V.
Bryant at the University of California, Irvine, demonstrated that the
cells in the blastema are equivalent to the cells in the developing limb
bud of the salamander embryo. This discovery suggested that the
construction of a limb by the blastema is essentially a recapitulation
of the limb formation that took place during the animal*s original
development. An important implication of this insight was that the same
genetic program is involved in both situations, and because humans make
limbs as embryos, in principle we should already have the necessary
programming to regenerate them as adults, too. It seemed, therefore,
that all scientists needed to do was figure out how to induce an
amputated limb to form a blastema.
One of us (Gardiner)*working with Tetsuya Endo of U.C. Irvine a few
years ago*took a minimalist approach to answering the basic question
of how to make a blastema. Instead of studying amputation sites on the
salamander, where a blastema would naturally form, we looked at simple
wounds on the side of a salamander limb, which would normally heal just
by regenerating the skin. Our idea was that such wounds are similar to
the site of an amputated mammalian limb that fails to generate a new
limb. If we could get an entire limb to grow where a simple
wound-healing response would typically occur, then we could further
dissect the regeneration process.
After we made a small incision in the salamander leg, epidermal cells
migrated to cover and seal the wound, as they would at an amputation
site, and fibroblasts from the dermis layer of the skin also moved in to
replace the missing skin. But if we carefully deviated a nerve to the
wound site, we could induce those fibroblasts to form a blastema
instead. Marcus Singer of Case Western Reserve University had already
demonstrated more than half a century ago that innervation was required
for a regeneration response, but our experiments clarified that unknown
factors provided by the nerve were influencing regeneration by altering
the behavior of resident fibroblasts.
These induced blastemas never progressed to the later stages of
regeneration to form a new limb, however. One more ingredient was
needed. The key to inducing a blastema that produced a new limb was to
graft a piece of skin from the opposite side of the limb to the wound
site, which allowed fibroblasts from opposite regions of the limb to
participate in the healing response. The resulting accessory limb was,
of course, growing out at an abnormal location, but it was anatomically
normal. So the basic recipe for making a blastema seemed relatively
simple: you need a wound epidermis, nerves and fibroblasts from opposite
sides of the limb. With this minimal view of limb regeneration in mind,
we began to focus on understanding the roles of the individual
ingredients.
We knew that the epidermis is derived from one of three layers of
primitive cells within an early developing embryo, the ectoderm, which
is also well known to provide signals that control the outgrowth of
limbs from limb buds on the embryo. Ectoderm cells gather in the bud to
form an apical ectodermal ridge (AER), which transiently produces
chemical signals that guide the migration and proliferation of the
underlying limb bud cells.
Although some of the critical signals from the epidermis have not yet
been identified, members of the family of fibroblast growth factors
(FGFs) are involved. The AER produces a number of FGFs that stimulate
the underlying cells of the limb bud to produce other FGFs, fueling a
feedback circuit of signaling between the AER and limb bud cells that is
essential for the outgrowth of a limb. A similar feedback circuit
spurred by the AEC is thought to function in the same way during limb
regeneration, and Hiroyuki Ide of Tohoku University in Japan discovered
that the progressive loss of regenerative ability in frog tadpoles is
associated with a failure to activate the FGF circuit. By treating older
nonregenerating tadpole limbs with FGF10, he was able to jump-start this
signaling circuit and stimulate partial regeneration of amputated
limbs.
The excitement this result inspired was tempered, however, by the fact
that the induced regenerates were abnormal, consisting of irregularly
placed limb parts, which raises the important issue of how regeneration
is controlled so that all the appropriate anatomical structures that are
lost when the limb is amputated are accurately replaced. It turns out
that the other primary cellular players, the fibroblasts, carry out this
function.
Location, Location, Location
Recall from the minimalist accessory-limb experiments that the presence
of fibroblasts per se was not sufficient for regeneration because
fibroblasts are present at the simple wound site that does not make a
new limb. It was the fibroblasts from the opposite side of the limb that
proved essential. That discovery illustrates the importance of cellular
position in triggering a regeneration response. In an embryo, the
sequence of events in limb development always begins with formation of
the base of the limb (the shoulder or hip) and is followed by
progressive building of more distal structures until the process
terminates with the making of fingers or toes. In salamander
regeneration, on the other hand (or foot), the site of amputation can be
anywhere along the length of the limb and regardless of where the wound
is located, only those parts of the limb that were amputated regrow.
This variable response indicates that cells at the amputation wound
edge must *know* where they are in relation to the entire limb. Such
positional information is what controls the cellular and molecular
processes leading to the perfect replacement of the missing limb parts,
and it is encoded in the activity of various genes. Examining which
genes are at work during these processes helps to reveal the mechanisms
controlling this stage of regeneration.
Although a large number of genes are involved during embryonic
development in educating cells about their position in the limb, the
activity of a gene family called Hox is critical. In most animals, cells
in the developing limb bud use the positional code provided by Hox genes
to form a limb, but then they *forget* where they came from as they
differentiate into more specialized tissues later on. In contrast,
fibroblasts in the adult salamander limb maintain a memory of this
information system and can reaccess the positional Hox code in the
process of limb regeneration.
During regeneration the fibroblasts bring this information with them as
they migrate across the wound to initiate blastema formation, and once
in the blastema, cells are able to *talk* to one another to assess
the extent of the injury. The content of this crosstalk is still largely
a mystery, but we do know that one outcome of the conversation is that
the regenerating limb first establishes its boundaries, including the
outline of the hand or foot, so that cells can use their positional
information to fill in the missing parts between the amputation plane
and the fingers or toes.
Because muscle and bone make up the bulk of a limb, we are also
interested in understanding where the raw material for those tissues
originates and what mechanisms control their formation. When the
regenerative response is initiated, one of the key early events involves
a poorly understood process called dedifferentiation. The term is
typically used to describe a cell*s reversion from a mature
specialized state to a more primitive, embryonic state, which makes it
capable of multiplying and serving as a progenitor of one or more tissue
types.
In the field of regeneration, the word was first used by early
scientists who observed under the microscope that the salamander stump
tissues, particularly the muscle, appeared to break down and give rise
to proliferative cells that formed the blastema. We now know that those
muscle-associated cells are derived from stem cells that normally reside
in the muscle tissue and not from dedifferentiation of muscle. Whether
or not dedifferentiation is actually happening in the case of every
tissue type within a regenerating limb has yet to be proved, although it
is clear that a variation of this theme does occur during regeneration.
Fibroblasts that enter the blastema and become primitive blastemal cells
have the ability to differentiate into skeletal tissues (bone and
cartilage) as well as to redifferentiate into the fibroblasts that will
form the interstitial meshwork of the new limb, for instance.
Returning to another of the central cellular players in blastema
formation, the epidermal cells, we can also pinpoint moments in the
regeneration process when it seems these cells are making a transition
to a more embryonic state. A number of genes active in the embryonic
ectoderm are critical for limb development, including Fgf8 and Wnt7a,
but as the ectoderm of the embryo differentiates to form the
multilayered epidermis of the adult, these genes are turned off. During
regeneration in the adult, the epidermal cells that migrate across the
amputation wound and establish a wound epidermis initially begin to
display gene activity, such as production of wound-healing keratin
proteins, that is not specifically related to regeneration. Later the
wound epidermal cells activate Fgf8 and Wnt7a, the two important
developmental genes. For practical purposes, then, the essential
definition of dedifferentiation*as it pertains to the epidermis and
other cell types*is the specific reactivation of essential
developmental genes.
Thus, our studies of salamanders are revealing that the regeneration
process can be divided into pivotal stages, beginning with the
wound-healing response, followed by the formation of a blastema by cells
that revert to some degree to an embryonic state, and finally, the
initiation of a developmental program to build the new limb. As we move
toward the challenge of inducing limb regeneration in humans, we rely on
these insights to guide our efforts. Indeed, the hardest things to
discover in science are those that do not already occur, and limb
regeneration in humans fits snugly into this category, although that
does not mean humans have no natural regenerative capacity.
Potential at Our Fingertips
One of the most encouraging signs that human limb regeneration is a
feasible goal is the fact that our fingertips already have an intrinsic
ability to regenerate. This observation was made first in young children
more than 30 years ago, but since then similar findings have been
reported in teenagers and even adults. Fostering regeneration in a
fingertip amputation injury is apparently as simple as cleaning the
wound and covering it with a simple dressing. If allowed to heal
naturally, the fingertip restores its contour, fingerprint and sensation
and undergoes a varying degree of lengthening. The success of this
conservative treatment of fingertip amputation injuries has been
documented in medical journals thousands of times. Interestingly, the
alternative protocol for such injuries typically included operating to
suture a skin flap over the amputation wound, a *treatment* that we
now know will inhibit regeneration even in the salamander because it
interferes with formation of the wound epidermis. The profound message
in these reports is that human beings have inherent regenerative
capabilities that, sadly, have been suppressed by some of our own
traditional medical practices.
It is not easy to study how natural human fingertip regeneration works
because we cannot go around amputating fingers to do experiments, but
the same response has been demonstrated in both juvenile and adult mice
by several researchers. In recent years two of us (Muneoka and Han) have
been studying the mouse digit-tip regeneration response in more detail.
We have determined that a wound epidermis does form after digit-tip
amputation, but it covers the regenerating wound much more slowly than
occurs in the salamander. We have also shown that during digit-tip
regeneration, important embryonic genes are active in a population of
undifferentiated, proliferating cells at the wound site, indicating that
they are blastema cells. And indirect evidence suggests that they are
derived from fibroblasts residing in the interstitial connective tissues
and in bone marrow.
To explore the roles of specific genes and growth factors during the
mouse-digit regeneration response, we developed a tissue culture that
serves as a model for fetal mouse-digit regeneration. With it, we found
that if we experimentally depleted a growth factor called bone
morphogenetic protein 4 (BMP4) from the fetal amputation wound, we
inhibited regeneration. In addition, we have shown that a mutant mouse
lacking a gene called Msx1 is unable to regenerate its digit tips. In
the fetal digit tip, Msx1 is critical to the production of BMP4, and we
were able to restore the regeneration response by adding BMP4 to the
wound in the Msx1-deficient mouse, confirming BMP4*s necessity for
regeneration.
Studies by Cory Abate-Shen and her colleagues at the Robert Wood
Johnson Medical School have also demonstrated that the protein encoded
by Msx1 inhibits differentiation in a variety of cell types during
embryonic development. That link to the control of differentiation
suggests that the protein plays a role in the regeneration response by
causing cells to dedifferentiate. Although Msx1 is not active during the
early dedifferentiation stages of salamander limb regeneration, its
sister gene Msx2 is one of the first genes reactivated during
regeneration and very likely serves a similar function.
The Human Challenge
The idea of regenerating a human limb may still seem more like fantasy
than a plausible possibility, but with insights such as those we have
been describing, we can evaluate in a logical stepwise manner how it
might happen. An amputated human limb results in a large and complex
wound surface that transects a number of different tissues, including
epidermis, dermis and interstitial connective tissue, adipose tissue,
muscle, bone, nerve and vasculature. Looking at those different tissue
types individually, we find that most of them are actually very capable
of regenerating after a small-scale injury.
In fact, the one tissue type within a limb that lacks regenerative
ability is the dermis, which is composed of a heterogeneous population
of cells, many of which are fibroblasts*the same cells that play such
a pivotal role in the salamander regeneration response. After an injury
in humans and other mammals, these cells undergo a process called
fibrosis that *heals* wounds by depositing an unorganized network of
extracellular matrix material, which ultimately forms scar tissue. The
most striking difference between regeneration in the salamander and
regenerative failure in mammals is that mammalian fibroblasts form scars
and salamander fibroblasts do not. That fibrotic response in mammals not
only hampers regeneration but can be a very serious medical problem unto
itself, one that permanently and progressively harms the functioning of
many organs, such as the liver and heart, in the aftermath of injury or
disease.
Studies of deep wounds have shown that at least two populations of
fibroblasts invade an injury during healing. Some of these cells are
fibroblasts that reside in the dermis, and the others are derived from
circulating fibroblastlike stem cells. Both types are attracted to the
wound by signals from immune cells that have also rushed to the scene.
Once in the wound, the fibroblasts migrate and proliferate, eventually
producing and modifying the extracellular matrix of the area. This early
process is not that dissimilar to the regeneration response in a
salamander wound, but the mammalian fibroblasts produce an excessive
amount of matrix that becomes abnormally cross-linked as the scar tissue
matures. In contrast, salamander fibroblasts stop producing matrix once
the normal architecture has been restored.
An exception to this pattern in mammals does exist, however. Wounds in
fetal skin heal without forming scars*yielding perfect skin
regeneration and indicating that the switch to a fibrotic response
arises with the developmental maturation of the skin. Although this
difference could reflect a change in the biology of the fibroblasts, it
is more likely a result of altered signaling from the extracellular
wound environment modulating the behavior of the fibroblasts, which in
turn suggests that therapeutically modifying those signals could change
the healing response. At the same time, the fact that limb amputations
during fetal stages of development do not result in regeneration of the
limb reminds us that scar-free wound healing is likely to be necessary
but not sufficient for regeneration.
To advance our understanding of what it will take to induce limb
regeneration in people, we are continuing our work with mice. Our
research group has already described a natural blastema in a mouse
amputation injury, and our goal within the next year is to induce a
blastema where it would not normally occur. Like the accessory-limb
experiments in salamanders, this achievement would establish the minimal
requirements for blastema formation. We hope that this line of
investigation will also reveal whether, as we suspect, the blastema
itself provides critical signaling that prevents fibrosis in the wound
site.
If we succeed in generating a blastema in a mammal, the next big hurdle
for us would be coaxing the site of a digit amputation to regenerate the
entire digit. The complexity of that task is many times greater than
regenerating a simple digit tip because a whole digit includes joints,
which are among the most complicated skeletal structures formed in the
body during embryonic development. Developmental biologists are still
trying to understand how joints are made naturally, so building a
regenerated mouse digit, joints and all, would be a major milestone in
the regeneration field. We hope to reach it in the next few years, and
after that, the prospect of regenerating an entire mouse paw, and then
an arm, will not seem so remote.
Indeed, when we consider all that we have learned about wound healing
and regeneration from studies in various animal models, the surprising
conclusion is that we may be only a decade or two away from a day when
we can regenerate human body parts. The striking contrast between the
behavior of fibroblasts in directing the regeneration response in
salamanders versus the fibrotic response leading to scarring in mammals
suggests that the road to successful regeneration is lined with these
cells. Equally encouraging is the recent discovery by Howard Y. Chang
and John L. Rinn of Stanford University that adult human fibroblasts,
like salamander fibroblasts, retain a memory of the spatial coordinate
system used to establish the body plan early in the embryo*s
development. Given that such positional information is re--quired for
regeneration in salamanders, its existence in human fibroblasts enhances
the feasibility of tapping into and activating developmental programs
necessary for regeneration.
Now, as we watch a salamander grow back an arm, we are no longer quite
as mystified by how it happens. Soon humans might be able to harness
this truly awesome ability ourselves, replacing damaged and diseased
body parts at will, perhaps indefinitely.