Tissue engineering is an emerging interdisciplinary field

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Tissue engineering is an emerging interdisciplinary field that applies the principles of
biology and engineering to the development of viable substitutes that restore,
maintain, or improve the function of human tissues or organs. This form of therapy
differs from standard therapies in that the engineered tissue becomes integrated
within the patient, affording a potentially permanent and specific cure of the disease
state.
Three general approaches have been adopted for the creation of new tissue:
1. Design and grow human tissues outside the body
for later implantation to repair or replace diseased
tissues. The most common example of this form of
therapy is the skin graft, which is used for treatment of
burns. Skin graft replacements have been grown and used
clinically for more than 10 years.
2. Implantation of cell-containing or cellfree devices that induce the regeneration
of functional human tissues. This approach
relies on the purification and large-scale
production of appropriate "signal" molecules,
like growth factors, to assist in biomaterialguided tissue regeneration. In addition, novel
polymers are being created and assembled
into three-dimensional configurations, to
which cells attach and grow to reconstitute
tissues. An example is the biomaterial matrix
used to promote bone re-growth for periodontal disease.
3. The development of external or internal
devices containing human tissues
designed to replace the function of
diseased internal tissues. This approach
involves isolating cells from the body, using
such techniques as stem cell therapy, placing
them on or within structural matrices, and
implanting the new system inside the body or
using the system outside the body. Examples
of this approach include repair of bone, muscle,
tendon, and cartilage, as well as cell-lined
vascular grafts and artificial liver that are being
developed in Pittsburgh.
Broadly defined, tissue engineering is the development and manipulation of
laboratory-grown molecules, cells, tissues, or organs to replace or support the
function of defective or injured body parts.
Although cells have been cultured, or grown, outside the body for many years, the
possibility of growing complex, three-dimensional tissues - literally replicating the
design and function of human tissue - is a recent development. The intricacies of this
process require input from many types of scientists, including the problem solving
expertise of engineers, hence the name tissue engineering.
Tissue engineering crosses numerous medical and technical specialties. Cell
biologists, molecular biologists, biomaterial engineers, computer-assisted designers,
microscopic imaging specialists, robotics engineers, and developers of equipment
such as bioreactors, where tissues are grown and nurtured, are all involved in the
process of tissue engineering.
Tissue engineers in the United States and abroad have set out to grow virtually every
type of human tissue - liver, bone, muscle, cartilage, blood vessels, heart muscles,
nerves, pancreatic islets, and more. Commercially produced skin is already available
for use in treating patients with diabetic ulcers and burns.
Many current medical therapies may be improved upon by
tissue engineering with significant financial savings. Take
organ transplantation, for example. In standard organ
transplantation, a mismatch of tissue types necessitates
lifelong immunosuppression, with its attendant problems of
graft rejection, drug therapy costs, and the potential for the
development of certain types of cancer. Furthermore, there
is always the potential for rejection of the tissue, and the
surgery, itself, always carries some risk.
As the field of tissue engineering progresses, it will inevitably provide many
improvements:


The costs of tissue harvest and postoperative patient costs will be significantly
reduced.
By actually designing replacements to mimic the native tissue being
reconstructed, the adequacy of tissue function will be optimized, leading to
improved patient care at less expense.
A Compelling Need
By Ricki Lewis
Field
Author: Ricki Lewis
Sidebars: A Tissue Survey
Over the past decade, tissue engineering has evolved from a hodgepodge of different disciplines
to a biotechnology field in its own right. A marriage of chemical engineering and cell biology, with
input from genetics and surgery, tissue engineering combines living cells, biochemicals, and
synthetic materials into implants that can function in the human body.
Some 30 companies and dozens of academic laboratories are pursuing tissue engineering, with
the ultimate goal of fashioning stand-ins for such varied tissues as pancreatic islets, liver, skin,
cartilage, bone, nervous tissue, bone marrow, and blood vessels. And although experts agree
that it will still be a few years until the family physician offers up replacement skin, engineered
tissues are already impacting basic cell culture research, taking it one step closer to mimicking
true in vivo conditions.
Today's tissue engineers are an eclectic group. The field "includes chemical engineers, chemists,
cell biologists, and surgeons. Many times you find teams, or people who can work in several
areas at once," observes Jeffrey Hubbell, a professor of chemical engineering at the California
Institute of Technology. Hubbell investigates synthetic polymers that can serve as scaffolds in
engineered tissues.
"People have had the idea for years to generate new tissue, but they have not had the
techniques, nor new synthetic materials, to make that a reality," notes Charles A. Vacanti,
chairman of the department of anesthesiology at the University of Massachusetts Medical Center
in Worcester. He is an editor of Tissue Engineering, a new journal published by New York-based
Mary Ann Liebert Inc. He is also a cofounder of a new professional organization, the Tissue
Engineering Society, with his brother Joseph Vacanti, a transplant surgeon at Children's Hospital
in Boston, and Robert Langer, a professor of chemical and biomedical engineering at the
Massachusetts Institute of Technology.
Charles Vacanti says he was stimulated to enter the field by an idea to combine polymers and
cells that his brother and Langer were tossing around eight years ago. His experience as an
anesthesiologist helped him recognize the need for engineered tissue. "I would peek over the
shoulders of orthopedic surgeons and watch them use bone grafts and bone powders," he
recalls. "I began thinking about my brother and Robert Langer's idea to create new tissue."
Charles Vacanti began working in Langer's lab at night, starting with cartilage. "Since then, we've
worked on about 10 different tissues," says Langer.
Motivating researchers in the field is the great demand for human spare parts. The cost stemming
from treatment and lost work hours for tissue and organ disease and injury in the United States
per year exceeds $400 billion, according to Langer and Charles Vacanti. And despite the speed
with which baseball hero Mickey Mantle and rock musician David Crosby obtained liver
transplants, fewer than 3,000 livers become available in the U.S. each year - as 30,000 people
die from liver failure.
The numbers are similar for other organs and tissues. Even the few existing organ replacements
or alternatives, such as skin grafts, ventricular-assist devices, and kidney dialysis, are hardly
ideal.
While clinical trials for some engineered tissues progress, the cell-synthetic combinations are also
finding uses in basic research. "In my lab, our long-term goal is to develop a blood vessel
substitute, but our initial goal is to develop a better model for use in cell-culture labs," says Robert
Nerem, a professor of mechanical engineering at the Georgia Institute of Technology. Such hot
cell biology areas as signal transduction, cell adhesion, and carcinogenesis will benefit from
engineered tissues, which can allow researchers to isolate steps in these complex processes.
For example, several researchers are manipulating a three-amino acid sequence (arginineglycine-aspartic acid), called the RGD sequence. This tripeptide is part of a common cell-surface
receptor protein which, like Velcro, enables cells to stick to each other and to an extracellular
matrix (substances cells secrete that surround them). By incorporating the RGD sequence into
synthetic polymers, chemical engineers can add a Velcro-like touch that attracts certain cells.
"We sometimes add the RGD sequence to our synthetic biodegradable polymers," notes Langer.
"People are finding that key amino acid sequences can be used to regulate cell growth. A lot of
information is apparently packed into a few amino acids."
Like many areas of biomedical engineering, tissue engineering is a field that existed before it had
a formal title. Many review articles date its formal debut to a series of National Science
Foundation meetings. First was a panel meeting early in 1987 when the name was brought up.
Then, in October, a forum on emerging technologies identified tissue engineering as an area for
rapid industrial development. At an NSF workshop on tissue engineering held at Lake Tahoe on
Feb. 26-29, 1988, a formal definition was drafted:
"Tissue engineering is the application of principles and methods of engineering
and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of
biological substitutes to restore, maintain, or improve tissue functions."
But long before tissue engineering had a name, researchers in more traditional fields were
experimenting with teaming cells and synthetic materials. Carl Wolf, director of the Blood Bank
Transfusion Service at New York Hospital Cornell Medical Center, a chemical engineer-turnedphysician, was one of them.
Before and during medical school, in the late 1960s and early 1970s, he worked at the DuPont
Experimental Station in Wilmington, Del. At first, he was combining hollow synthetic fibers conduits for nutrients and wastes - with kidney cells to mimic renal function. Then, when blood
clotting became a problem, he had the idea to add liver cells, which secrete the anticoagulant
heparin. Realizing that the hollow fibers with liver cells could emulate liver function, he turned his
interest toward that organ.
Later he found a hepatitis research group at the New York Blood Center that was growing liver
cells, and he went to work on a bioengineered liver. "At first I tried growing cells in fibers, lining
them, with only moderate success. It occurred to me that maybe cells could function just as well
growing on the outside of the fibers - which is the geometry that is currently being tried clinically,"
says Wolf.
Great ideas sometimes spark in several minds at once. At the same time that Wolf was nurturing
his liver hollow-fiber bioreactor, Richard Knazek at the National Institute (now Institutes) of Health
was doing the same thing, and patented the invention (R.A. Knazek, Science, 178:65, 1972). And
at the University of Massachusetts Medical School, William L. Chick was combining insulinsecreting pancreatic islet cells with hollow fibers to treat diabetes in dogs (W.L. Chick, Diabetes,
20:327, 1971; W.L. Chick, Science, 187:847, 1975).
Then came AIDS, and Wolf had to put his research interests aside as he dealt with the mounting
crisis of HIV-contaminated blood. "That put an end to my research in tissue engineering," he
says. "I advanced the field as far as I could. I showed how to grow cells on hollow fibers, and how
to hook them into animals. But the field needed a major developmental effort to take it from
research to engineering."
That push came in the mid-1980s, when tissue engineering began to find its niche in the biotech
industry. Organogenesis Inc. of Canton, Mass., was formed in 1985, and Advanced Tissue
Sciences Inc. in La Jolla, Calif., in 1986. Both companies are racing to market their skin
substitutes. Providence, R.I.-based CytoTherapeutics came on the scene in 1988, and focuses
on central nervous system implants (see accompanying story). Several other companies are also
pursuing tissue engineering.
Building tissue entails several steps. The first is biological - identifying cell types that constitute a
tissue. "The biggest challenge is to get the right structure for a tissue with many cell types,"
Hubbell points out. "For example, cartilage is easy, because it is not vascularized, and consists of
chondrocytes [cartilage cells] in a matrix that's not highly organized. Skin also has a relatively low
structural level. Larger, vascularized constructs, such as pancreas, liver, and kidney, are more
difficult to model."
Researchers must also select synthetic materials, usually polymers, that are biodegradable and
compatible with cells. In addition, cells are often stripped of surface molecules that could
stimulate an immune response. Synthetics have molecules added that can foster cell adhesion.
A step less studied in tissue engineering is to understand the physical constraints on tissue
formation and maintenance. How will pressure from surrounding structures affect implanted
tissue? How can a tissue be engineered so that each cell is only a short distance from a blood
supply, as occurs in vivo? How do shear and tension affect cell specialization? These
considerations are where engineering comes in.
Looking at tissue structure and function from an engineering perspective enabled J.H. David Wu,
an associate professor of chemical engineering, microbiology, and immunology at the University
of Rochester, to develop a long-term bone marrow culture that may make bone marrow
transplants easier, cheaper, and more successful. The procedures are performed to treat
inherited or acquired blood disorders or to increase tolerance to chemotherapy and radiation
therapy.
Wu noticed that the conventional way to culture bone marrow cells - in petri dishes or culture
flasks - was nothing like the real thing. So he took cues from the anatomy of bone marrow. "In the
body, bone marrow is a spongy filler of bone," Wu explains. "When blood flows through bone
marrow, it takes with it mature blood cells, which egress into blood vessels from bone marrow. In
the body, bone marrow makes about 10 different types of blood cells. But in a petri dish, it makes
only two."
Wu developed a bioreactor that nurtures bone marrow cells in a sponge-like material consisting of
porous bovine collagen microspheres. "With this method, you start to see all the cell types. By
taking an engineering approach, we are moving into a completely different way to culture bone
marrow," he says.
An enthusiastic response to the new journal and society are evidence of the field's growth,
according to Vacanti. Using lists from existing societies to which people doing this research might
belong, as well as by searching citations, Vacanti sent postcards to 1,000 people, inquiring about
interest in forming a society. Within weeks, he had 500 responses. With his brother and Robert
Langer, he began planning the Tissue Engineering Society. "I found very quickly that there was a
big need for the journal and, talking to people at national meetings, also learned there was a big
need for a society," he says.
Even though engineered tissue as a treatment isn't yet a medical reality, there are already
benefits for life scientists. Concludes Robert Nerem: "Tissue engineering won't enter the clinic for
a decade, except perhaps for skin. But in the meantime, there will be significant payoff in terms of
the impact of these constructs in the ability to do basic research. What we are seeing is the very
beginning of an industry."
Ricki Lewis is a biology textbook author and geneticist based in Scotia, N.Y.
(The Scientist, Vol:9, #15, pg.12 , July 24, 1995)
(Copyright © The Scientist, Inc.)
[email protected]
RESEARCH
Stem Cells Tapped to Replenish
Organs
Embryonic or adult? The superior source depends on the tissue
By Douglas Steinberg
Credit: Eric Laywell
Editors Note: This is the second of two articles on issues
raised by recent stem cell discoveries. The first article
appeared in the November 13 issue
"All politics is local" was a famous maxim of Thomas "Tip"
O'Neill, the late speaker of the House of Representatives, and
the same can be said of medically useful stem cells.
Progenitor cells may prove to be more or less pluripotent in the
lab, but if they don't succeed on a local level in the body, they
won't cure anything. They must be capable of being coaxed
into differentiating reliably into the cell types that populate
particular organs.
How much can embryonic stem cells (ESCs) and adult stem
cells (ASCs) replenish tissues of the brain, pancreas, liver,
heart, and blood? So far, researchers have manipulated ESCs
to generate a broad span of cell types. ASCs have yielded a
narrower range, partly because several subtypes haven't been
isolated yet.
The phenomenon of transdifferentiation, however, promises to
extend the capabilities of ASCs. And as studies proliferate in
the wake of discoveries and the issuance of new guidelines by
the National Institutes of Health, the relative advantages and
disadvantages of ESCs and ASCs could change considerably An astrocyte monolayer that can
be coaxed into becoming
within the next few years.
multipotent neural stemlike cells
Brain
Goal: To replace neurons that have died as a result of degenerative diseases or stroke.
Ronald D.G. McKay and his Laboratory of Molecular Biology at the National Institute of
Neurological Disorders and Stroke can efficiently generate dopaminergic and serotonergic
functional neurons in vitro from mouse ESCs.2 They can get ASCs, in the form of mesencephalic
precursor cells, to induce functional recovery when transplanted into parkinsonian rats. 3 But
according to McKay, these ASCs stop generating dopaminergic neurons in culture after a week or
so.
The yield improves if the cells are grown under low-oxygen conditions, which are characteristic of
the fetal environment.4 Still, McKay notes that his lab's experience thus far with several types of
ASCs is that "they don't turn into dopaminergic neurons with any kind of efficiency." Referring to a
1999 paper from the Karolinska Institute that reported such a result,5 he wonders whether the
final yield is "really a dopaminergic cell or not."
One problem besetting such research is the uncertain identity of ASCs in the mammalian brain.
Last year, a Karolinska team led by Jonas Frisén announced that the ependymal cells lining the
brain's ventricles were neuronal ASCs.6 Five months later, a Rockefeller University group headed
by Arturo Alvarez-Buylla countered that subventricular zone (SVZ) astrocytes were the true
neuronal ASCs. This group also rejected the ependymal-cell hypothesis after finding that those
cells neither formed neurospheres, nor accumulated nucleoside labels, as they would if they
divided.7 The New York Times ran a story on the ensuing brouhaha.8
Credit: Eric Laywell Alvarez-Buylla, who just moved to the neurosurgery
department of the University of California, San
Francisco, says that the conflict may arise, in part,
because SVZ astrocytes "interact very, very closely
with the ependymal cells." But he maintains that
ependymal cells only serve to create a niche where
neurogenesis can occur. His lab is currently
examining two signaling systems that seem to prompt
SVZ astrocytes into becoming neurogenic.9
Last June, Frisén bolstered his theory with a paper
showing that neural stem cells had broad
differentiation potential.10 The authors couldn't verify
that most of their experiments actually involved
ependymal cells. But when ependymal-cell-derived
neurospheres were injected into the amniotic cavities
A clone of newly generated neurons derived of chick embryos, the cells showed broad
differentiation potential (the data, at footnote 16,
from a monolayer of astrocytic stemlike
weren't published). Frisén now says he has additional,
cells
unpublished lines of evidence indicating that
ependymal cells are neural stem cells.
His theory may need that support. Derek van der
Kooy and his colleagues at the University of Toronto weren't able to get ependymal cells to make
neurons in vitro.11 A similar negative finding appears in an upcoming paper describing a study led
by Eric D. Laywell and Dennis A. Steindler, professors of anatomy and neurobiology at the
University of Tennessee in Memphis.12
They and their colleagues, on the other hand, confirmed Alvarez-Buylla's hypothesis by observing
that SVZ astrocytes could give rise to neurons, as identified by the expression of ß-III tubulin and
other markers. (Functional studies of the neurons are now under way.) In a significant extension
of that hypothesis, they found that astrocytes from cerebral cortex, cerebellum, and spinal cord
could also turn into neurons--but only if the astrocytes were derived in the first two postnatal
weeks.
"This correlates with what we believe to be the maturation of the astrocyte in the nervous
system," notes Steindler. "The end of this critical period in astrocyte multipotency coincides with
the end of a period in which the brain's regenerative responses are far more successful than
those in the more mature brain."
Pancreas
Credit: Russ Lante, University of Florida, Health
Goal: To replace insulin-producing islet ß cells destroyed in
Sciences Center
some types of diabetes.
Stem cell research involving the pancreas seemed to score
two home runs this year. In February, Bernat Soria and
colleagues at the Universidad Miguel Hernandez in San Juan,
Spain, reported that they had obtained insulin-secreting cells
from mouse ESCs by using antibiotic selection under the
control of the insulin gene's regulatory regions.13 Soria says he
is now trying to replicate his results using human ESCs. (A
poster at a recent diabetes meeting, meanwhile, is said to
have announced that human ESCs differentiate spontaneously
into insulin-positive cells.)
A month after the Soria paper came out, a team of researchers
led by Ammon B. Peck, a professor of pathology,
immunology, and laboratory medicine at the University of
Florida College of Medicine in Gainesville, reported a second
major advance. They claimed to have reversed diabetes in
non-obese diabetic (NOD) mice by transplanting islets
generated in vitro from pancreatic ASCs, which had not been
previously isolated.14 NOD mice are the best current model for
autoimmune diabetes.
Nora D. Sarvetnick, a professor of immunology at Scripps
Ammon Peck
Research Institute, is puzzled by Peck's results. "Unless you
immunosuppress the mouse"--which wasn't done--"the mouse
is just going to reject the ß cells," she contends. Peck
responds that cells grown in culture, such as his ASC-derived islets, sometimes exhibit lower
antigenicity for unknown reasons.
Credit: J. Berndt Susan Bonner-Weir, an associate professor of
medicine at Harvard Medical School, objects that the
amount of insulin in Peck's ASC-derived islet cells
was "orders of magnitude" too low. "What they were
putting in [the NOD mice] would have been a very
minuscule amount," she says, though she concedes
that more insulin might have been made if the islet
cells differentiated further inside the mice. BonnerWeir's own work involves expanding human
pancreatic duct cells in vitro, then turning them into
insulin-producing islet cells.15 She calls the duct cells,
which are differentiated, "functional stem cells"
because they undergo scores of doublings in culture
Susan Bonner-Weir
and help to regrow pancreas after a portion is
removed.
Liver
Goal: To develop a plentiful source of hepatocytes for regenerating damaged livers and treating
some metabolic diseases.
Another functional stem cell is the hepatocyte. "For liver repopulation purposes and
transplantation, the best cell type is the differentiated hepatocyte," says Markus Grompe, a
professor of molecular and medical genetics and pediatrics at Oregon Health Sciences
University. He adds that in transplants, hepatocytes are "far superior" to liver stem cells, whose
existence has been established only in the past 12 months or so.
The major source of hepatocytes for therapeutic purposes, however, is human cadavers. More
accessible and plentiful are the liver stem cells residing in the bone marrow, discovered by Neil
D. Theise, an associate professor of pathology at New York University School of Medicine, and
colleagues. Their proof: The Y chromosome pops up in some hepatocytes after male marrow
transplants into females.
Are these new liver cells functional? In a small-scale study of human transplants,16 "We show
such extensive engraftment that it's hard to avoid the conclusion that this is a part of physiological
regeneration," asserts Theise. The next test is to use bone-marrow transplants to correct
defective liver function in animal models of some human metabolic diseases. Grompe and a team
of researchers published a paper this month reporting such a finding in a mouse model of
tyrosinemia.17
The roles played by ASCs in the liver are still far from clear. Intrahepatic oval cells have recently-and grudgingly--won full acceptance as stem cells, particularly after injury. (Theise proposes that
oval cells ultimately derive from the bone marrow.) Apparently no one has yet generated liver
cells from ESCs. The growth factors "are just absolutely not known," notes Grompe.
Heart
Goal: To replace cardiomyocytes that have died during heart attacks.
Several years ago, the lab of Loren J. Field, a professor of medicine and pediatrics at Indiana
University School of Medicine in Indianapolis, derived relatively pure cardiomyocyte cultures from
transfected mouse ESCs.18 The cardiomyocytes weren't identical to their adult counterparts. But
according to Field, experimental data suggest that under appropriate humoral and neuronal
stimulation, a cardiomyocyte derived from ESCs "will adapt the characteristics typical for the adult
cell."
Transdifferentiation
Researchers will have to understand
transdifferentiation better before they can
The number of heart muscle cells in a mouse is
several orders of magnitude lower than the
number in a human. Now that his lab has
refined its methods, Field is optimistic that "with
bio-processing and growth factors, we can
produce sufficient cells for therapeutic
applications." To address the low efficiency at
which the cardiomyocytes seed into recipient
hearts, he is testing such strategies as blocking
apoptosis, making the cells more resistant to
ischemia, and boosting their capacity to divide.
Geron Corp., based in Menlo Park, Calif., and a
few academic labs have already shown that
cultured human ESCs can give rise to
cardiomyocytes. Meanwhile, the presence of
ASCs in the heart itself still hasn't been proven.
"If they exist, they aren't doing their job," Field
says, noting the heart's limited capacity to heal
after injury. Other researchers have reported
finding ASCs for cardiomyocytes in other parts
of the body such as the bone marrow, but no
such claim has yet won wide acceptance.
deploy adult stem cells (ASCs) as broadly and
Blood
Richard C. Mulligan, a professor of genetics at
effectively as possible. Transdifferentiation is
the phenomenon whereby a muscle ASC, say,
can give rise to a blood cell.
Margaret A. Goodell, who studies stem cells
at Baylor College of Medicine's Center for Cell
& Gene Therapy, foresees that once biologists
begin to "rationalize" the recent spate of
observations of this phenomenon, "it won't
turn out to be just this wild free-for-all where
anything can differentiate into anything." Rules
discovered over the last 20 years, she adds,
"must have some meaning because otherwise
you wouldn't get the development of a very
highly organized animal."
Harvard Medical School, has proposed
Goal: To develop a limitless source of blood
cells for transfusions.
Over the past 30 years, a small army of
researchers has investigated the culture
conditions under which hematopoietic ASCs
preferentially give rise to myeloid or lymphoid
lineages. (Relatively pure cultures of red blood
cells have been the most elusive to produce.)
Gordon Keller, a professor at Mount Sinai
School of Medicine's Institute for Gene Therapy
and Molecular Medicine, has succeeded at
differentiating mouse ESCs into a variety of
blood cell types, though he admits that
generating lymphocytes is still a problem. His
lab has developed the requisite protocols by trial
and error over the past decade.19
When removed from conditions that keep them
in an undifferentiated state, ESCs form clusters
of differentiating cells called embryoid bodies.
"At that point, we take the cells from the
embryoid body and put them into cultures
containing cytokines that stimulate the growth
and maturation of blood-cell progenitors," Keller
recounts. "Alternatively, we can first isolate the
blood-cell progenitors from the embryoid bodies
by using antibodies to specific cell-surface
markers and then put them into culture."
Keller is now searching within embryoid bodies
for the hematopoietic stem cell equivalent to the
hematopoietic ASC that other labs have isolated
alternative hypotheses that could help explain
transdifferentiation. One theory is that ASCs in
various organs all originate from ASCs in bone
marrow; these ASCs then adopt organspecific traits after being seeded in local
environments. The other theory is that ASCs
arise independently in various organs but
share phenotypic and functional
characteristics. Thus, ASCs from one organ
can generate mature cells of another organ
because the ASCs of both organs have a
common origin and/or exhibit certain common
features.
In a 1999 Nature paper, a team headed by
Mulligan and his Harvard colleague, Louis M.
Kunkel, reported that injecting muscle-derived
ASCs into irradiated mice led to reconstitution
of the recipients' hematopoietic compartment.1
ASCs with this capability were designated
muscle SP ("side population") cells. Like
hematopoietic SP cells, muscle SP cells
resisted staining by a Hoechst dye. The two
SP cell types weren't identical, however.
In unpublished work since then, "we've
in bone marrow. This putative stem cell in the embryoid body has been harder to find, he says,
because it "appears to be more immature than the one in adult bone marrow." His approach is to
transplant candidate stem cells into mice with drug-damaged hematopoietic systems and then to
observe whether blood-cell re-population occurs. When might his methods boost human blood
supplies for transfusions? "Some years away" is all that Keller will predict.
Douglas Steinberg is a freelance writer in New York.
References
1. D. Steinberg, "Stem cell discoveries stir debate," The Scientist, 14[22]:1,14-5, Nov. 13, 2000.
2. S.-H. Lee et al., "Efficient generation of midbrain and hindbrain neurons from mouse embryonic
stem cells," Nature Biotechnology, 18:675-9, June 2000.
3. L. Studer et al., "Transplantation of expanded mesencephalic precursors leads to recovery in
parkinsonian rats," Nature Neuroscience, 1:290-5, 1998.
4. L. Studer et al., "Enhanced proliferation, survival, and dopaminergic differentiation of CNS
precursors in lowered oxygen," Journal of Neuroscience, 20:7377-83, Oct. 1, 2000.
5. J. Wagner et al., "Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing
neural stem cells by type 1 astrocytes," Nature Biotechnology, 17:653-9, 1999.
6. C.B. Johansson et al., "Identification of a neural stem cell in the adult mammalian central
nervous system," Cell, 96:25-34, 1999.
7. F. Doetsch et al., "Subventricular zone astrocytes are neural stem cells in the adult mammalian
brain," Cell, 97:703-16, 1999.
8. N. Wade, "Brain stem cell is discovered, twice," New York Times, p. F3, June 15, 1999.
9. One paper is in press. For the other, see J.C. Conover et al., "Disruption of Eph/ephrin
signaling affects migration and proliferation in the adult subventricular zone," Nature
Neuroscience, 3:1091-7, November 2000.
10. D.L. Clarke et al., "Generalized potential of adult neural stem cells," Science, 288:1660-3,
June 2, 2000.
11. B.J. Chiasson et al., "Adult mammalian forebrain ependymal and subependymal cells
demonstrate proliferative potential, but only subependymal cells have neural stem cell
characteristics," Journal of Neuroscience, 19:4462-71, 1999.
12. E.D. Laywell et al, "Identification of a multipotent astrocytic stem cell in the immature and
adult mouse brain," Proceedings of the National Academy of Sciences (PNAS), in press.
13. B. Soria et al., "Insulin-secreting cells derived from embryonic stem cells normalize glycemia
in streptozotocin-induced diabetic mice," Diabetes, 49:157-62, February 2000.
14. V.K. Ramiya et al., "Reversal of insulin-dependent diabetes using islets generated in vitro
from pancreatic stem cells," Nature Medicine, 6:278-82, March 2000.
15. S. Bonner-Weir et al., "In vitro cultivation of human islets from expanded ductal tissue,"
PNAS, 97:7999-8004, July 5, 2000.
16. N.D. Theise et al., "Liver from bone marrow in humans," Hepatology, 32:11-6, July 2000.
17. E. Lagasse et al., "Purified hematopoietic stem cells can differentiate into hepatocytes in
vivo," Nature Medicine, 6:1229-34, November 2000.
18. M.G. Klug et al., "Genetically selected cardiomyocytes from differentiating embryonic stem
cells form stable intracardiac grafts," Journal of Clinical Investigation, 98:216-24, 1996.
19. See, e.g., M. Kennedy et al., "A common precursor for primitive erythropoiesis and definitive
haematopoiesis," Nature, 386:488-93, 1997.
Precursor Cells to the Rescue?
Two reports out this month suggest that
less-than-fully differentiated cells-whether embryonic or adult--could help
humans recover from a host of nervous
system ailments, ranging from motor
neuron diseases to brain cancer.
In a study announced at the Society for
Neuroscience meeting in New Orleans
Nov. 4-9, researchers from Johns
Hopkins University School of Medicine
Left: Immunohistological staining for epithelialspecific cytokeratins of a female liver allograft in a
male recipient. Right: Fluorescent in situ
hybridization for X and Y chromosomes.
and Harvard Medical School infected
rodents with Sindbis virus, which causes limb paralysis by attacking the motor neurons that
thread from the spinal cord to the muscles. The team then injected embryonic germ cells
(EGCs), primordial cells that are pluripotent like stem cells, into the cerebrospinal fluid (CSF)
at the base of the animals' spines. The EGCs were pretreated with growth factors to nudge
them onto the path of neural differentiation, thereby preventing them from developing into
tumors.
Within several weeks, the EGCs had migrated to the ventral horn of the spinal cord, which
contains the cell bodies of motor neurons. And by eight weeks after injection, 11 out of 18
animals had regained the ability to place the soles of one or both of their hind feet on the
ground. Yet, only about 6 percent of the migrating EGCs seemed to have differentiated into
neurons, as indicated by expression of cell-surface markers.
Earlier animal studies showed that applying stem cells to the site of a traumatic spinal-cord
injury leads to some functional recovery. The newly reported experiments were the first
involving a diffuse disease that affects the whole spinal cord and the first in which primordial
cells were delivered via CSF, according to lead researcher Douglas A. Kerr, an assistant
professor of neurology at Hopkins. In ongoing work, "We're trying to characterize why the
animals recovered," he says. "And we're also trying to trick the cells prior to implantation into
really thinking that they are to be motor neurons. Presumably then we might even see a
better functional recovery."
Before Kerr and his colleagues turned to EGCs, their study used human neural stem cells
(NSCs) derived from a fetus' telencephalon by Evan Y. Snyder, an assistant professor of
neurology at Harvard Medical School.1 Kerr recalls that these NSCs restored some function
to a handful of rodents, but that they showed no effect in later experiments on a larger group
of animals.
Snyder and a Harvard team, meanwhile, used the same NSCs in a newly published study
on brain cancer.2 After the researchers implanted glioblastoma cells into rodents, the
animals developed intracranial tumors. NSCs, which were implanted several days later,
infiltrated and surrounded the tumors, and chased down malignant cells that were migrating
into normal tissue. Tumor targeting occurred even when the NSCs were introduced far from
a tumor, such as into the vein of an animal's tail.
When NSCs expressed cytosine deaminase, an enzyme that converts a non-toxic pro-drug
into a chemotherapeutic agent, one mouse's tumor shrank about 80 percent. The
researchers found that the NSCs neither differentiated nor turned tumorigenic in the
recipient rodents.
One message of the study, says Snyder, is that "if there's pathology, not only can there be
very dramatic, extensive [NSC] migration, but it happens along nonstereotypical,
unpredicted pathways." Why do NSCs track brain tumor cells? He suggests some
possibilities: Some oncologists view brain-tumor cells as NSCs "gone bad," and these two
similar cell types might respond to the same cues. In addition--or alternatively--tumors or the
brain cells that they're killing might secrete factors that attract stem cells.
Snyder and his colleagues have been holding talks with the Food and Drug Administration
about using NSCs as adjunctive therapy to treat brain tumors, which are now almost always
incurable. He notes that NSCs could be equipped with transgenes that fight cancer by
promoting differentiation or blocking angiogenesis.
--Douglas Steinberg
References
1. J.D. Flax et al., "Engraftable human neural stem cells respond to developmental cues,
replace neurons, and express foreign genes," Nature Biotechnology, 16:1033-9, 1998.
2. K.S. Aboody et al., "Neural stem cells display extensive tropism for pathology in adult
brain: evidence from intracranial gliomas," Proceedings of the National Academy of
Sciences, 97:12846-51, Nov. 7, 2000.
Tissue Engineering
Illustrated below is one approach to tissue engineering. To create a living replacement
tissue, a small number of cells can ideally be harvested from the patient using a biopsy
and then cultured to the appropriate numbers in the laboratory. These cells can then be
grown within a three-dimensional natural or synthetic scaffold and in the presence of
appropriate growth and differentiation factors. If provided with the appropriate conditions
and signals, the cells will secrete various matrix materials to create an actual living tissue
that can be used as a replacement tissue to be implanted back into the defective site in the
patient. The scaffold should ultimately degrade to prevent certain risks that can occur
with the long-term presence of any foreign material in the body. If cells from the patient
are used, then there will be no immune rejection response to the implanted tissue.
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