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Bionic Human: Chapter 11 Assignment
Stem cells and tissue engineering
Differences Between Embryonic and Adult Stem Cells
Embryonic stem cells can become ALL cell types of the body
because they are pluripotent. Adult stem cells were originally
thought to be limited to differentiating into different cell types
of their tissue of origin. However, evidence suggests that adult
stem cell plasticity (see below) does indeed exist, increasing the
number of cell types a given adult stem cell can become.
Large numbers of embryonic stem cells can be grown in culture
relatively easily, while adult stem cells are rare in mature
tissues and methods for expanding their numbers in cell culture
are improving, but have not yet been optimized.
Plasticity: Defined as the ability of stem cells from one adult
tissue to generate the differentiated cell types of another tissue.
Scaffold: The scaffold or three-dimensional (3-D) construct
provides the necessary support for cells to proliferate and
maintain their function. Ideally a scaffold should have the
following characteristics:
• Three- dimensional and highly porous with an
interconnected pore network for cell growth and flow transport
of nutrients and metabolic waste
• Biocompatibile and Bioresorbable with a controllable
degradation and reabsorption rate to match cell/tissue growth in
vitro and/or in vivo.
• Suitable surface chemistry for cell attachment proliferation

and differentiation
• Mechanical properties to match those of the tissues at the
site of implantation
Instructions: Read Chapter 11 and review the slides to answer
the following questions. Additionally, a paper listed on
blackboard can provide for further assistance.
1. Name two sources of stem cells, either embryonic stem cells
or adult stem cells.
2. What is a tissue engineering scaffold?
3. Give three examples of materials that can be used as a
scaffold.
4. Name two types of polynucleotides found in our cells.
The human genetic information is stored in _________
5. What is pluripotency?

6. Name three polymers that are used in medicine.
7. Name two important considerations for an implantable
material.
ISSN:1369 7021 © Elsevier Ltd 2006DECEMBER 2006 |
VOLUME 9 | NUMBER 1226
Scaffolds for
stem cells
Today, most people know at least something about stem cells.
Embryonic
stem cells enjoy regular mentions in news programs and
magazines,
probably because of the controversial way in which they are
generated
but also because of their huge potential in medicine. Here we
distinguish
between and define types of stem cells, discuss techniques used
so far
to create various cells and tissues from stem cells, and discuss
how
three-dimensional supports and stem cells have been and should
be used
to encourage the development of functional replacement tissue.
Nicholas D. Evans1*, Eileen Gentleman1, and Julia M. Polak2

1Department of Materials, Royal School of Mines, Imperial
College London, South Kensington, London SW7 2AZ, UK
2Tissue Engineering and Regenerative Medicine, Department of
Chemical Engineering, Room 144, Roderic Hill Building,
Imperial College, South
Kensington Campus, London SW7 2AZ, UK
*E-mail: [email protected]
Stem cells can be thought of as versatile, unspecialized cells
that
have the potential either to divide to make more stem cells or to
differentiate into one or more cell type, usually in response to
some kind of signal. Ultimately, these cells are used in the hope
of addressing the shortfall in the quantity of tissue available for
transplantation, either alone, as may be the case for replacing
lost
pancreatic beta cells in type 1 diabetes mellitus1, or in
combination
with a scaffold, as may be necessary in the engineering of bone
tissue2. The term ‘stem cell’ refers to a rather confusing
assortment
of different and distinct cell types all sharing this property, but
for
simplicity stem cells are usually divided into adult stem cells

and
embryonic stem cells.
Stem cells
Adult stem cells
Adult stem cells have been used for many years now, with
particular
success in the treatment of cancers of the blood system.
Hematologists
often point out that they pioneered stem cell biology long
before the
more recent explosion in scientific and public interest3.
Throughout the
1950s and 1960s, these scientists demonstrated that
transplantations
of ‘hematopoietic stem cells’ (HSCs), isolated from the bone
marrow,
could generate a new immune system composed of many distinct
specialist cell types in organisms in which the host immune
system had
been destroyed4. This culminated in 1963 when Mathé
demonstrated
the long term survival of a leukemia patient treated with HSCs5.
Bone
marrow transplantation is now a routine medical procedure.

Following these successes, Friedenstein et al.6 noticed another
cell
type in bone marrow explants, initially called the fibroblast
colony-
forming cell because it stuck down on cell culture plastic, that
was
later shown to be a stem cell7,8. They are now referred to as
marrow
stromal cells or mesenchymal stem cells (MSCs) (Fig. 1). These
cells
resemble cells of the connective tissue (fibroblasts) and, in
contrast
to HSCs, can be grown easily in cell culture dishes. By
changing the
composition of the medium in which they are grown, MSCs can
mailto:[email protected]
Scaffolds for stem cells REVIEW
DECEMBER 2006 | VOLUME 9 | NUMBER 12 27
be selectively differentiated into bone cells (osteocytes), fat
cells
(adipocytes), and cartilage cells (chondrocytes)8. This property
has made

them an attractive choice for bone and cartilage tissue
engineering,
especially since they may be used to treat the person from
whom they
were isolated – as an ‘autologous’ transplant. There are also
numerous
examples of evidence in the literature that these cells can
differentiate
into other lineages, including heart cells9 and neurons10. But
they
do have limitations – they can only divide a finite number of
times
(depending on the age of the donor)11, which limits their
supply, and
they may accumulate genetic changes over time12.
Stem cells are also known to be distributed around the body
in various other ‘niches’. For example, neural stem cells can be
isolated from brain tissue, grown in vitro, and induced to
differentiate
into the three cell types of the brain – neurons, astrocytes, and
oligodendrocytes13,14. They also appear to be capable of
turning into

other cell types – after injection into a developing mouse
blastocyst,
they can be found later in the adult organism in several tissues,
including heart, kidney, and liver15. Similar stem cells are also
thought
to reside in other tissues as a repair mechanism against injury,
for
example in the skin16. Again, however, these stem cells cannot
be grown
easily in vitro and are thought to have a limited replicative
capacity.
Embryonic stem cells
Embryonic stem cells (ESCs), on the other hand, are renowned
for their
ability to divide indefinitely and their capacity to differentiate
into most,
if not all, of the tissues of the body. This makes them a
potentially far
more versatile cell type than adult stem cells. In mammals, they
were
first isolated in 1981 from the blastocyst of the mouse, a ball of
cells
formed several days following fertilization. Because of
technical and

ethical hurdles, it was not until 1998 that Thomson and
colleagues17
were able to do the same in humans. Human ESCs can now be
routinely
cultured as preserved ‘pluripotent’ cells, which retain their
ability to
divide indefinitely in an undifferentiated state and, when
stimulated
with the right signals, to differentiate into all of the tissues of
the adult
(Fig. 2). This raises the exciting possibility that they may be
able to
provide an unlimited source of cells for tissue replacement.
Following this advance, many groups rushed to make clinically-
important cell types using human ESCs as a starting point. It
was
known that human ESCs would differentiate spontaneously into
many
cell types, both in vitro in free-floating structures analagous to
the
early embryo called ‘embryoid bodies’ and following
implantation
into experimental animals17, but methods were needed to turn
them

selectively into a cell type of interest. The simplest and most
common
strategies involved simply growing ESCs in a medium designed
for the
required cell type. For example, Bielby et al.18 grew human
ESCs in a
medium containing β-glycerophosphate, vitamin C, and
dexamethasone,
which is used routinely for the growth of osteoblasts in cell
culture
experiments, and demonstrated the formation of bone nodules
and
cells that expressed bone-specific genes. Other researchers have
used
similar methods, usually by including a variety of growth
factors in the
medium, to make pancreatic β cells19, neurons20,
cardiomyocytes21,
lung cells22,23, and even eggs24 and sperm25. Another simple
strategy
involves culturing human ESCs in the presence of the target cell
or a cell type postulated to have a role in differentiation. In this
way, Vats et al.26, Mummery et al.27 and Van Vranken et al.28
have

shown that human ESCs can be differentiated into chondrocytes,
cardiomyocytes, and pneumocytes respectively. Genetic
manipulation
is also a useful technique for directing the differentiation of ES
cells.
Kim and colleagues29 introduced the gene for nurr1 into ESCs,
and
demonstrated differentiation into dopamine-producing neurons
and
an improvement in the condition of Parkinson’s disease-
afflicted rats
following their implantation. Tai et al.30 have shown that
increased
numbers of osteoblasts could be produced by ESCs over-
expressing
osterix. Extracellular matrix – the proteinaceous amalgam in
which cells
grow – is also thought to be an important factor in the
differentiation of
ESCs. For example, Coraux et al.31 showed that the culture of
ESCs on a
matrix derived from skin fibroblasts could generate a tissue that
looked
remarkably like real skin.

The literature on the directed differentiation of ESCs into
various
cell types is now vast, with over 1600 papers published since
1998,
most of which use variations or combinations of the methods
given
Fig. 1 Diagram demonstrating how adult stem cells can be used
in tissue
engineering. Marrow is removed from the adult bone and placed
in a culture
dish. Adherent mesenchymal stem cells can then be expanded or
directed to
differentiate into bone, cartilage, or fat cells.
REVIEW Scaffolds for stem cells
DECEMBER 2006 | VOLUME 9 | NUMBER 1228
above. Despite this, however, the induction of differentiation to
a
specific cell type remains a largely hit-and-miss affair, and
ESCs appear
to have a malicious tendency to differentiate into a host of other
cell types in addition to the cell type of interest. So most

reviewers
suggest that it is probably necessary to select the cell type of
interest
for most applications, either by sorting the cells using
fluorescent
antibodies or genetic markers, or by engineering a lethal marker
into
the cells that can be switched on should they not turn into the
right
kind of cell32-34. Perhaps for this reason, ESC research to date
has lain
firmly in the domain of cell biologists, who perform
differentiation
assays on cells grown on two-dimensional surfaces in cell
culture
dishes and flasks. Cellular differentiation and tissue
development is,
however, an inherently three-dimensional process and so to
investigate
differentiation and tissue formation in vitro fully, it may be
necessary
to turn to the field of tissue engineering, where three-
dimensional cell
culture systems have been used for many years.

Tissue engineering
Tissue engineers often focus their efforts on providing a three-
dimensional environment, or scaffold, for cell attachment and
growth,
and hope that, by mimicking the in vivo environment, cells can
be
coaxed into creating a desired tissue type. The ultimate aim of
tissue
engineering is to make a three-dimensional cell-containing
scaffold
that can be implanted in the body to cure a disease or repair a
defect
(Fig. 3). The standard in vivo culture system – where cells are
grown in
a monolayer on a charged, flat, plastic surface – cannot
replicate
the complexity of the cells’ natural environment and rarely
supports
the assembly of cells into a functioning tissue. Providing an
appropriate scaffold that will lead to the development of a
functional
tissue is certainly not a simple matter, however, and tissue
engineers

have approached the problem in many ways, using a variety of
materials.
Conventional scaffolds
Biomedical implants have been used since ancient times – for
example,
a Brazilian group recently reported that the ancient Incas
successfully
used Au plates to repair cranial defects35. Until the last few
decades of
the 20th century, the criteria used in choosing materials for
implants
has fundamentally changed very little and usually implant
materials
were chosen that were functional because of their inertness.
Since the
discovery in the 1960s that some glass ceramics actively bond
to living
bone, however, the focus has shifted away from inert materials
and
toward materials that are bioactive – those that deliberately
elicit a
specified response from the body. Currently, most scaffolds
provide a
three-dimensional environment in which tissue can grow and

develop,
so that it is able to reproduce the functions of the tissue it is
intended
to replace. Some scaffolds may be designed to be implanted
without
any cellular component36 – instead they are designed to
encourage
tissue ingrowth and de novo tissue synthesis in vivo – while
most are
intended to have some kind of cellular component engineered in
vitro
before implantation (an example can be found elsewhere37).
The latter
strategies require that cells have access to nutrients and space to
grow.
Fig. 2 (a) Photomicrograph of undifferentiated human ESCs
(H1) grown on a mouse fibroblast feeder layer. (b) Diagram
explaining the derivation and
differentiation of ESCs. ESCs can be derived from the inner cell
mass of a preimplantation blastocyst and expanded in culture on
fibroblast feeder layers. Colonies of
ESCs can be directed to differentiate into cell types from the
three germ layers.
(b)(a)

Fig. 3 Diagram outlining the potential for tissue engineering.
Stem cells can be derived from embryos or adult tissues and
expanded in culture. They can then be
either seeded directly on a scaffold or differentiated in culture
and sorted to obtain a purified population of a target cell type
before seeding on the scaffold. Cell-
seeded scaffolds can then be grown in culture to develop a
desired tissue prior to implanting in the body.
For this reason most scaffolds, regardless from which material
they are
made, are constructed with some kind of porous network and
cultured
with cells in a manner that encourages nutrient transport (Fig.
4). For
instance, inorganic materials such as bioactive glasses and
calcium
phosphates have been used extensively for bone tissue
engineering
because of the similarities to and their ability to bond with
bone’s
natural mineral backbone. Bioactive glasses can be sintered in

powder
form to create porous networks38 or, when in solution, can
simply
be ‘foamed’ using soap and then gelled to make sol-gels39.
Similarly,
porosity can be engineered into polymers, such as polyesters
(which
Fig. 4 Schematic illustration demonstrating four methods for
achieving nutrient transport in an engineered tissue construct.
(a) Construct is placed in static culture
where it is reliant on simple diffusion for delivery of nutrients.
(b) Construct is placed in either an environment where the
media is mixed, such as a spinner flask,
or in a rotating bioreactor where it achieves a zero-gravity state
as fluid is moved around it. (c) Construct is placed in a system
that forces fluid and nutrients to
be continuously perfused directly through it. (d) Small channels
are engineered into the construct allowing nutrients to be
delivered in a similar manner to blood
vessels in the body.
(a) (b) (c) (d)

have the advantage of being biodegradable), either by foaming
the
polymer solution40 or by molding the dissolved polymer around
lumps
of another material such as salt, allowing the polymer to harden
and
then leaching out the salt with water41. Porous networks can
also be
engineered into natural molecules – for example, collagen gels
can be
freeze-dried before cell seeding42.
Alternatively, hydrogels can be used as scaffolds for cell
growth and
cell delivery. Since the gelling process is often nontoxic, cells
can be
introduced into the solution prior to gelation. In the case of
alginate,
a natural polymer made up of chains of guluronic and
manuronic acid,
calcium is usually added to cell/gel solutions, which crosslinks
these
chains and hardens the gel43. Likewise, collagen gels can be
hardened

by altering the pH of the solution44,45 (Fig. 5) and
poly(ethene) glycol
can be solidified using light46. Hydrogels have different
mechanical
properties from other scaffolds, so the material must be selected
on
the basis of its properties, keeping in mind the intended
application.
But all of these scaffolds have their disadvantages. Inorganic
scaffolds such as ceramics and glasses tend to be too brittle and
weak
to be used in load-bearing applications, and even bioactive
glasses,
discovered more than 30 years ago, are limited to non-load-
bearing
applications such as the replacement of small bones in the
middle
ear47. Artificial polymers, on the other hand, may be viewed by
the
body as foreign material because they lack sticky surface
molecules
for cell adhesion. Their degradation products are, in the case of
polyesters, acidic, and though not directly toxic, may create a
possibly

unphysiological acidic microenvironment. This is particularly
important
and often overlooked in bone tissue engineering – the natural
mechanism by which bone is degraded in vivo by osteoclasts
involves
the formation of an acidic microenvironment! Collagen may be
a
better bet in this case, as the natural mechanism of bone
formation
involves the mineralization of a collagen scaffold created by
osteoblasts
– unsurprisingly collagen scaffolds are readily mineralized in
tissue-
engineering experiments48. Another problem with porous
scaffolds is
that because cells are seeded onto the internal porous matrix of
the
scaffold it becomes arguable whether the cells experience a
truly three-
dimensional environment – they merely ‘see’ a slightly curved
two-
dimensional surface. This can be solved to some extent by
decreasing

the pore size and adding surface texture, or embedding cells in a
soft
extracellular matrix, but then problems arise as to how to keep
deeply
embedded cells supplied with nutrients. Most importantly in the
case of
stem cells, there are challenges involved in providing the
correct signals
to encourage differentiation and to pattern cells as they
differentiate
into an organized tissue.
Micro- and nanopatterned scaffolds
To solve these problems, various groups have begun to
investigate
scaffold patterning – both at microscale resolution, possibly
including
channels so that cells can be supplied with nutrients and areas
where
different cell-types can be deposited, and at nanoscale
resolution,
where cells are supplied with the correct ligands to enhance and
direct
their function and to induce differentiation.
To address the first challenge, a number of new technologies

are being investigated. Photopolymerizable poly(ethene) glycol
hydrogels have been used as a material for encapsulating cells
in
three dimensions – these gels can be mixed with cells as a
liquid and
light can be used to solidify them46. Recently, several groups
have
used this property to add an element of patterning into
hydrogels49-51.
In these cases, a layer of cell-containing gel is selectively
exposed
to light in a pattern dictated by a light-blocking mask.
Following
gelation of this layer, another layer of cells, possibly containing
another cell type, can be poured over this solid mold and gelled
using another pattern. To illustrate this technique, Liu and
Bhatia50
encapsulated cells in recognizable shapes and layered different
cell
types in geometric patterns (Fig. 6). Another interesting
technique
involves the use of photolithography, a technique used
extensively

to make electronic components. This simply involves exposing
the
(b)(a)
Fig. 6 Illustration of patterned cell-containing hydrogels. (a)
HepG2 cells,
a liver cancer cell line, encapsulated in various three-
dimensional shapes
in photopolymer ized PEG hydrogels. (b) Cells labeled with red
or green
dyes, partitioned in distinct areas within a three-dimensional
hydrogel layer.
(Reprinted with permission from50. © 2002 Springer.)
Fig. 5 Epifluorescence micrograph of a short collagen
fiber/collagen hydrogel
composite seeded with rat skin fibroblasts prior to hardening of
the gel. Live
cells appear green and have long projections into the gel matrix,
while dead
cells are small and round and stain red. The collagen fibers
autofluoresce
orange.

surface of a ‘photoresist’ mounted on a Si wafer to ultraviolet
light.
The exposed areas can be hardened – again in a pattern
determined
by a mask – and the patterned surface can be used to mold a soft
substrate. This technique has been used extensively for
microfluidic
applications, where poly(dimethyl siloxane) (PDMS) is indented
and
adhered to a glass slide to make a series of channels (the
technique has
been reviewed elsewhere52). But more recently, tissue
engineers have
started to realize that this could be an excellent way of
introducing
something akin to a vascular system in a biomaterial. For
example,
Shin et al.53 simulated a vascular network in a PDMS
microfluidic
device and showed the growth of endothelial cells – blood
vessel cells

– on the walls of the ‘vascular tree’ (Fig. 7), while Tan and
Desai54
made a fibroblast-containing scaffold based on collagen. The
ability to
develop three-dimensional, perfusable scaffolds has great
potential in
both tissue engineering and bioreactor technology as it could
provide
a way to keep deeply-embedded cells supplied with nutrients
within a
scaffold.
Another possible way of patterning scaffolds more intricately
might
involve rapid prototyping (RP). Put simply, RP encompasses a
range
of different techniques, all of which have the property of
producing a
physical object based on a computer design55. Most of these
devices
are analogous to printers and can print scaffolds using a variety
of
materials. Arguably, the most interesting, however, are devices
that
appear to be able to print cells and matrices in

combination56,57, which
could allow precise control over tissue microstructure in the
future.
Developing technologies such as these allow us to control the
immediate surroundings of cells in a three-dimensional
environment
more precisely, and may provide a more authentic environment
in
which to direct the differentiation of cells to form a coherent
tissue.
But these approaches do not directly consider the chemical
interactions
that go on between a cell and its substrate (Fig. 8). To address
this
‘nanoscale’ problem, tissue engineers are beginning to fabricate
bioactive scaffolds, where the surface of the scaffold is
engineered to
stimulate cell function.
One such method involves using self-assembling amphiphilic
peptides. These molecules are engineered with hydrophilic
heads and
hydrophobic tails that, under the correct conditions, can self-
assemble

into a network of ‘nanofibers’ with the heads sticking out into
solution
and the tails hidden in the core of the thread (Fig. 9)58.
Networks
F ig. 7 Cells growing in microfabricated networks in PDMS.
Channels are
constructed by selectively exposing a photoresist to light to
create an intricate,
raised relief pattern. This relief pattern is then used to indent
PDMS, which
is then adhered to a glass slide. Cells and medium can be
perfused through
the channels and cells adhere to the walls. The cells here are
HMEC-1, an
immortalized endothelial cell line. (Reprinted with permission
from53. © 2004
Springer.)
Fig. 8 Diagram demonstrating the relationship between cells
and scaffolds. The microenvironment created around a cell
adherent to a tissue-engineering scaffold
is complicated. Nutrient transport brings growth factors,
ligands, and other signals that can bind to cell receptors. The
degrading scaffold can likewise release
chemical messengers that bind to membrane receptors and

influence intracellular communication and cellular processes
such as gene transcription. Cells also
attach to the scaffold via integrin receptors. Integrin receptors
are closely connected to the cell’s cytoskeleton and relay
further information to the cell thereby
affecting cell function.
such as these have the advantage of porosity and living cells can
be
combined with such materials before the scaffold is fabricated.
More
importantly, peptides common to the extracellular matrix of the
tissue
under investigation can be engineered into the hydrophilic
heads of
such molecules, which can improve cell attachment and promote
differentiation and function. Since the meshwork is so fine, the
internal
surface area of the scaffold is very large and cells are exposed
to a
high density of these ligands from all directions, which may

encourage
three-dimensional growth. Another fabrication method currently
under
investigation for making very fine scaffold networks is
electrospinning,
in which tiny threads of material can be fabricated to provide a
porous
mesh. This is accomplished by extruding a material from a fine
nozzle
using electrostatic force to form fibers between 3 nm and 5 µm
in
diameter. Again, extracellular matrix ligands can be attached to
such
fibers, or the fibers can be made out of biologically derived
materials
such as collagen59.
Such micro- and nanopatterned scaffolds, therefore, introduce
a greater level of control over the fine structure of a scaffold
than
conventional scaffolds and may allow control over cell
patterning and
cell differentiation. This level of control may be invaluable if a
scaffold

is to be used in combination with stem cells.
Scaffolds and stem cells
Stem cells, of course, are reliant on the extracellular
environment
not only to survive but also to develop into a functional tissue.
So
increasingly, tissue engineers are beginning to use the
composition of
scaffolds to persuade stem cells to differentiate. Arinzeh et
al.60 have
shown that adjusting the ratio of hydroxyapatite to tri-calcium
phosphate could influence the degree to which osteogenic
differentiation of MSCs occurs, while others have begun to
engineer
bioactive factors into porous scaffolds. For instance, Kim et
al.61 have
created a polyester scaffold that slowly exudes vitamin C and
β-glycerophosphate and demonstrated an increase in
osteogenesis
from MSCs, while Yang et al.62 have demonstrated increased
osteogenic differentiation in a polylactic acid (PLA) scaffold
spiked
with a bone-specific growth factor. Alternatively, mechanical
force can

be used to stimulate differentiation – Altman et al.63 have
recently
shown that applying a mechanical force to a collagen-gel
scaffold can
encourage MSCs to differentiate into ligament tissue.
Micro- and nanopatterned scaffolds have been investigated less
well in regard to stem cells, although two recent studies
highlight
their attractiveness. Silva and colleagues64 included a five
amino
acid, laminin-specific cell-binding domain (which binds to
specific
integrins on cell surfaces) at the hydrophilic head of their
amphiphiles,
and showed that neural stem cells could be induced to
differentiate
into neurons when cultured within the network. In contrast,
cells
grown in control scaffolds without the laminin-specific domain
or on
two-dimensional tissue culture plastic coated with laminin
solution
differentiated much less. This was hypothesized to be largely as

a
result of the density of the cell-binding ligands to which the
cells
were exposed, indicating clearly the importance of extracellular
matrix
in influencing cell function. In a similar study, Hosseinkhani et
al.65
replaced the laminin-specific domain in the amphiphilic
molecule
with the amino acid sequence, arginine-glycine-aspartate
(RGD), a
common cell-binding domain in many extracellular matrix
proteins,
especially collagen. They then showed that the differentiation of
MSCs
to osteoblasts is significantly enhanced compared with
amphililic
nanofibers without this sequence on to two-dimensional
controls.
So far, remarkably few studies have been published on the
effect of
three-dimensional environments and scaffolds on ESC
differentiation.
In two rare examples, Levenberg and colleagues66,67 have

shown that
human ESCs embedded in an extracellular matrix gel called
Matrigel
can be differentiated in three dimensions on conventional
polyester
scaffolds. In these cases, several structures that resemble
primitive
tissues were generated, depending on the content of the growth
medium. The authors also show that tissues grown in three
dimensions
express higher levels of differentiation-associated proteins than
those
on coated two-dimensional surfaces. Interestingly, another
group has
Fig. 9 Illustration of how amphiphilic peptides self-assemble to
produce
nanofibers. (a) The chemical structure of each peptide,
including a long
hydrophobic tail (1) and the three amino acid RGD motif at the
head (5). (b)
and (c) show how these peptides assemble to form a fiber.
(Reprinted with
permission from58. © 2001 AAAS.)

(b)
(a)
(c)
recently reported that the chondrogenic differentiation of human
ESCs
in a PEG hydrogel is dependent upon whether or not the
hydrogel
contains adhesive RGD sites, illustrating the importance of the
cell
matrix and microenvironment in ESC differentiation68.
The use of more novel, patterned scaffolds should provide ESC
biologists with an important new tool to stimulate and model
differentiation in vitro. For instance, nanopatterned scaffolds,
such as
those using amphiphilic peptides, could be used to partition
cells within
a mixed population of ESC-derived cells, based on the
specificity of the

ligands to which different cells bind. In this way, scaffolds
could play a
role in directing tissue organization, not only with the aim of
producing
tissue for transplant but also for studying differentiation in
vitro.
Similarly, ESCs could be compartmentalized within scaffolds to
study
cell-cell interactions and their effect on cell differentiation and
tissue
formation. Such scaffolds will undoubtedly find exciting
applications in
the study of ESC differentiation in the future.
Conclusions
With stem cells, we are presented with a versatile material with
which
we may be able to rebuild many structures found in the body.
But the
challenge of how to construct three-dimensional tissues from
them
still remains. Recently, biologists and materials scientists have
realized
that scaffolds can and should be designed that direct and
enhance cell

function and differentiation. We have only scratched the surface
of
how these scaffolds can be used in concert with stem cells,
however,
and this provides us with great encouragement that they may
provide
us with the power to construct three-dimensional functional and
intricate replicas of human tissue in the near future.
Acknowledgments
NDE acknowledges the support of Medical Research Council,
UK for funding.
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3. Jansen, J., et al., J. Cell Mol. Med. (2005) 9, 37
4. Ford, C. E., et al., Nature (1956) 177, 452
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Scaffolds for stem cellsStem cellsAdult stem cellsEmbryonic
stem cellsTissue engineeringConventional scaffoldsMicro- and
nanopatterned scaffoldsScaffolds and stem
cellsConclusionsAcknowledgmentsREFERENCES
Stem Cells and
Regenerative Medicine
Chapter 11
Learning Objectives

types of Stem Cells
e used in
regenerative medicine and tissue engineering
Through understanding how
these animals regenerate
limbs we can develop an
understanding of why we are
not able to do so, and
potentially learn to overcome
those “roadblocks” to
regeneration.

Salamander -
Epimorphosis
The Salamander’s Trick
The “blastema cells” (shown in light blue) are essentially stem
cells that arose from the normal limb cells “de-differentiating”
or
returning to a primitive embryonic-like state.
The salamander’s trick is that it can make it’s cells “go back in
time”, something that human cells are not believed to be
capable
of doing.
The basic concept of a stem cell
1. A stem cell can make copies of itself.
2. A stem cell can differentiate, i.e. become a
specialized type of cell. Differentiation is a
series of steps from initial commitment to
becoming a functional, specialized cell.

http://www.stemcellsforhope.com/Stem%20Cell%20Therapy.ht
m
Characteristics of Stem Cells
Some Definitions
• Somatic Cell: a mature, differentiated cell, i.e. a
skin cell.
• Differentiated (cell): committed to being a
specialized cell.
• Undifferentiated (cell): retains the potential to
become multiple specialized cell types.
• Stem Cell: primitive, undifferentiated cells
– Self-renewing: can give rise to copies of itself.
– Multipotent: can give rise to multiple (but not all) cell
types.
– Pluripotent: can give rise to all cell types of the body.

Some Definitions
• Regeneration: re-growth of lost or damaged
cells, tissues or organs.
• Regenerative Medicine: process of
replacing or regenerating human cells,
tissues or organs to restore or establish
normal function through biomedical
interventions that may involve the use of
stem cells.
Cell Identity
• All cells in the body have an identical genotype
(genetic makeup), i.e. the same set of genes.
21,000 protein-coding genes.
• Why do cells have different phenotypes
(observable characteristics)? What makes a liver
cell a liver cell? A brain cell a brain cell?

• When a gene is “expressed” the result is the
production of the protein it codes for.
– Muscle cells produce myosin which drives
contraction.
– Pancreatic beta cells produce insulin.
Cell Identity
• Genes are turned “on” or “off”, and depending
on the combination a cell will take on certain
functional characteristics.
• Differential gene expression determines cell
identity (or phenotype) and is controlled by
signals in the cell’s environment.
This is the point of intervention for the
biomedical scientist. We manipulate the
environment to coax a stem cell into thinking it
needs to become the desired type of cell.

1 genotype, 2 phenotypes
The caterpillar and the butterfly are the same
organism. There is only one set of genes, or genotype,
yet there are 2 wildly different forms, or phenotypes,
which can emerge.
Important for understanding diversity of life and also
for understanding how to control the behavior of cells.
Stem Cell Repair Damaged
Heart
• http://www.cbsnews.com/videos/heart-
patient-sees-results-in-stem-cell-study
Classes of Stem Cells
• Embryonic Stem Cells - the infamously debated
cells. Embryonic stem cells are pluripotent,

which means they can give rise to ALL cell types
of the body.
– They are derived from the blastocyst of a
developing embryo.
Classes of Adult Stem Cells
– Hematopoietic Stem Cells: derived from
bone marrow, can give rise to all of the
blood cells and immune system cells.
– Mesenchymal Stem Cells: derived
primarily from bone marrow, but also from
fat tissue (peripheral vs visceral), can give
rise to multiple cell types – but not all (see
next slides).
– The most abundant reservoir of stem cells
in an adult human is the bone marrow.

Classes of Adult Stem Cells
– Stem cells have been found in most adult
organs (brain, heart, lung) however they are
much lower in number & therefore difficult
to harvest and potentially use for therapy.
These cells can give rise to the tissue from
the organ in which they are found.
• Cord Blood Stem Cells – essentially blood
stem cells, with some evidence of potential
for other tissues.
Hematopoietic (Adult) Stem Cells
http://www.allthingsstemcell.com/category/hematopoietic-stem-
cells/
Used to treat blood and immune system disorders, as well as to
replenish the bone marrow following chemotherapy in cancer
patients.

Cord Blood Stem Cells
• Cord blood stem cells are believed to be largely hematopoietic
in
nature, although they have also been shown the possibility of
forming other types of cells such as nerve or liver cells.
• Big advantage is that each person could have a “bank” of their
own stem cells to use later in life when diseases arise.
Cord blood stem cells turning
into nerve cells.
Cord blood at the time of
harvest from an infant.
Mesenchymal (Adult) Stem Cells
The idea of (adult) stem cell therapy
for bone and cartilage repair
• Autologous cells – the
patient’s own.

• Bone marrow-derived
mesenchymal stem cells
are harvested from the
marrow of the pelvic bone.
• Cells are differentiated into bone or
cartilage cells “in vitro”, i.e. outside
of the body in the laboratory.
• New bone or cartilage cells are
used for therapy. http://www.miamiherald.com/living/health-
fitness/article68194662.html
Embryonic Stem Cells
• Obtained from embryo 4-5 days after
fertilization.
• These stem cells are “pluripotent”, able to
differentiate into the 220 cell types of the
body.
• Embryonic stem cells can also propagate

themselves indefinitely.
Potential of Embryonic Stem Cells
http://www.stemcellsforhope.com/images/StemPic1-small.JPG
Safety Issues with
Embryonic Stem Cells
• Teratoma: Malignant teratoma is a type of cancer made of
cysts
that contain one or more of the three layers of cells found in a
developing fetus at the gastrula stage. These layers are called
ectoderm, mesoderm, and endoderm.
https://www.youtube.com/w
atch?v=kTHoTMr_0U8
Safety Issues with Embryonic
Stem Cells
• If un-differentiated embryonic stem cells
are injected into an animal they form

teratomas.
– Therefore they must be differentiated into the
desired cell type prior to therapeutic use.
– This implies that precise purification is
needed to remove any lingering
undifferentiated cells. Even one cell could
lead to the formation of a teratoma.
Alternative sources of
pluripotent stem cells
• There are 2 additional ways to produce
pluripotent cells that do not involve
fertilization of an egg.
1. Therapeutic cloning (has never happened
in humans). Requires an egg cell and the
nucleus of a normal adult cell.
2. Induced pluripotency (has been done in
humans) – involves genetic manipulation of

an adult somatic cell (a cell forming the body
of an organism).
Induced pluripotent stem cells
• Very new technology
• Utilizes a gene therapy approach
to force a normal, somatic cell (a
differentiated cell of the body) to
express 4 particular genes (only
4!!!) that “reprogram” the somatic
cell into an induced pluripotent
stem cell.
"Shinya yamanaka10" by National Institutes of
Health -
http://nihrecord.od.nih.gov/newsletters/2010/02_19_
2010/story1.htm. Licensed under Public Domain via
Commons -
https://commons.wikimedia.org/wiki/File:Shinya_ya

manaka10.jpg#/media/File:Shinya_yamanaka10.jpg
Shinya Yamanaka (山中伸弥)
The 2012 Nobel Prize in
Physiology "for the discovery
that mature cells can be
reprogrammed to become
pluripotent”.
Stimulus-triggered acquisition of
pluripotency (STAP)
• STAP - an alleged
method of generating
pluripotent stem cells
Haruko Obokata (小保方晴子)
She and her colleagues had demonstrated a
surprisingly simple way of turning ordinary
body cells – she used mouse blood cells –
into something very much like embryonic
stem cells. All you need to do is drop them

into a weak bath of citric acid, let them soak
for half an hour.
Nobody can reproduce her result,
including herself
Obokata probably had falsified
and fabricated data
STAP cells were actually
embryonic stem cells, and the
mixup was probably not
accidental
On August 5, 2014, Obokata's
mentor and co-author Yoshiki
Sasai committed suicide
Obokata, Haruko;
Wakayama, Teruhiko;
Sasai, Yoshiki; et al.
(2014). "Stimulus-

triggered fate conversion
of somatic cells into
pluripotency". Nature 505
(7485): 641–647.
https://en.wikipedia.org/wiki/Nature_(journal)
Everyday Stem Cells:
Regeneration of Intestinal Lining
The mechanism of intestinal
regeneration (new surface
every 2-3 days) involves
stem cells which reside in a
“niche”. They are constantly
dividing to make more stem
cells and also enough cells
to reform the intestinal lining.
Liver Regeneration
• A (healthy) liver will completely regenerate following

surgical removal of up to 2/3 of the liver mass (liver
cells only live about 150 days).
• This regenerative process is driven by proliferation,
i.e. multiplication, of pre-existing liver cells, and does
not involve activity of stem cells as in the intestine.
• The problem: With aging and disease (i.e. cirrhosis)
the liver loses it’s ability to regenerate.
Diseases involving cellular death in organs
that do not easily regenerate
• Alzheimer’s, Parkinson’s disease - brain cells
• Retinitis pigmentosa - retinal cells
• Diabetes - pancreatic cells
• Muscular dystrophy – muscle cells
• Heart failure and other cardiovascular
diseases - heart muscle/vascular cells
At the current time the only foreseeable solution to these
problems is replacement of the dead cells with healthy

functional cells derived from stem cells. In many cases
only embryonic stem cells have currently been shown to
form the needed cell type, for example retina and certain
types of brain cells.
Macular Degeneration
• A chronic eye disease causing
vision loss in the center of the
field of vision. Dry macular
degeneration caused by
deterioration of macula in the
center of the retina. No
treatments available.
• Wet macular degeneration is
caused by blood vessels growing
under retina leaking blood and
fluid. May be treated with
medication or lasers to destroy

blood vessels.
Clinical trials so far in the U.S. involving the use
of human embryonic stem cells
From the horse’s mouth:
https://clinicaltrials.gov/ct2/results?ter
m=human+embryonic+stem+cells&recr
=Open
The first safe, and reportedly effective use of a
human embryonic stem cell-based therapy was for
the treatment of macular degeneration.
https://www.youtube.com/watch?v=ewEdR5_V-v0
The Ethical Debate
• At what moment does a life begin?
– A deeply personal opinion
• Left over fertilized eggs from in vitro
fertilization will be discarded.

– Is this a waste?
• One must consider these issues before
deciding whether they support human
embryonic stem cell research.
Therapeutic Cloning: Dolly the Sheep
http://nyti.ms/16Xxmq8
Tissue Engineering
and Regenerative Medicine
Why Tissue Engineering?
• Examine the challenges of organ transplantation -
(The Clinical Problem!!!)
• Surgical methods are advanced and highly
successful when suitable donor organs are

available, but…
– Limited donor availability – genetic matching;
size matching. Should be a healthy organ.
– Immune rejection (not a long-term solution)
Why Tissue Engineering?
• The idea is to utilize the patients own cells outside
of the body in combination with scaffolds and
bioreactors to create tissues that can be implanted
without the problem of immune rejection.
Why outside the body?
• Remember our cells have the capacity to grow and
regenerate but there are roadblocks to regeneration
in the body, i.e. inflammation. Outside of the body
we have control over the environment and therefore
cell behavior.

Tissue Transplantation
• Autografts – from same organism (no immune rejection)
– Middle 1/3rd of patellar tendon graft for ACL reconstruction
– Hip bone fragments for bridges in spinal fusion
– Coronary vessel bypass – utilize healthy vessels from the
lower extremities
– Stem cells from the bone marrow!!
• Allografts – from same species (one human to another)
– Traditional organ transplants
• Xenografts – tissue from another species (animal to human)
– Porcine (pig) heart valves
– Bovine bone for packing defects
– Encapsulated porcine pancreatic islets
Porcine heart valve
From Veterinary Transplant Services
Tissue Engineered Heart Valves
Autologous Cells + Scaffolds + Bioreactors
Pig heart valves (Gold
Standard):
1. Prepared with

glutaraldehyde, a toxic
fixing agent that gives
the tissue a leathery
strength.
2. They calcify over time.
Biodegradable
scaffold
(PGA polymer)
Seeding of
autologous
cells,
culture in
vitro
Bioreactor
allows for flow
conditions
that simulate

natural heart
valve fluid
forces.
Tissue Engineered Heart Valves
(still in the development stages)
Porcine heart valves
(xenografts) calcify over
time, as part of the
immune-inflammatory
response, giving them a
limited life, typically 5-10
years.
Tissue-engineered heart valves
(autografts) tested in animals do
not show long term signs of
calcification.
https://www.youtube.com/watch
?v=7p8VrGr-Drk

Shape?
Example of an Engineered Xenograft:
Alginate-encapsulated Pig Islets
for treatment of Diabetes
• Alginate is a natural polymer derived from seaweed.
• Tunable pore size: big enough for movement of nutrients
and insulin, but small enough to prevent penetration of
antibodies or immune cells.
http://www.biotechlearn.org.nz/focus_stories/pig_cell_transplan
ts/images/encapsulated_pig_islet_diagram
http://www.voxy.co.nz/national/lct-begins-trials-
implant-pig-tissue-auckland-diabetics/5/18932
Tissue Engineering
Tissue engineering is the
use of a combination of
cells, engineering and
materials methods, and

suitable biochemical and
physical factors to
improve or replace
biological functions.
The Tissue Engineering Paradigm
Implantation
& Integration
3-dimensional
culture
(Scaffolds)
Mechanical
stimulation
Chemical
stimulation
Autologous
Tissue Cells
Patients own

healthy tissue
Stem
Cells
Cell
Proliferation
Differentiation
Extracellular
Matrix Production
Tissue
Remodeling
Homeostasis
Homeostasis
• Homeostasis (homeo = same, stasis = stable) - tendency of
tissues
& organs within the body to remain functionally stable
through coordinated interactions with other parts of body.
• In the context of tissue engineering, the way tissues
participate in homeostasis is through the blood, i.e.
vascularization is a central challenge.

Classic example: blood glucose regulation “Engineer’s View”
of Homeostasis
Homeostasis
https://www.youtube.com/watch?v=G-nffUdhwjE
The Trinity (+1) of Tissue Engineering
1. Cells classes of stem cells
(see previous lecture), or somatic cells, i.e.
epidermal and dermal cells from the patient
for skin grafts.
Remember that certain (healthy) tissues such as skin,
liver, and intestine have the capacity to proliferate.
2. Scaffolds
3. Bioactive molecules
--------------------
4. Bioreactors

The idea of a “Scaffold”
• Tissues are composed of cells (bricks) and
extracellular matrix (mortar). Extracellular
matrix provides support and structure, but
should be thought of as a continuum of
fibers within a fluid gel.
• The term "scaffold” in tissue engineering
and regenerative medicine represents an
engineered equivalent of the natural
extracellular matrix.
Scaffold for tissue engineered
heart valve
Hybrid valve at various
stages of development.
Nitinol structure along with
(A) smooth muscle,
(B) fibroblast/myofibroblast,

(C) endothelial cells
all encapsulated in collagen as the
first, second, and third layers,
respectively.
Polymeric
https://www.researchgate.net/publication/5894568_Prosthetic_h
eart_valves_Catering_for_the_few/figures?lo=1
http://kheradvar.eng.uci.edu/research.php?cat=hybridvalve
The idea of a “Scaffold”
• Scaffolds have several essential
functions:
– Provides a three-dimensional space for
new tissue development.
– Delivers cells and maintains their
localization at the site of implantation.
– Directs macroscopic size/shape of new
tissue, i.e. if a vascular graft is needed the
scaffold should be a tubular structure with

the appropriate diameter to match.
The Extracellular Matrix
(Nature’s Scaffold)
1. Structural support -
acts as a malleable
“mortar” or “glue” that
holds cells together in
tissue.
2. Composition – the most
common components
are collagens (> 20
different types of
collagen proteins).
3. Information - each
tissue’s extracellular
matrix is unique.

Another control element
for the scientist.
Ideal scaffold designs should incorporate all of these
aspects, taking care to match the design parameters as
closely to the target tissue as possible.
Some tissues are primarily
extracellular matrix
• Connective Tissues such
as tendons and ligaments
are primarily composed of
collagen type I fibers.
• Cartilage is mostly
extracellular matrix,
composed of collagen
type II and
glycosaminoglycans
(sugar polymers) with a

very high water content.
The Tissue Engineering Paradigm:
Utilize scaffolding materials to arrange cells
in a 3-dimensional structure
http://www.nature.com/nature/journal/v414/n6859/fig_tab/4141
18a0_F1.html
Cells isolated from patient
and maintained in culture in
vitro to expand population.
Cells seeded in vitro into a
3-D “scaffold” and treated
with various chemical
stimulants to promote
tissue development.
“Tissue construct” is
implanted once cellular

development has been
established.
The scaffold eventually
degrades, being replaced
by remodeled tissue.
Engineering the Scaffolds
Natural or synthetic biomaterials.
Synthetic polymers:
e.g. poly(glycolic acid) [PGA], poly(lactic acid) [PLLA]
Natural materials:
usually composed of ECM components (e.g., collagen,
elastin, fibrin, or de-cellularized tissues, such as a heart
valve free of cells)
--------------------------------------------------------------------------
-------

Engineering the Scaffolds
The scaffold material should be biocompatible and
satisfy these basic requirements:
1. Favorable cell attachment.
2. Mechanical properties to match the target tissue.
3. Biodegradable - we want the cells to remodel the tissue
and produce their own extracellular matrix.
4. No adverse reactions or toxicity to degradation
products.
--------------------------------------------------------------------------
-----
• Interconnecting pores are a key design feature of
scaffolds to allow for movement of nutrients and cells
throughout the structure.

Delivery of oxygen and nutrients
(and removal of waste products)
http://www.sciencedirect.com/science/article/pii/S13509462110
00279
Capillaries are 25-100 micrometers in all tissues. A cell is
~ 10-20 microns in diameter on average, some much larger.
When attempting to construct 3D tissues outside of body,
scaffold must account for this transport of nutrients and
waste by having an open & interconnected pore structure.
• Angiogenesis – the growth of new blood vessels
from pre-existing blood vessels.
• Once an engineered tissue is implanted in the body,
angiogenesis must occur in order for the tissue to
remain viable and participate in homeostasis.
Cancer has evolved mechanisms to promote
angiogenesis – this is a required step for the
tumor to grow and eventually spread.
Tissue Engineers attempt to

grow blood vessels within
tissue in vitro, so they can
“connect’ upon implantation.
https://youtu.be/M4jMLXE-CBc
https://www.youtube.com/watch?v=O5r-T6ANKto
Use preexisting blood vessels
https://www
.sciencedail
y.com/relea
ses/2008/0
1/08011314
2205.htm
http://www.nyt
imes.com/201
2/09/16/healt
h/research/sci
entists-make-

progress-in-
tailor-made-
organs.html
for skin grafts.
2. Scaffolds
--------------------
4. Bioreactors
Growth Factors (Bioactive Molecules)

typically used in regenerative strategies
http://rsif.royalsocietypublishing.org/content/8/55/153.full.pdf+
html
Signals in the cell’s environment:
Extracellular Matrix and Growth Factors
html
Incorporating growth factors into scaffolds
html
Schematic of two
tissue engineering
approaches using
synthetic ECMs to
present growth
factors to tissues.
(a) Physically encapsulated bioactive factors can be released
from synthetic

ECMs to target specific cell populations to migrate and direct
tissue
regeneration.
(b) Alternatively, growth factors can be chemically bound to the
material system,
making them available to cells that infiltrate the material.
for skin grafts.
2. Scaffolds
--------------------
4. Bioreactors

Bioreactors
• Bioreactor - a manufactured or engineered
device or system that supports a biologically
active environment. In the context of Tissue
Engineering, a bioreactor improves cell growth
and tissue formation in 3-dimensional tissue
constructs.
Bioreactors
• Tissue Engineers utilize bioreactors for 2
general purposes:
– To improve mass transport, i.e. delivery of
oxygen and nutrients and the removal of waste
products – this replaces the function of the
vasculature in body. We need to keep tissue alive!!!
– To provide mechanical cues that “condition” the
tissue for it’s in vivo function, i.e. flow of liquid
medium through engineered blood vessels,

mechanical stretch of engineered muscle, ventilation
of engineered lungs. Being alive isn’t enough, we
need to “train” the tissue to perform the required
function.
Examples of bioreactors in
tissue engineering
For mechanical stretching of engineered
muscle tissue:
https://www.youtube.com/watch?v=XmDeaP6n9vA
For pulsatile flow perfusion of engineered
blood vessels:
https://www.youtube.com/watch?v=5jb7ed2iCJs
For a low pulsatile flow that grants the correct
opening and closing of the valve without high
shear stresses:
https://www.youtube.com/watch?v=UFZzX-X-DzY

Successful implantation of
tissue engineered bronchus
Successful implantation of a
Tissue Engineered Trachea
http://www.youtube.com/watch?v=_GyQWAiDu0w
Tissue Engineered Bladder
(Tengion company)
http://bmb.oxfordjournals.org/content/97/1/81.full
Scaffold without cells New Bladder sutured
in place
Wrapped in “fibrin

glue” and omentum.
1. Scaffold composed of PLGA (synthetic)
and collagen (natural) is shaped into the
required size for the patients bladder.
2. Cells isolated from the patients bladder
are cultured on the scaffold for ~ 8 weeks.
3. New “engineered” bladder is implanted
by anastomosis and sutured into place.
4. New bladder is wrapped in fibrin glue
and omentum to enhance vascularization.
The Tissue Engineering and Stem Cell industry
Currently Available Tissue
Engineered “Products”
• Bone grafts: InfuseTM (Medtronic)
• Wound healing grafts/dressings: ApligraftTM (Organogenesis)
• Cell banking services: cord blood

• RESTORETM small intestine submucosa (duPont)
-------------------------------------
Countless therapies (and eventually products/services) are
still in the research and development stages.
This is due to the complexity of organs such as pancreas,
liver, lungs, hearts, eyes, etc
It is impossible to put a timeline, much depends on funding
for basic research – but we are looking at decades,
maybe 10 years for some applications, perhaps 30 years
or longer for others.
http://www.ted.com/talks/anthony_atala_printing_a_human_kid
ney?language=en
Printing a human kidney

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