Monitoring Stem Cell Research
The President's Council on Bioethics
Recent Developments in Stem Cell Research and Therapy
Research using human and animal stem cells
is an extremely active area of current biomedical inquiry.
It is contributing new knowledge about the pathways of normal
and abnormal cell differentiation and organismal development.
It is opening vistas of new cell transplantation therapies
for human diseases. Although the availability of a variety
of human stem cells is relatively recent-the isolation of
human embryonic stem cellsi
was first reported only in 1998-much is happening in both
publicly funded and privately funded research centers around
the world. It is difficult for anyone to stay abreast of all
the results now rapidly accumulating.
To help us fulfill our mandate to "monitor stem cell research,"
the President's Council on Bioethics asked several experts
to survey the recent published scientific literature and to
contribute articles on various areas of stem cell research
to this report (see articles by Drs. Gearhart,1
Ludwig and Thomson,2
in the Appendices). These reviews and the present chapter
emphasize peer-reviewed, published work with human stem cells
through July 2003. Interested readers should also consult
the wide variety of other review articles that have appeared.8
This chapter should be read in conjunction with the commissioned
review articles cited above. It draws on their findings, as
well as on the Council's own monitoring activities, but it
makes no attempt to summarize all the complexity of stem cell
research or the vast array of results. Rather we offer here
some general observations and specific examples that might
help non-scientist readers understand the overall state of
present human stem cell research, its therapeutic promise,
and some of the problems that need to be solved if the research
is to yield sound knowledge and clinical benefit. To that
end, we highlight the importance of well-characterized, stable
preparations of stem cells for obtaining reproducible experimental
results, and we identify several problems that must be solved
before these requirements can be fully met. This chapter then
describes, by way of illustration and example, some of the
better-characterized adult and embryonic stem cells. It also
indicates some of the specific investigations that are being
conducted with their aid. Finally, it considers how human
stem cells are being used to explore their potential for treating
disease, using experiments in animal models of Type-1
diabetes as an example, and it points out some of
the difficulties that must be overcome before stem cell-based
remedies may be available to treat human diseases.
We confine our attention here to newly identified types
of human stem cells and their potential use in research and
future medical treatment. Accordingly, we do not consider
those stem cell types that are already well established in
medical practice and research. Specifically, we will not examine
those preparations of bone marrow cells that have been clinically
used for some years to treat various forms of anemia and cancer.9
Neither will we deal with hematopoietic (blood-forming)
stem cells that have been isolated and purified from
bone marrow and are now being intensively studied.10
Although these developments lie beyond the scope of this report,
the demonstrated usefulness of these cells for research and
therapy encourages many researchers to expect similar benefits
from the newer stem cells that we shall consider here.
I. Stem Cells and Their Derivatives
The adult human body, and all its differentiated cells, tissues,
and organs, arise from a small group of cells contained within
the early embryo at the blastocyst stage
of its development. During in vivo embryonic
development, these cells, constituting the inner cell
mass (ICM), will divide and differentiate in concert
with each other and with the whole of which they are a part,
eventually producing the specialized and integrated tissues
and organs of the body. But when embryos are grown [using
in vitro fertilization (IVF)] in a laboratory
setting, these ICM cells may be removed and isolated,
and under appropriate conditions some will proliferate in
vitro and become embryonic stem cell lines.
These embryonic stem cells are capable of becoming many different
types of differentiated cells if stimulated to do so in vitro
[see endnote 2 for references]. However, it is not yet clear
that the cells that survive the in vitro selection process
to become embryonic stem cells have all of the same
biological properties and potentials as the ICM cells of the
blastocyst.7 In particular, it is not known for
certain that human embryonic stem cells in vitro can give
rise to all the different cell types of the adult body.ii
As noted in the Introduction to this report, stem cells are
a diverse class of cells, which can now be isolated from a
variety of embryonic, fetal, and adult tissues. Stem cells
share two characteristic properties: (1) unlimited or prolonged
self-renewal (that is, the capacity to maintain a pool
of stem cells like themselves), and (2) potency for differentiation,
the potential to produce more differentiated
cell types-usually more than one and, in some cases, many.iii
When stem cells head down the pathway toward
differentiation, they usually proceed by first giving rise
to a more specialized kind of stem cell (sometimes called
"precursor cells" or "progenitor cells"), which can in turn
either proliferate through se
lf-renewal or produce fully specialized or differentiated
cells (see Figure 1).
1. Schematic Diagram of Some Stages in Cell Differentiation
At the top of the figure is an undifferentiated stem cell;
in the central box are more "specialized" stem cells (or "precursor
cells" or "progenitor cells"); at the bottom are various differentiated
cells that are derived from the specialized stem cells. Dashed
arrows indicate symmetrical (in the sense that both the daughter
cells are stem cells) cell divisions thatproduce more stem
cells (self-renewal). Solid arrows indicate asymmetric cell
divisions that produce moredifferentiated daughter cells.
(There may also be self-renewal with asymmetric division-not
shown here-in which one daughter cell initiates a differentiation
pathway while the other remains a stem cell.) Differentiation
signals can be supplied by both soluble proteins and by specific,
cell-surface binding sites. Some of the specialized stem cells
inside the dashed box, for example, mesenchymal stem cells,
can be isolated from tissues after birth and correspond to
adult stem cells. Scientists are currently investigating
whether, at least in some cases, the process can be reversed,
that is, whether specialized cells may, on appropriate signals,
dedifferentiate to become precursor or even fully undifferentiated
The terminology used to describe different stem cell types
can be confusing. As used in this chapter, stem cells are
self-renewing, cultured cells, grown and preserved in vitro,
that are capable-upon exposure to appropriate signals-of differentiating
themselves into (usually more than one) specialized cell types.
Stem cells may be classified either according to their origins
or according to their developmental potential.
Stem cells may be obtained from various sources: from embryos,
from fetal tissues, from umbilical cord blood, and from tissues
of adults (or children). Thus, depending on their origin,
stem cell preparations may be called adult stem cells,iv
embryonic stem cells, embryonic germ cells,
or fetal stem cells. Adult stem cells [see (4)]
are cells derived from various tissues or organs in humans
or animals that have the two characteristic properties of
stem cells (self-renewal and potency for differentiation).
Embryonic stem cells (ESCs) [see (2)] are derived from
cells isolated from the inner cell mass of early embryos.
Embryonic germ cells (EGCs) [see (1)] are stem cells
derived from the primordial germ cells of a fetus.
Fetal stem cells (not further discussed in this chapter,
but included for the sake of completeness) are derived from
the developing tissues and organs of fetuses; because they
come (unlike EGCs) from already differentiated tissues, they
are (like adult stem cells) "non-embryonic," and may be expected
to behave as such.
Depending on their developmental potential, cells
may be called pluripotent, multipotent,
or unipotent. Cells that can produce all the cell types
of the developing body, such as the ICM cells of the blastocyst,
are said to be pluripotent. The somewhat more specialized
stem cells, of the sort found in the developed organs or tissues
of the body, are said to be multipotent if they produce
more than one differentiated tissue cell type, and unipotent
if they produce only one differentiated tissue cell type.
We introduce in this chapter an additional term: stem
cell preparation. A stem cell preparation
is a population of stem cells, prepared, grown, and preserved
under certain conditions. Because different laboratories (or
even the same one) can have different preparations of the
same type of stem cell, it is important to recognize the potential
differences between particular preparations of embryonic stem
It will sometimes be important to call attention to this fact,
by speaking of a "preparation of ES cells" (or a preparation
of adult stem cells) rather than of "ES cells," pure and simple.
We will use the term "stem cell preparations" when we are
speaking of a diverse group of stem cell cultures, when we
are speaking of stem cell cultures that contain an admixture
of other types of cells, or when the developmental homogeneity
of the stem cells in the population has not been defined.
Adult and embryonic stem cell populations have also been
called "stem cell lines." In the past, the term "cell line"
denoted a cell population (usually of cancer cells containing
abnormal chromosome numbers or structure, or both)
that could grow "indefinitely" in vitro. Embryonic and some
adult stem cell preparations are capable of prolonged growth
beyond 50 population doublings in vitro while retaining their
characteristic stem cell properties and initially with no
change in the chromosome numbers and structure. It is not
yet known whether any preparation of human ES cells (generally
believed to be much longer-lived than adult stem cells) will
continue to grow "indefinitely," without breaking down.
Under the influence of various cell-differentiation signals,
embryonic stem cells differentiate into numerous distinct
types of more specialized cells. Some of these are specialized
stem cells that can also self-renew, while retaining their
ability also to differentiate into multiple cell types. Recent
research has led to the isolation of an increasing number
of adult (non-embryonic) stem cells (dashed box area of Figure
1) from such tissues as bone marrow (for example, hematopoietic
and mesenchymal stem cells), brain (for example,
neural stem cells) and other tissues [see (4)]. Although these
stem cell preparations differ from one another in their future
fates, they tend to be grouped together (especially in the
public policy debates) under the name "adult stem cells,"
even though they may have been obtained from children or even
from umbilical cord blood obtained at the time of
Subsequent exposure to additional differentiation signals
can cause these specialized stem cells to differentiate further,
so that they finally give rise to the variety of differentiated
cells that make up the adult body (labeled A-D in Figure 1).
At each stage of the differentiation process, specific sets
of genes are expressed (or "turned on") and other
sets are repressed (or "turned off"), to produce the specific
proteins that give each cell its distinctive properties. At
each stage along the way, proteins called transcription
factors play key roles in determining which sets of genes
are expressed and repressed, and therefore what sort of a
cell the newly differentiated cell will become.
II. Reproducible Results Using Stem
Cell Preparations and Their Derivatives
A major goal of scientific research is the acquisition of
reliable knowledge based on experiments that yield reproducible
results. Reproducible results are possible only if the materials
used in experiments remain constant and stable. To obtain
reproducible results in experiments using stem cells, it is
essential to produce, preserve, characterize, and continually
re-characterize preparations of stem cells in ways that increase
the likelihood that the cells used to repeat experiments will
remain unchanged-a technically challenging task. The tendency
of stem cells in vitro to differentiate spontaneously into
more specialized cells makes the task of obtaining homogeneous
and stable stem cell preparations especially challenging,
and much basic research is needed to learn how to control
the fate of these cells. Failure to control the cells may
yield experimental results that are difficult or impossible
to reproduce. The following more specific observations make
clear the dimensions of this difficulty.
A. Initial Stem Cell Preparations Can Contain Multiple
Isolation of adult stem
cells from source tissues such as bone marrow, brain, or muscle
initially yields a heterogeneous cell preparation. The initial
preparation contains the several cell types found in the source
tissue, and it may also include red blood cells, white blood
cells, and (possibly) circulating stem cells, owing to the
presence of blood flowing through the tissue in question.
Initial mixtures of cells may then be treated in various ways
to remove unwanted contaminating cells, thereby increasing
the proportion of stem cells in the preparation. But
seldom, if ever, does one produce an adult stem cell preparation
that is 100 percent stem cells, unless the adult stem cell
preparation has been "single-cell cloned" in vitro
The way in which human embryonic stem cells have been
produced from ICM cells also raises a question about the "species
homogeneity" of the initial cell preparations. In the past,
human embryonic stem cells were isolated and maintained
by in vitro growth on top of irradiated (so that they no longer
divide) "feeder layers" of mouse cells.
It is thought that the feeder cells secrete factor(s) that
enable the stem cells to divide while maintaining a relatively
undifferentiated state. Although the mouse cells have been
treated to prevent their cell division, should any of them
happen to survive, human embryonic stem cells prepared in
this way may contain some viable mouse cells.vi
More recently, several groups have shown that it is possible
to grow ESCs on feeder layers of human cells, including fibroblasts
obtained from skin biopsies, or without any feeder cell layer
One way to be certain that human embryonic stem cell preparations
do not contain any mouse feeder cells is through "single cell
cloning" (see below).
B. Genetically Homogenous Stem Cells through Single
Some preparations of stem
cells growing in vitro have been "single cell cloned," that
is, grown as a population derived from a single stem
cell. By placing a cylinder over a single cell located with
a microscope, scientists are able to isolate within the cylinder
all the progeny produced by subsequent cell divisions beginning
from this single cell. The result is a stem cell preparation
in which all the cells are descended from the original single
cell. The cells within the cylinder are then harvested and
grown to greater numbers in vitro, and the resulting stem
cell preparation is said to be "single cell cloned." The stem
cells within a "single cell cloned" population are, at least
to begin with, genetically homogeneous because they
are all derived from the same original cell. Some of the ESC
preparations produced prior to August 9, 2001 have been "single
C. Expansion in Vitro, Preservation, and Storage
Reproducible results require that preparations of stem cells,
even if genetically homogenous when first isolated, remain
stable over time and during preservation. This, too, is not
a simple matter with stem cells, despite the fact that the
self-renewal characteristic of human embryonic and adult stem
cells enables them-unlike differentiated cells from many human
tissues-to be grown in large numbers in vitro while maintaining
their essential stem cell characteristics. After such expansion,
many, presumably identical, vials of the cells can be frozen
and preserved at very low temperatures. Frozen stem cell preparations
can later be thawed and grown again in vitro to produce larger
numbers of cells.
As with all dividing cells, stem cells are subject to a very
small but definite chance of mutation during DNA replication;
thus, prolonged growth in vitro could introduce genetic
heterogeneity into an originally homogeneous population.
During this process of repeated expansion and preservation,
subtle changes in the growth conditions or other variables
may give rise to "selective pressures" that can increase the
heterogeneity in a stem cell preparation by favoring the multiplication
of advantaged cell variants in the population. It is not known
at present how many of the 78 human ESC preparations, designated
as eligible for federal funding under the current policy,
have developed genetic variants that may make them unsuitable
for further research.
Whether several cycles of freezing and thawing change the
phenotypic characteristics of stem cell preparations
needs detailed study. However, the practical advantages of
preserving stem cell preparations by freezing are too large
to ignore. Such preservation makes it possible to repeat an
experiment many times with a very similar stem cell preparation.
It would also make it possible, should stem cell based therapies
be developed in the future, to treat multiple patients with
a common, well-characterized cell preparation derived from
a single initial stem cell sample.
D. Chromosome Changes
In addition to the possible loss of homogeneity in stem cell
preparations owing to variability in growth conditions or
to freezing and thawing, there is the possibility of variation
being introduced during the processes of growth and cell division.
Normal human stem cells (like all human somatic cells) have
46 chromosomes. During the copying of chromosomal DNA and
the separation of daughter chromosomes at cell division, rare
mistakes occur that lead to the formation of abnormal chromosomes
or maldistribution of normal ones. Cells with abnormal chromosomes
or chromosome numbers can progress to malignancy, so retention
of the normal human chromosome number and structure is an
essential characteristic of useful human stem cell preparations.
The most studied preparations of human stem cells generally
have normal human chromosome numbers and structure.3
Nevertheless, vigilance is needed, for even a small number
of chromosomally abnormal cells could end up causing cancer
in future clinical trials of stem cell based therapies.
E. Developmental Heterogeneity of Stem Cell Preparations
The in vitro growth conditions and the presence
of specific chemicals or proteins, or both, in the culture
medium can influence the differentiation pathway taken
by stem cells as they start to differentiate. Thus, even initially
homogeneous, "single cell cloned" stem cell preparations may
become developmentally heterogeneous over time, with
respect to the percentage of cells in the preparation that
are in one or another differentiated state. For example, a
stem cell preparation after growth in vitro under specific
conditions might contain 75 percent fully differentiated (insulin-producing)
cells and 25 percent partially differentiated cells. The biological
properties of the fully differentiated cells and the partially
differentiated cells are likely to be different. If such a
cell preparation is used in research, or transplanted into
an animal model of human disease and a biological effect is
observed, one must do additional experiments to determine
whether the effect was due to the fully differentiated cells
or to the partially differentiated cells (or perhaps to both
acting together) in the now mixed preparation.
F. Microbial Contamination
Stem cell preparations originally isolated from humans and
expanded in vitro may also be variably contaminated with human
viruses, bacteria, fungi and mycoplasma.
ESC preparations isolated using mouse feeder cell layers might
also be contaminated with mouse viruses. Specific tests need
to be performed on the source tissue and periodically on the
resulting stem cell preparations to rule out the presence
of these contaminants. Some of these contaminants can also
multiply when stem cells are grown in vitro, and their presence
can influence the results obtained when stem cell preparations
are used in subsequent experiments. The presence of such contaminants
can also potentially affect the reproducibility of the results
of experiments in which stem cell preparations are studied
in vivo in experimental animals.
In summary, there are numerous challenges to obtaining and
preserving the uniform and stable preparations of stem cells
necessary for reliable research and, eventually, for safe
and effective possible therapies. Researchers must address
multiple factors in order to maximize the probability of obtaining
reproducible results with human stem cell preparations. Human
stem cell preparations that are
are most likely to yield experimental results that will be reproducible.
Preparations with these properties will be the most useful both
in basic research and in investigations of possible clinical
well-characterized as to the absence of cellular, viral,
bacterial, fungal, and mycoplasma contaminants, and
III. Major Examples
of Human Stem Cells
In this section we discuss major examples of human stem cells
that meet many of the criteria listed above. Among human adult
stem cells, we focus on mesenchymal stem cells (MSCs),4
multipotent adult progenitor cells (MAPCs),3
and neural stem cells, and among human embryonic stem
cells, on ESC2
cells. For information on the wide variety of other human
stem cell preparations isolated from adult tissues, see reference
(4) (Appendix K).
Further research on some of these other adult stem cell preparations
may demonstrate that they can also be "single cell cloned,"
expanded considerably by growth in vitro with retention of
normal chromosome structure and number, and preserved by freezing
and storage at low temperatures. At that point, it would be
very important to compare the properties of these other adult
stem cells, and the more differentiated cells that can be
derived from them, with the already characterized human embryonic
and adult stem cell preparations.
A. Human Adult Stem Cells
1. Human Mesenchymal Stem Cells.
Bone marrow contains at least two major kinds of stem cells,
hematopoietic stem cells10 that give rise to the
red cells and white cells of the blood, and mesenchymal stem
that can be reproducibly isolated and expanded in vitro, and
that can differentiate in vitro into cells with properties
of cartilage, bone, adipose (fat), and muscle cells.14
The characteristics (morphology, expressed proteins,
and biological properties) of these cells have been somewhat
difficult to specify, because they appear to vary depending
upon the in vitro culture conditions and the specific cell
However, there is a recent report indicating that MSCs, if
isolated using three somewhat different methods, give rise
to stem cell preparations whose properties are very similar
to one another.16
Using dual antibody staining and fluorescence-activated cell
sorting, Gronthos and colleagues17
isolated human MSCs in almost pure form and expanded them
substantially in vitro. Thus, human MSC preparations isolated
in different laboratories by different methods may have similar
but not identical properties.
A molecular analysis of genes expressed in a single-cell-derived
colony of MSCs provided evidence for the activity of genes
also turned on in bone, cartilage, adipose, muscle, hematopoiesis-supporting
stromal, endothelial, and neuronal cells.15
These results are surprising in that MSCs derived from a single
cell appear to be expressing genes associated with multiple
major cell lineages. It is possible that different
cells within the colony had already entered into distinct
differentiation pathways, resulting in a developmentally
heterogeneous population composed of several different cell
Mesenchymal stem cells are important for research and therapy
for several reasons. First, because they can be differentiated
in vitro into multiple cell types, they make possible detailed
research on the molecular events underlying differentiation
cartilage, and fat cell lineages. Second, they have recently
been shown to support the in vitro growth of human embryonic
stem cells.19 Thus,
they could replace the mouse feeder cells used previously,
obviating the need to satisfy FDA requirements for xenotransplantation,
should the ESCs or their derivatives ever be used in human
clinical research or transplantation therapy. Third, clinical
studies are already underway in which MSCs are co-transplanted
with autologous hematopoietic stem cells into cancer patients
to replace their blood cell-forming system, destroyed by radiation
or high dose chemotherapy.20
It is believed that the MSCs will support the repopulation
of the bone marrow by the injected hematopoietic stem cells.
In addition, injecting allogeneic MSCs (MSCs from a genetically
different human donor) may also prove valuable in modulating
the immune system to make it more accepting of foreign tissue
grafts [see Itescu review, reference (5)]. Finally, MSCs have
the potential for cell-replacement therapies in injuries involving
bone, tendon, or cartilage and possibly other diseases. They
are, in fact, already being tested as experimental therapies
for osteogenesis imperfecta,21
metachromatic leukodystrophy, and
These last two studies are of great interest, since allogeneic
MSCs were used and no serious adverse immune reactions were
2. Multipotent Adult Progenitor Cells (MAPCs).
Verfaillie and coworkers recently described the isolation
of MAPCs from rat, mouse, and human bone marrow [see (3) and
references cited therein]. Like MSCs, MAPCs can also be differentiated
in vitro into cells with the properties of cartilage, bone,
adipose, and muscle cells. In addition, there is evidence
for the in vitro differentiation of human MAPCs into functional,
a potential that has not so far been shown for MSCs. There
is increasing interest in MAPCs, both as potential precursors
of multiple differentiated tissues and, ultimately, for possible
autologous transplantation therapy.
The relationship between human MSCs and the human MAPCs described
by Verfaillie and coworkers [see (3)] needs to be clarified
by further research. Both kinds of cells are isolated from
bone marrow aspirates as cells that adhere to plastic. Each
can be differentiated in vitro into cells with cartilage,
bone, and fat cell properties. They express several of the
same cell antigens, but are reported to differ in
a few others.3
MAPCs have to be maintained at specific, low cell densities
when grown in vitro, otherwise they tend to differentiate
It remains important that the isolation and properties of
MAPCs be reproduced in additional laboratories.
3. Human Neural Stem Cells.
The nervous system is made up of three major types of cells;
neurons or nerve cells proper, and two kinds of supporting
or glial cells (oligodendrocyte, astrocyte).
Stem cells capable of differentiating into one or more of
these neural cell lineages can be isolated from brain tissue
(particularly the olfactory bulb and lining of the ventricles)24,25
and grown in vitro. In the presence of purified growth-factor
proteins, the population of cells can be expanded by growth
in vitro as round clumps of cells called neurospheres. However,
many neurospheres grown in culture are developmentally heterogeneous
in that they contain more than one neural cell type, and the
number of self-renewing cells is frequently low (less than
Although neural stem cells are still insufficiently understood,
they are already proving valuable in basic research on neural
development. The ability to grow reproducible neural stem
cells in vitro has facilitated identification of important
neural stem cell growth factors and their cellular receptors.
For example, human neural stem cells from the developing human
brain cortex, expanded in culture in the presence of leukemia
inhibitory factor (LIF), allowed growth of a self-renewing
neural stem cell preparation for up to 110 population doublings.
Withdrawal of LIF led to decreased expression of about 200
which were specifically identified through use of "gene chips"
manufactured by Affymetrix. These genes are presumably involved
in promoting or preserving the stem cell's capacity for self-renewal
in the undifferentiated state. The number and specificity
of the molecular changes characterized in these experiments
powerfully illustrate the usefulness of neural and other stem
cell preparations in basic biomedical research.
Human neural stem cells are also being injected into animals
to test their effects on animal models of human neurological
disease. To track the fate of the introduced human cells,
they must first be modified or "marked" in ways that permit
their specific detection.ix
Marked human neural stem cells are easily tracked after they
are injected into experimental animals, making it possible
to determine whether they survive and migrate following injection.
Studies of this type have provided evidence that human neural
cells can migrate extensively in the brain after injection.28
In addition, such cells can be injected into animal models
of human diseases such as intracerebral hemorrhage and Parkinson
Disease (PD) to study their effect on the progression
of the disease.29
Although human neural stem cells may not yet be as well characterized
as MSCs or ESCs, they are being actively studied with the
hope that they can be used in future treatments for devastating
neurological diseases such as Alzheimer Disease and PD.
4. Adult Stem Cells from Other Sources.
Prentice [see (4)] has summarized a large amount of recent
information on preparations of stem cells isolated from amniotic
fluid, peripheral blood, umbilical cord blood, umbilical
cord, brain tissue, muscle, liver, pancreas, cornea,
salivary gland, skin, tendon, heart, cartilage, thymus,
dental pulp, and adipose tissue.
Studies of many of the stem cell preparations from these sources
are just getting started, and further work is needed to determine
their biological properties and their relatedness to other
stem cell types. In some cases, the long-term expandability
in vitro of these stem cells has not been demonstrated. Yet,
the demonstration that they can be isolated from such tissue
compartments in animals should spur the search for similar
human stem cell types.
As Prentice also reports,4
many attempts have already been made using various preparations
of adult stem cells to influence or alter the course of diseases
in animal models. Despite the fact that the stem cell preparations
used are not well characterized, and reproducible results
have yet to be obtained, preliminary findings are sometimes
encouraging. It is of course not yet clear whether the injected
cells are functioning as stem cells, fusing with existing
host cells, or stimulating the influx of the host's own stem
cells into the target tissue.x
But, if reproduced, these preliminary findings may point the
way to future therapies, even in the absence of precise knowledge
of the mechanism(s) of cellular action.
B. Human Embryonic Stem Cells
1. Human Embryonic Stem Cells (ESCs).
Human embryonic stem cells have been isolated from the inner
cell masses of blastocyst-stage human embryos in multiple
laboratories around the world.xi
There is great interest in understanding the properties of
these cells because they hold out the promise of being able
to be differentiated into a large number of different cell
types for possible cell therapies, as contrasted with the
more limited number of cell types available by differentiation
of specific adult stem cell preparations. As of July 2003,
12 ESC preparations (up from 2 such preparations a year earlier)
out of a total of 78 "eligible" preparations of human ESCs
were available for shipment to recipients of U.S. federal
The review by Ludwig and Thomson2
lists more than 40 peer-reviewed human ESC primary research
papers that have been published since the initial publication
Although isolated from different blastocyst-stage human embryos
in laboratories in different parts of the world, ESCs have
a number of properties in common. These include the presence
of common cell surface antigens (recognized by binding of
specific antibodies), expression of the enzymes alkaline phosphatase
and telomerase, and production of a common gene-regulating
transcription factor known as Oct-4. At least 12 different
preparations of ESCs have been expanded by growth in vitro,
frozen and stored at low temperature, and at least partially
Some of these ESC preparations have been "single-cell cloned."
Human ESCs have been differentiated in vitro into neural
(neurons, astrocytes, and oligodendrocytes), cardiac (synchronously
contracting cardiomyocytes), endothelial (blood vessels),
hematopoietic (multiple blood cell lineages), hepatocyte (liver
cell), and trophoblast (placenta) lineages.2
In the case of neural and cardiac lineages, similar results
have been obtained in different laboratories using different
preparations of ESCs, thus fulfilling the "reproducible results"
criterion described above. For other lineages, the results
described have not yet been reproduced in another laboratory.
2. Embryonic Germ Cells.
Human embryonic germ cells are isolated from the primordial
germ tissues of aborted fetuses. Gearhart1
has summarized the results of recent research with human and
mouse EG cells. One study focused on regulation of imprinted
genes in EG cells: it showed "that general dysregulation of
imprinted genes will not be a barrier to their (EG cell) use
in transplantation studies."30
addition, Kerr and coworkers31
showed that cells derived from human EG cells, when introduced
into the cerebrospinal fluid of rats, became extensively
distributed over the length of the spinal cord and expressed
markers of various nerve cell types. Rats paralyzed by virus-induced
nerve-cell loss recovered partial motor function after transplantation
with the human cells. The authors suggested that this could
be due to the secretion of transforming growth factor-&alpha
and brain-derived growth factor by the transplanted cells
and subsequent enhancement of rat neuron survival and function.
Until recently, work with human EG cells came primarily from
one laboratory. Recently the isolation and properties of human
EG cells have been independently confirmed.32
Because human EG cells share many (but
not all) properties with ESCs, these cells offer another important
avenue of inquiry.
3. Embryonic Stem Cells from Cloned Embryos (Cloned ESCs).
Although it has yet to be accomplished in practice, somatic
cell nuclear transfer (SCNT) could create cloned human
embryos from which embryonic stem cells could be isolated
that would be genetically virtually identical to the person
who donated the nucleus for SCNT: hence cloned ESCs
[see (7)]. In theory, using such cloned embryonic stem cells
from individual patients might provide a way around possible
immune rejection (see below), though in practice this could
require individual cloned embryos for each prospective patient-a
daunting task. And clinical uses might require a separate
FDA approval for every single cloned stem cell line or its
The ability to produce cloned mouse stem cells and genetically
modify them in vitro has made possible an experiment
demonstrating the potential of cloned human embryonic
stem cells in the possible future treatment of human
genetic diseases. Rideout et al.33
used a mutant mouse strain that was deficient in immune system
function. They produced a cloned mouse embryonic stem
cell line carrying the mutation, and then specifically repaired
that gene mutation in vitro. The repaired cloned stem cell
preparation was then differentiated in vitro into bone marrow
precursor cells. When these precursor cells were injected
back into the genetically mutant mice, they produced partial
restoration of immune system function.
Production of cloned human embryonic stem cell preparations
remains technically very difficult and ethically controversial.
Recently however, Chen and coworkers34
have reported that fusion of human fibroblasts with enucleated
rabbit oocytes in vitro leads to the development
of embryo-like structures from which cell preparations with
properties similar to human embryonic stem cells can be isolated.
This work needs to be confirmed by repetition in other laboratories.
In addition, further work is needed to decisively settle
the question of whether rabbit (or human egg donor) mitochondrial
DNA and rabbit (or human egg donor) mitochondrial
proteins persist in the embryonic stem cell preparations.
Persistence of these foreign mitochondrial proteins in these
human ESC-like preparations could possibly increase the probability
of immune rejection of the cloned cells, thus limiting their
clinical application, although the immune reaction might not
be as severe as that to foreign proteins produced under the
direction of chromosomal genes. The presence of foreign or
aberrant mitochondria also carries the risk of transmitting
mitochondrial disease (caused by defects in mitochondrial
DNA) that could be detrimental to the cells and to the recipient
into whom they might eventually be transplanted.
IV. Basic Research Using Human Stem
Human stem cells are proving useful in basic research in
several ways. They are useful in unraveling the complex molecular
pathways governing human differentiation. For example, because
ESCs can be stimulated in vitro to produce more differentiated
cells, this transition can be studied in greater detail and
under better-controlled conditions than it can be in vivo.
In the best circumstances, these differentiated cells can
be grown as largely homogeneous cell populations, and their
gene expression profiles can be compared in detail.
Also, stem cell preparations can be used to produce populations
of specialized cells that are not easily obtained in other
ways. In one case, for example, this approach has provided
large quantities of human trophoblast-like cells that have
not been previously available.35
In addition, cultures of differentiated cells derived from
stem cells could be used to test new drugs and chemical compounds
for toxicity and mutagenicity.36
As experience with these differentiated derivatives of human
ESCs grows, it may become possible to reduce or eliminate
the use of live animals in such testing protocols.
In the near future, the differentiated state of various human
cell types will be characterized not just by a few biological
markers, but by the pattern and levels of expression of hundreds
or thousands of genes. Integration of this knowledge with
the catalog of all human genes produced during the Human Genome
Project will gradually give us knowledge of which genes are
key regulators of human development and which genes are central
to maintaining the stem cell state.37
Increased understanding of the molecular pathways of human
cell differentiation should eventually lead to the ability
to direct in vitro differentiation along pathways that yield
cells useful in medical treatment. In addition, when the normal
range of gene expression patterns is known, researchers can
then determine which genes are expressed abnormally in various
diseases, thus increasing our understanding of and ability
to treat these diseases.
A group of stem cell researchers has recently outlined a
set of important research questions, which, once answered
will greatly enhance our understanding of human embryonic
stem cells and their potential fates and possible uses.38
They include the following:
- What is the most effective way to isolate and grow ESCs?
- How is the self-renewal of ESCs regulated?
- Are all ESC lines the same?
- How can ESCs be genetically altered?
- What controls the processes of ESC differentiation?
- What new tools are needed to measure ESC differentiation
in vitro and in vivo?
V. Human Stem Cells and the Treatment
A major goal of stem cell research is to provide healthy
differentiated cells that, once transplanted, could repair
or replace a patient's diseased or destroyed tissues. In pursuit
of this goal, one likely approach would start by isolating
stem cells that could be expanded substantially in vitro.
A large number of the cultivated stem cells could then be
stored in the frozen state, extensively tested for safety
and efficacy as outlined above, and used as reproducible starting
material from which to prepare differentiated cell preparations
that will express the needed beneficial properties when they
are transplanted into patients with specific diseases or deficiencies.
To make more concrete both the potential of this approach
and the obstacles it faces, we will summarize, as a case study
example, some current information on the properties of cells
derived from human stem cell populations that have been used
in an animal model of Type-1 diabetes. But before doing so,
we discuss an obstacle to any successful program of stem cell-based
transplantation therapy: the problem of immune rejection of
the transplanted cells.
A. Will Stem Cell-Based Therapies Be Limited by
Much of the impetus
for human stem cell research comes from the hope that stem
cells (or more likely, differentiated cells derived from them)
will one day prove useful in cell transplantation therapies
for a variety of human diseases. Such cell transplantation
would augment the current practice of whole organ transplantation.
To the extent that the healing process works with in vitro
derived cells, the need for organ donors and long waiting
lists for organ donation might be reduced or even eliminated.
Will the recipient (patient) accept or reject the transplanted
human cells? In principle, the problem might seem avoidable
altogether: adult stem cells could be obtained from each individual
patient needing treatment. They could then be grown or modified
to produce the desired (autologous and hence rejection-proof)
transplantable cells. But the logistical difficulties in processing
separate and unique materials for each patient suggest that
this approach may not be practical. The cost and time required
to produce sufficient numbers of well-characterized cells
suitable for therapy suggest that it will be cells derived
from one or another unique stem cell line that will be used
to treat many (genetically different) individual patients
(allogeneic cell transplantation).
When allogeneic organ or tissue transplantation is currently
done using, for example, bone marrow, kidney, or heart, powerful
immunosuppressive drugs-carrying undesirable side
effects-must be used to prevent immunological rejection of
the transplanted tissue.5
Without such immunosuppression, the patient's T-lymphocytes
and natural killer (NK) cells recognize surface molecules
on the transplanted cells as "foreign" and attack and destroy
the cells. Also, in whole organ transplantation donor
T-lymphocytes and NK cells, entering the recipient with the
transplanted organ, can also destroy the tissues of the transplant
recipient (called "graft versus host" disease).
Are the differentiated derivatives of human stem cells as
likely to incite immune rejection, when transplanted, as are
solid organs? Do their surfaces carry those protein antigens
that will be recognized as "foreign"? Experiments have been
done to examine human ESC and MSC preparations growing in
vitro for the expression of surface molecules known to play
important roles in the immune rejection process. Drukker and
coworkers39 showed that embryonic stem cells
in vitro express very low levels of the immunologically crucial
major histocompatibility complex class I (MHC-I) proteins
on their cell surface. The presence of MHC-I proteins increased
moderately when the ESCs became differentiated, whether in
vitro or in vivo. A more pronounced increase in MHC-I antigen
expression was observed when the ESCs were exposed to gamma-interferon,
a protein produced in the body during immune reactions. Thus,
under some circumstances, human ESC-derived cells can express
cell surface molecules that could lead to immune rejection
upon allogeneic transplantation.
Similarly, Majumdar and colleagues showed that human mesenchymal
stem cells in vitro express multiple proteins on their cell
surfaces that would enable them to bind to, and interact with,
T-lymphocytes. They also observed that gamma-interferon increased
expression of both human leukocyte antigen (HLA) class I and
class II molecules on the surface of these MSCs.40
These results indicate that it will probably not be possible
to predict, solely on the basis of in vitro experiments, the
likelihood that transplanted allogeneic MSCs would trigger
immune rejection processes in vivo.
Many further studies in this area are badly needed. At this
time there is insufficient information to determine which,
if any, of the approaches to get around the rejection problem
will eventually prove successful.
B. Case Study: Stem Cells in the Future Treatment of Type-1
1. The Disease and Its Causes.
The human body converts the sugar glucose into cell energy
for heart and brain functioning, and indeed, for all bodily
and mental activities. Glucose is derived from dietary carbohydrates,
is stored as glycogen in the liver, and is released again
when needed into the bloodstream. A protein hormone called
insulin, produced by the beta cells in the islets
of the pancreas, facilitates the entrance of glucose from
the bloodstream into the cells, where it is then metabolized.
Insulin is critical for regulating the body's use of glucose
and the glucose concentration in the circulating blood.
The body's failure to produce sufficient amounts of insulin
results in diabetes, an extremely common metabolic disease
affecting over 10 million Americans, often with widespread
and devastating consequences. In some five to ten percent
of cases, known as Type-1 diabetes (or "juvenile diabetes"),
the disease is caused by "autoimmunity," a process in which
the body's immune system attacks "self." xiv
T-lymphocytes attack the patient's own insulin-producing
beta cells in the pancreas. Eventually, this results in destruction
of ninety percent or so of the beta cells, resulting in the
With a deficiency or absence of insulin the blood glucose
becomes elevated and may lead to diabetic coma, a fatal condition
if untreated. Chronic diabetes, both Type-1 and the much more
common Type-2 diabetes (which is not autoimmune, but largely
genetic), causes late complications in the retina, kidneys,
nerves, and blood vessels. It is the leading cause of blindness,
kidney failure, and amputations in the U.S. and a major cause
of strokes and heart attacks.
Type-1 diabetes is a devastating, lifelong condition that
currently affects an estimated 550,000-1,100,000 Americans,41
including many children. It imposes a significant burden on
the U.S. healthcare system and the economy as a whole, over
and above the disabilities and impairments borne by individual
sufferers. Recent estimates suggest that treatment of all
forms of diabetes costs Americans a total of $132 billion
At 5-10 percent of all diabetes cases, the costs of Type-1
diabetes can be estimated as $6.5-13 billion per year.
2. Current Therapy Choices and Outcomes.
The current treatment of Type-1 diabetes consists of insulin
injections, given several times a day in response to repeatedly
measured blood glucose levels. Although this treatment is
life-prolonging, the procedures are painful and burdensome,
and in many cases they do not adequately control blood glucose
concentrations. Whole pancreas transplants can essentially
cure Type-1 diabetes, but less than 2,000 donor pancreases
become available for transplantation in the U.S. each year,
and they are primarily used to treat patients who also need
a kidney transplant. Like all recipients of donated organs,
pancreas transplant recipients must continuously take powerful
drugs to suppress the immunological rejection of the transplanted
In addition to treatment with whole pancreas transplantation,
small numbers of Type-1 diabetes patients have been treated
by transplantation of donor pancreatic islets into the liver
of the patient coupled with a less intensive immunosuppressive
treatment (the Edmonton protocol).43
Expanded clinical trials of this procedure are currently underway.
Scientists are also evaluating methods of slowing the original
autoimmune destruction of pancreatic beta cells that produces
the disease in the first place.
Whole pancreas and islet cell transplants ameliorate Type-1
diabetes, but there is nowhere near enough of these materials
to treat all in need. To overcome this shortage, people hope
that human stem cells can be induced-at will and in bulk-to
differentiate in vitro into functional pancreatic beta cells,
available for transplantation. Of course, it would still be
crucial to prevent immunological destruction of the newly
transplanted stem cell-derived beta cells.
3. Stem Cell Therapy for Type-1 Diabetes?
Initial experiments in mice suggested that insulin-producing
cells could be obtained from mouse embryonic stem cells following
in vitro differentiation.44 Can this approach be
extended to human stem cells? A number of attempts have been
made, with promising initial findings, yet they are not easily
evaluated, partly because the criteria for characterizing
the cells are not standardized. In a recent paper, Lechner
and Habener provided a list of six criteria to define the
characteristics of pancreas-derived "beta-like" cells that
could be potentially useful in treatment of Type-1 diabetes.45
We have used those criteria to facilitate assessment of the
current state of progress toward development of functional
"beta-like" cells that might eventually be tested in Type-1
diabetes patients. Table 1 summarizes and compares the properties
of human cell preparations recently produced in research seeking
this objective by Abraham et al.,46Zulewski
Assady et al.,48
Zhao et al.,49
and Zalzman et al.,50
and tested in mouse models of human diabetes.
Table 1: Comparison of Insulin-Producing
Cells Derived from Human Stem Cells
Clonally Isolated / Marked?
Examination to Ensure Endogenous Insulin Production
et al, 2002 (46); Zulewski et al, 2001 (47)
Clonally isolated adult
stem cells (derived from adult pancreatic islets)
Insulin mRNA(+); Insulin
protein (+); No ultra-structural examination
Clonally isolated embryonic
Insulin mRNA (+)
Insulin protein (+); No ultrastructural
examination; possible insulin uptake from serum
al, 2002 (49)
Uncloned cadaver islets
(cultured in vitro)
(+); Insulin protein (+);
insulin secretory granuoles (+)
High blood glucose
concentrations reversed in STZ/SCID mice
et al, 2003 50)
Cloned fetal liver cells:
immortalized with human telomerase and transduced with
Human and rat PDX-1
(+); GK (-); GLUT-2 (-)
Insulin mRNA (+); Insulin
No ultra- structural examination
High blood glucose
concentrations reversed in STZ/NOD-SCID mice; high blood
glucose returned upon graft removal
No tumors at 3 months
Beta-cell-specific markers: PDX-1: (a.k.a
IPF-1), a regulatory gene important for beta-cell function;
Glucokinase (GK), an enzyme that detects high levels of
glucose and modulates insulin release; GLUT-2, a protein
associated with glucose-responsive insulin secretion. CK-19
is a marker for pancreatic duct cells. Insulin production
criteria: synthesis of messenger RNA for insulin or preproinsulin;
tests for the presence of insulin protein; and ultrastructural
studies (electron microscopy) to determine the presence
of typical insulin secretory granules. In addition, the
glucose-responsiveness of insulin production and release,
an essential characteristic of normal beta-cell function,
was assessed in a number of the studies described above.
Both mouse models of Type-1 diabetes used mice that had
a condition known as Severe Combined Immunodeficiency (SCID)
and were treated with streptozotocin (STZ), a drug that
induces selective destruction of the insulin-producing cells.
The mice in the Zalzman study were also born with a form
of mouse diabetes, and are called Non-Obese Diabetic (NOD)
As the results described in Table 1 indicate, cells derived
from some human stem cells transplanted into specific strains
of mice mimicking major aspects of Type-1 human diabetes51
were able to reverse high blood glucose concentrations. Although
these results are encouraging, the transplant rejection question
remains unanswered because the likely immune rejection of
the transplanted human cells was prevented in these experiments
by using special strains of immunodeficient mice
that lack the capacity to recognize and attack foreign cells.
No tumors were observed in the transplanted mice, but the
experiments were terminated after about three months, an insufficient
time for much tumor development to occur. Because many Type-1
diabetes patients are children and because a largely effective
therapy (insulin injection) is currently available, the introduction
of islet cell transplant therapy will need a high degree of
certainty that the introduced cells or their derivatives will
not become malignant over the course of the patient's life.
Stringent tests of the cancer-causing potential of candidate
cell preparations will be required, including multi-year studies
in animals that live longer than mice or rats. Long-term follow-up
of children and adult patients who had received bone marrow
transplants many years ago has revealed an increased risk
of severe neurologic complications52
and a variety of types of cancer.53
C. Therapeutic Applications of Mesenchymal Stem
Before stem cell based therapies
are used to treat human diseases, they will have to gain approval
through the Food and Drug Administration (FDA) regulatory
process. The first step in this process is filing an Investigational
New Drug (IND) application. As of July 2003, four IND applications
have been filed for clinical applications of mesenchymal stem
cells. The disease indications include: (1) providing MSC
support for peripheral blood stem cell transplantation in
cancer treatment, (2) providing MSC support for cord blood
transplantation in cancer treatment, (3) using MSCs to stimulate
regeneration of cardiac tissue after acute myocardial infarction
(heart attack), and (4) using MSCs to stimulate regeneration
of cardiac tissue in cases of congestive heart failure. The
first two applications are currently in Phase II of the regulatory
process, with pivotal Phase III trials scheduled to begin
VI. Private Sector Activity
In the United States, much of the basic research on animal
stem cells and human adult stem cells has been publicly
funded. Yet before 2001, research in the U.S., using human
ESCs could only be done in the private sector (the locus also
of much research on animal and human adult stem cells). The
current state of knowledge about human ESCs (and also about
human MSCs) reflects pioneering and on-going stem cell research
funded by the private sector in the U.S.54,55
For example, the work that led to the 1998 reports of the
first isolation of both ESCs and EGCs, was funded by Geron
Corporation. Embryonic and adult stem cell research is today
vigorously pursued by many companies and supported by several
private philanthropic foundations,56
and the results of some of this research have been published
in peer-reviewed journals.57
Private sector organizations have pursued and been awarded
patents on the stem cells themselves and methods for producing
and using them to treat disease. As noted above, at least
one company (Osiris Therapeutics) has protocols under review
at the FDA for clinical trials with MSCs. It seems likely
that private sector companies will continue to play large
roles in the future development of stem cell based therapies.
VII. Preliminary Conclusions
While it might be argued that it is too soon to attempt to
draw any conclusions about the state of a field that
is changing as rapidly as stem cell research, we draw the
following preliminary conclusions regarding the current state
of the field.
Human stem cells can be reproducibly isolated from a variety
of embryonic, fetal, and adult tissue sources. Some human
stem cell preparations (for example, human ESCs, EGCs, MSCs,
and MAPCs) can be reproducibly expanded to substantially larger
cell numbers in vitro, the cells can be stored frozen and
recovered, and they can be characterized and compared by a
variety of techniques. These cells are receiving a large share
of the attention regarding possible future (non-hematopoietic)
stem cell transplantation therapies.
Preparations of ESCs, EGCs, MSCs, and MAPCs can be induced
to differentiate in vitro into a variety of cells with properties
similar to those found in differentiated tissues.
Research using these human stem cell preparations holds promise
for: (a) increased understanding of the basic molecular process
underlying cell differentiation, (b) increased understanding
of the early stages of genetic diseases (and possibly cancer),
and (c) future cell transplantation therapies for human diseases.
The case study of developing stem cell-based therapies for
Type-1 diabetes illustrates that, although insulin-producing
cells have been derived from human stem cell preparations,
we could still have a long way to go before stem cell-based
therapies can be developed and made available for this disease.
This appears to be true irrespective of whether one starts
from human embryonic stem cells or from human adult stem cells.
The transplant rejection problem remains a major obstacle,
but only one among many.
Human mesenchymal stem cells are currently being evaluated
in pre-clinical studies and clinical trials for several specific
Much basic and applied research remains to be done if human
stem cells are to achieve their promise in regenerative medicine.58
This research is expensive and technically challenging, and
requires scientists willing to take a long perspective in
order to discover, through painstaking research, which combinations
of techniques could turn out to be successful. Strong financial
support, public and private, will be indispensable to achieving
i. In this chapter, technical
terms that are defined in the Glossary are underlined when
they are used for the first time.
ii. It is also not known
whether stem cells, either human or animal, when cultured
in vitro apart from the embryonic whole from which they
were originally derived, will function in all respects like
cells do when they act as parts of a developing organic
iii. Some stem cells,
however, give rise to only one type of specialized cell.
For example, one type of stem cell found in the epidermis
(skin) apparently gives rise only to keratinoctyes (cells
that produce the protein keratin, found in hair and nails).
iv. As already noted
in Chapter 1, "adult stem cells" is something of a misnomer.
The cells are not themselves "adult." As non-embryonic stem
cells, they are, however, partially differentiated and many
of them are multipotent. (See discussion in the text that
v. Embryonic stem cell
cultures prepared from different embryos of a single inbred
mouse strain are more likely to have closely similar biological
properties than will ESC cultures from genetically different
individual human beings.
vi. The issue of possible
mouse virus contamination is dealt with in Section F, below.
vii. As of November 2003,
reports were available about the chromosome patterns of
only 21 out of the 78 ESC preparations designated as eligible
for federal funding; 11 of the 12 preparations currently
available have had their chromosome patterns characterized,
and they appear normal. However, a recent publication, presenting
results from two different laboratories, reports abnormalities
in chromosome number and structure in some samples of three
different human ESC preparations. Two of these ESC preparations
are among the twelve preparations currently available for
federal funding. [Draper, J.S., et al., "Recurrent gain
of chromosomes 17q and 12 in cultured human embryonic stem
cells," Nature Biotechnology December 7, 2003, advance
viii. The terms "stromal
stem cells," "mesenchymal stem cells," and "mesenchymal
progenitor cells" have all been used by different authors
to describe these cells.
ix. Stem cell preparations
are frequently transduced in vitro with foreign genes that,
when expressed, produce readily visualized proteins, such
as Green Fluorescent Protein (GFP).
x. In a recent review
article on adult stem cell plasticity, Raff [see (8)] discusses
the phenomenon of spontaneous cell fusion masquerading as
xi. According to published
reports, laboratories in Australia, Britain, China, India,
Iran, Israel, Japan, Korea, Singapore, Sweden and the United
States have isolated ESC preparations.
xiii. Previous work
had shown that variation in imprinted gene expression was
observed in cloned mice, and that it might be partly responsible
for their subtle genetic defects. So it was reassuring that
the pattern of imprinted gene expression appeared to be
normal in EG cells.
xiv. Normally the immune
system protects against infectious and toxic agents and
surveys for cancer cells with the intent of destroying them,
but does not attack one's own tissues. There are many other
autoimmune diseases such as multiple sclerosis and lupus
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7. Jaenisch, R.,
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Bianco, P., et al., "Bone marrow stromal cells: nature,
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180-192 (2001); Martinez-Serrano, A., et al., "Human neural
stem and progenitor cells: in vitro and in vivo properties,
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Annual Review of Cell and Developmental Biology 19:
9. Storb, R., "Allogeneic
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10. Kondo, M.,
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growth of human inner cell masses and embryonic stem cells,"
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Amit, M. and Itskovitz-Eldor, J., "Derivation and spontaneous
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M. K., et al., "Characterization and Differentiation of
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M. F. et al., "Multilineage potential of adult human mesenchymal
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M., et al., "Adult mesenchymal stem cells: Potential for
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15. Tremain, N.,
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16. Lodie, T. A.,
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18. Qi, H., et
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21. Horwitz, E.
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22. Koc, O. N.,
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24. Pagano, S.
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27. Wright, L.
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28. See, for example,
Englund, U., et al., "Transplantation of human neural progenitor
cells into the neonatal rat brain: extensive migration and
differentiation with long-distance axonal projections,"
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and integrate after intravenous transplantation in adult
rats with transient forebrain ischemia," Neuroscience
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29. See, for example,
Jeong, S., et al., "Human neural cell transplantation promotes
functional recovery in rats with experimental intracerebral
hemorrhage," Stroke 34: 2258-2263 (2003); Liker,
M., et al., "Human neural stem cell transplantation in the
MPTP-lesioned mouse," Brain Research 971: 168-177
30. Onyango, P.,
et al., "Monoallelic expression and methylation of imprinted
genes in human and mouse embryonic germ cell lineages,"
Proceedings of the National Academy of Sciences of the
United States of America 99: 10599-10604 (2002).
31. Kerr, D. A.,
et al., "Human Embryonic Germ Cell Derivatives Facilitate
Motor Recovery of Rats with Diffuse Motor Neuron Injury,"
The Journal of Neuroscience 23: 5131-5140 (2003).
L., et al., "Derivation of Human Embryonic Germ Cells: An
Alternative Source of Pluripotent Stem Cells," Stem Cells
21: 598-609 (2003).
33. Rideout, W.,
et al., "Correction of a genetic defect by nuclear transplantation
and combined cell and gene therapy," Cell 109: 17-27
(2002); Tsai, R. Y. L., et al., "Plasticity, niches and
the use of stem cells," Developmental Cell 2: 707-712
(2002); For political and legislative aspects of the debate
relative to these articles, see Daly, G., "Cloning and Stem
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34. Chen, Y., et
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39. Drukker, M.,
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40. Majumdar, M.
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41. American Diabetes
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45. Lechner, A.
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284: E259-266 (2003). The criteria that these authors
outlined were as follows:
The stem or progenitor cell should
be clonally isolated or marked; "enrichment" of a certain
cell type alone is not sufficient.
In vitro differentiation to a fully
functional beta cell should be unequivocally established.
Insulin expression per se does not make a particular cell
a beta cell. The expression of other markers of beta cells
(e.g. Pdx1/Ipf1, GLUT2, and glucokinase) or other endocrine
islet cells should be demonstrated.
Ultrastructural studies should confirm
the formation of mature endocrine cells by identification
of characteristic insulin secretory granules.
The in vitro function of endocrine
cells, differentiated from stem cells, should be reminiscent
of the natural counterparts. For beta cells, this would
imply a significant glucose-responsive insulin secretion,
adequate responses to incretin hormones and secretagogues,
and the expected electrophysiological properties.
In vivo studies in diabetic animals
should demonstrate a reproducible and durable effect of
the stem/progenitor-derived tissue on the attenuation
of the diabetic phenotype. It should also be demonstrated
that removal of the stem cell-derived graft after a certain
period of time leads to reappearance of the diabetes.
For future clinical use, the tumorigenicity
of stem/progenitor tissue should be determined.
Additionally, immune responses toward
the transplanted cells should be examined.
46. Abraham, E.
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47. Zulewski, H.,
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48. Assady, S.,
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51. For a useful
summary of the advantages and limitations of rodent models
of diabetes see: Atkinson, M. A. and Leiter, E. H., "The
NOD mouse model of type 1 diabetes: As good as it gets?"
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52. Faraci, M.,
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stem cell transplantation in children," Neurology
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53. Baker, K. S.,
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54. Pursley, W.
H., Presentation at the September 4, 2003, meeting of the
President's Council on Bioethics, Washington, D.C., available
55. Okarma, T.,
Presentation at the September 4, 2003, meeting of the President's
Council on Bioethics, Washington, D.C., available at www.bioethics.gov.
56. See presentations
from the Juvenile Diabetes Research Foundation International
and the Michael J. Fox Foundation at the September 4, 2003,
meeting of the President's Council on Bioethics, Washington,
D.C., available at www.bioethics.gov.
57. See, for example,
Carpenter, M. K., et al., "Characterization and Differentiation
of Human Embryonic Stem Cells," Cloning and Stem Cells
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potential of adult human mesenchymal stem cells," Science
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mesenchymal stem cells: Potential for muscle and tendon
regeneration and use in gene therapy," Journal of Musculoskeletal
and Neuronal Interactions 2: 309-320 (2002).
58. Daley, G. Q.,
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