Research using human and animal stem cellsi
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 cells 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
Verfaillie,3
Prentice,4
Itescu5, 6
and Jaenisch7
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.
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 self-renewal or produce fully specialized or differentiated cells (see Figure 1).
Figure 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 that produce more stem
cells (self-renewal). Solid arrows indicate asymmetric cell
divisions that produce more differentiated 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 stem cells.
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
cells.v
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 undergoing
genetic changes.
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 childbirth.
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.
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 Cell Types
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 (see below).
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
at all.11
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 Cell Cloning
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 cell cloned.”12
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 13 vii 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
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
and EGC1
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 cells,10
which give rise to the red cells and white cells of the blood,
and mesenchymal stem cells,viii
which 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
preparation.15
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
types.
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
into bone,18
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 Hurler syndrome.22
These last two studies are of great interest, since allogeneic
MSCs were used and no serious adverse immune reactions were
noted.
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,
hepatocyte-like cells,23
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
into MSCs.3
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
five percent).26
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
genes,27
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 research grants.xii
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 in 1998.
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
characterized.13
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
xiii
In 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-a 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 derivatives.
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.
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.36As
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 that, once answered, will
greatly enhance our understanding of human embryonic stem
cells and their potential fates and possible uses.38
They include the following:
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 Immune Rejection?
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 Diabetes?
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
diabetic state.
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
per year.42 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 fewer 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
pancreas.
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.,46
Zulewski et al.,47
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
References |
Cell Source:
Clonally Isolated / Marked? |
Beta-cell
markers |
Ultrastructural
Examination to Ensure Endogenous Insulin Production |
Glucose-responsive
Insulin Secretion? |
In vivo
studies |
Tumorigenicity? |
| Abraham
et al, 2002 (46); Zulewski et al, 2001 (47) |
Clonally isolated adult
stem cells (derived from adult pancreatic islets) |
PDX-1 (+) |
Insulin mRNA(+); Insulin
protein (+); No ultra-structural examination |
Not assessed |
None |
Not assessed |
| Assady et al, 2001(48) |
Clonally isolated embryonic
stem cells |
PDX-1 (-); GK (+); GLUT-2 (+) |
Insulin mRNA (+) |
No |
None |
Not assessed |
| Zhao et
al, 2002 (49) |
Uncloned cadaver islets
(cultured in vitro) |
CK-19 (+) |
Preproinsulin mRNA
(+); Insulin protein (+); electron microscopy insulin secretory granuoles (+) |
Yes |
High blood glucose
concentrations reversed in STZ/SCID mice |
Not assessed |
| Zalzman
et al, 2003 50) |
Cloned fetal liver cells: immortalized with human telomerase and transduced with rat PDX-1 |
Human and rat PDX-1
(+); GK (-); GLUT-2 (-) |
Insulin mRNA (+); Insulin protein (+); No ultra- structural examination |
Yes |
High blood glucose
concentrations reversed in STZ/NOD-SCID mice; high blood
glucose returned upon graft removal |
No tumors at 3 months
after transplantation |
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) mice.
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 Cells (MSCs)
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 in 2004.54
D. Evaluating the Different Types of Stem Cells
A major unresolved issue at present involves the therapeutic potential of human adult stem cells compared with embryonic stem cells. The answer may well be different for different diseases and for patients of different ages. For example, in treating an elderly patient with Parkinson’s Disease, the use of adult stem cells may be appropriate even if these cells may have a more limited number of cell divisions remaining. On the other hand, treating a child with Type I Diabetes, one may want to use embryonic stem cells because of their potentially greater longevity, or other factors. The only valid way to resolve these questions is by instituting rigorous therapeutic trials which test the efficacy of the different types of stem cells in treating a variety of different diseases to determine their comparative efficacy. Clearly, such trials would be a long-term endeavor, since it would take years to obtain answers to these very critical questions.
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.
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
human diseases.
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
success.
__________________
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 whole.
ii. 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 follows shortly.)
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 as of that time 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 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 online publication.]
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
cell plasticity.
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.
xii. For current information on available
and eligible ESC preparations see http://stemcells.nih.gov/registry/index.asp.
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 some forms of thyroiditis and lupus erythematosis.
__________________
1. Gearhart, J., “Human
Embyronic Germ Cells: June 2001-July 2003. The Published Record,”
Paper prepared for the President’s Council on Bioethics,
July 2003. [Appendix H]
2. Ludwig, T. E. and Thomson, J. A., “Current Progress in Human Embryonic Stem Cell Research,” Paper prepared for the President’s Council on Bioethics, July 2003. [Appendix I]
3. Verfaillie, C., “Multipotent Adult Progenitor Cells: An Update,” Paper prepared for the President’s Council on Bioethics, July 2003. [Appendix J]
4. Prentice, D., “Adult Stem Cells,” Paper prepared for the President’s Council on Bioethics, July 2003. [Appendix K]
5. Itescu, S., “Stem Cells and Tissue Regeneration: Lessons from Recipients of Solid Organ Transplantation,” Paper prepared for the President’s Council on Bioethics, June 2003. [Appendix L]
6. Itescu, S., “Potential
Use of Cellular Therapy For Patients With Heart Disease,”
Paper prepared for the President’s Council on Bioethics,
August 2003. [Appendix M]
7. Jaenisch, R., “The Biology of
Nuclear Cloning and the Potential of Embryonic Stem Cells
for Transplantation Therapy,” Paper prepared for the
President’s Council on Bioethics, July 2003. [Appendix
N]
8. See, among others, Bianco, P., et al., “Bone marrow stromal cells: nature, biology and potential applications,” Stem Cells 19: 180-192 (2001); Martinez-Serrano, A., et al., “Human neural stem and progenitor cells: in vitro and in vivo properties, and potential for gene therapy and cell replacement in the CNS,” Current Gene Therapy 1: 279-299 (2001); Nir, S., et al., “Human embryonic stem cells for cardiovascular repair,” Cardiovascular Research 58: 313-323 (2003); Raff, M., “Adult stem cell plasticity: fact or artifact?” Annual Review of Cell and Developmental Biology 19: 1-22 (2003).
9. Storb, R., “Allogeneic hematopoietic stem cell transplantation – Yesterday, today and tomorrow,” Experimental Hematology 31: 1-10 (2003).
10. Kondo, M., et al., “Biology of Hematopoietic Stem Cells and Progenitors: Implications for Clinical Application,” Annual Review of Immunology 21: 759-806 (2003) and references cited therein.
11. Xu, C., et al., “Feeder-free growth of undifferentiated human embryonic stem cells,” Nature Biotechnology 19: 971-974 (2001); Richards, M., et al., “Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells,” Nature Biotechnology 20: 933-936 (2002); Amit, M., et al., “Human Feeder Layers for Human Embryonic Stem Cells,” Biology of Reproduction 68: 2150-2156 (2003); Richards, M., et al., “Comparative Evaluation of Various Human Feeders for Prolonged Undifferentiated Growth of Human Embryonic Stem Cells,” Stem Cells 21: 546-556 (2003).
12. Amit, M., et al., “Clonally derived Human Embryonic Stem Cell Lines Maintain Pluripotency and Proliferative Potential for Prolonged Periods of Culture,” Developmental Biology 227: 271-278 (2000); Amit, M. and Itskovitz-Eldor, J., “Derivation and spontaneous differentiation of human embryonic stem cells,” Journal of Anatomy 200: 225-232 (2002).
13. Carpenter, M. K., et al., “Characterization and Differentiation of Human Embryonic Stem Cells,” Cloning and Stem Cells 5: 79-88 (2003).
14. Pittenger, M. F. et al., “Multilineage potential of adult human mesenchymal stem cells,” Science 284: 143-147 (1999); Pittenger, M., et al., “Adult mesenchymal stem cells: Potential for muscle and tendon regeneration and use in gene therapy,” Journal of Musculoskeletal and Neuronal Interactions 2: 309-320 (2002).
15. Tremain, N., et al., “MicroSAGE Analysis of 2,353 Expressed Genes in a Single-Cell Derived Colony of Undifferentiated Human Mesenchymal Stem Cells Reveals mRNAs of Multiple Cell Lineages,” Stem Cells 19: 408-418 (2001).
16. Lodie, T. A., et al., “Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction,” Tissue Engineering 8: 739-751 (2002).
17. Gronthos, S., et al., “Molecular and cellular characterization of highly purified stromal stem cells derived from bone marrow,” Journal of Cell Science 116: 1827-1835 (2003).
18. Qi, H., et al., “Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells,” Proceedings of the National Academy of Sciences of the United States of America 100: 3305-3310 (2003).
19. Cheng, L., et al., “Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture,” Stem Cells 21: 131-142 (2003).
20. Koc, O. N., et al., “Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal cells in advanced breast cancer patients receiving high-dose chemotherapy,” Journal of Clinical Oncology 18: 307-316 (2000).
21. Horwitz, E. M., et al., “Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone,” Proceedings of the National Academy of Sciences of the United States of America 99: 8932-8937 (2002).
22. Koc, O. N., et al., “Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH),” Bone Marrow Transplantation 30: 215-222 (2002).
23. Schwartz, R. E., et al., “Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells,” Journal of Clinical Investigation 109: 1291-1302 (2002).
24. Pagano, S. F., et al., “Isolation and characterization of neural stem cells from the adult human olfactory bulb,” Stem Cells 18: 295-300 (2000).
25. Liu, Z., and Martin, L. J., “Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human,” Journal of Comparative Neurology 459: 368-391 (2003).
26. Pevny, L., and Rao, M. S., “The stem-cell menagerie,” Trends in Neurosciences 26: 351-359 (2003).
27. Wright, L. S., et al., “Gene expression in human neural stem cells: effects of leukemia inhibitory factor,” Journal of Neurochemistry 86: 179-195 (2003).
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,” Experimental Neurology 173: 1-21 (2002); Chu, K., et al., “Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia,” Neuroscience Letters 343: 129-133 (2003).
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 (2003).
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).
32. Turnpenny, 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 Cells—Handicapping the Political and Scientific Debates,” New England Journal of Medicine 349: 211-212 (2003).
34. Chen, Y., et al., “Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes,” Cell Research 13: 251-264 (2003).
35. Xu, R. H., et al., “BMP4 initiates human embryonic cell differentiation to trophoblast,” Nature Biotechnology 20: 1261-1264 (2002).
36. Rohwedel, J., et al., “Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity, and embryotoxicity studies: present state and future prospects,” Toxicology In Vitro 15: 741-753 (2001).
37. Sato, N., et al., “Molecular signature of human embryonic stem cells and its comparison with the mouse,” Developmental Biology 260: 404-413 (2003); Ramalho-Santos, M., et al., “‘Stemness’: Transcriptional Profiling of Embryonic and Adult Stem Cells,” Science 298: 597-600 (2002); Ivanova, N. B., et al., “A Stem Cell Molecular Signature,” Science 298: 601-604 (2002).
38. Brivanlou, A. H., et al., “Stem cells. Setting standards for human embryonic stem cells,” Science 300: 913-916 (2003).
39. Drukker, M., et al., “Characterization of the expression of MHC proteins in human embryonic stem cells,” Proceedings of the National Academy of Sciences of the United States of America 99: 9864-9869 (2002).
40. Majumdar, M. K., et al., “Characterization and functionality of cell surface molecules on human mesenchymal stem cells,” Journal of Biomedical Science 10: 228-241 (2003).
41. American Diabetes Association, “Facts and Figures,” http://diabetes.org/main/ info/facts/facts.jsp (accessed June 23, 2003).
42. Hogan, P., et al., “Economic Costs of Diabetes in the US in 2002,” Diabetes Care 26: 917-932 (2003).
43. Ryan, E. A., et al., “Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol,” Diabetes 50: 710-719 (2001).
44. Soria, B., et al., “Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice,” Diabetes 49: 157-162 (2000); Lumelsky, N., et al., “Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets,” Science 292: 1389-1394 (2001); Hori, Y., et al., “Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells,” Proceedings of the National Academy of Sciences of the United States of America 99: 16105-16110 (2002).
45. Lechner, A. and Habener, J. F., “Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus,” American Journal of Physiology - Endocrinology and Metabolism 284: E259-266 (2003). The criteria that these authors outlined were as follows:
46. Abraham, E. J., et al., “Insulinotropic hormone glucagons-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells,” Endocrinology 143: 3152-3161 (2002).
47. Zulewski, H., et al., “Multipotential Nestin-Positive Stem Cells Isolated From Adult Pancreatic Islets Differentiate Ex Vivo Into Pancreatic Endocrine, Exocrine and Hepatic Phenotypes,” Diabetes 50: 521-533 (2001).
48. Assady, S., et al., “Insulin production by human embryonic stem cells,” Diabetes 50: 1691-1697 (2001).
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50. Zalzman, M., et al., “Reversal of hyperglycemia in mice using human expandable insulin-producing cells differentiated from fetal liver cells,” Proceedings of the National Academy of Sciences of the United States of America 100: 7253-7258 (2003).
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?” Nature Medicine 5: 601-604 (1999).
52. Faraci, M., et al., “Severe neurologic complications after hematopoietic stem cell transplantation in children,” Neurology 59: 1895-1904 (2002).
53. Baker, K. S., et al., “New Malignancies After Blood or Marrow Stem-Cell Transplantation in Children and Adults: Incidence and Risk Factors,” Journal of Clinical Investigation 21: 1352-1358 (2003).
54. Pursley, W. H., Presentation at the September 4, 2003, meeting of the President’s Council on Bioethics, Washington, D.C., available at www.bioethics.gov.
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 5: 79-88 (2003), and Pittenger, M. F. et al., “Multilineage potential of adult human mesenchymal stem cells,” Science 284: 143-147 (1999), and Pittenger, M. F., et al., “Adult 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., et al., “Realistic Prospects for Stem Cell Therapeutics,” Hematology American Society for Hematology Education Program: 398-418 (2003).
