Stem cell research has been hailed for the potential
to revolutionize the future of medicine with the ability to regenerate
damaged and diseased organs. On the other hand, stem cell research
has been highly controversial due to the ethical issues concerned
with the culture and use of stem cells derived from human embryos.
This article presents an overview of what stem cells are, what roles
they play in normal processes such as development and cancer, and
how stem cells could have the potential to treat incurable diseases.
Ethical issues are not the subject of this review.1
In addition to offering unprecedented hope in treating many
debilitating diseases, stem cells have advanced our understanding
of basic biological processes. This review looks at two major
aspects of stem cells:
I. Three processes in which stem cells play a central role in an
organism, development, repair of damaged tissue, and cancer resulting
from stem cell division going awry.
II. Research and clinical applications
of cultured stem cells: this includes the types of stem cells
used, their characteristics, and the uses of stem cells in studying
biological processes, drug development and stem cell therapy;
heart disease, diabetes and Parkinson's disease are used as examples.
What are stem cells?
Stem cells are unspecialized cells that have two defining properties: the ability to differentiate into other cells and the ability to self-regenerate.
The ability to differentiate is the potential to develop
into other cell types. A totipotent stem cell (e.g.
fertilized egg) can develop into all cell types including the
embryonic membranes. A pleuripotent stem cell
can develop into cells from all three germinal layers (e.g cells
from the inner cell mass). Other cells can be oligopotent, bipotent
or unipotent depending on their ability to develop into few, two
or one other cell type(s).2
Self-regeneration is the ability of stem cells to divide and
produce more stem cells. During early development, the cell division
is symmetrical i.e. each cell divides to gives rise to daughter
cells each with the same potential. Later in development, the
cell divides asymmetrically with one of the daughter cells produced
also a stem cell and the other a more differentiated cell.
Number of cell types
Example of stem cell
Cell types resulting
Zygote (fertilized egg),
All cell types
All except cells of the
Cultured human ES cells
Cells from all three germ
skeletal muscle,cardiac muscle,
liver cells, all blood cells
5 types of blood cells (Monocytes,
macrophages, eosinophils, neutrophils, erythrocytes)
Cartilage cells, fat cells,
stromal cells, bone-forming cells
2 types of astrocytes, oligodendrocytes
Bipotential precursor from
murine fetal liver
B cells, macrophages
Mast cell precursor
cell e.g. Red blood cell
No cell division
Table 1: Differential potential
ranges from totipotent stem cells to nullipotent cells.
from information in sources shown
I. Stem cells are central to three processes in an organism:
development, repair of adult tissue and cancer.
A. Stem cells in mammalian development
The zygote is the ultimate
stem cell. It is totipotent with the ability to produce all the
cell types of the species including the trophoblast and the embryonic
membranes. Development begins when the zygote undergoes several
successive cell divisions, each resulting in a doubling of the
cell number and a reduction in the cell size. At the 32- to 64-cell
stage each cell is called a blastomere.2
The blastomeres stick together to form a tight ball of cells called
a morula. Each of these
cells retains totipotential. The next stage is the blastocyst which consists
of a hollow ball of cells; trophoblast cells along
the periphery develop into the embryonic membranes and placenta
while the inner cell mass develops into the fetus. Beyond the
blastocyst stage, development is characterized by cell migration in addition
to cell division. The gastrula is composed of three germ layers: the ectoderm,
mesoderm and endoderm. The outer layer or ectoderm gives rise to
the future nervous system and the epidermis (skin and associated
organs such as hair and nails). The middle layer or mesoderm gives rise to
the connective tissue, muscles, bones and blood, and the endoderm (inner layer)
forms the gastrointestinal tract of the future mammal.
Early in embryogenesis, some cells migrate to the primitive gonad or genital ridge. These are the precursors to the gonad of the organism and are called germinal cells. These cells are not derived from any of the three germ layers but appear to be set aside earlier.
Stem cells in late development
As development proceeds, there is a loss of potential and a gain of specialization, a process called determination. The cells of the germ layers are more specialized than the fertilized egg or the blastomere. The germ layer stem cells give rise to progenitor cells (also known as progenitors or precursor cells). For example, a cell in the endoderm gives rise to a primitive gut cell (progenitor) which can further divide to produce a liver cell (a terminally differentiated cell).
|Hierarchy of stem cells during
differentiation.2at each stage, differential potential decreases and specialization increases.
(* These are also called transit-amplifying cells)
Role of Progenitor Cells in Development
While there is consensus in the literature that a
progenitor is a partially specialized type of stem cell, there
are differences in how progenitor cell division is described.
For instance, according to one source,3
when a stem cell divides at least one of the daughter cells it
produces is also a stem cell; when a progenitor cell undergoes
cell division it produces two specialized cells. A different source,2
however, explains that a progenitor cell undergoes asymmetrical
cell division, while a stem cell undergoes symmetrical cell division.
The apparent inconsistency of these two versions illustrates the diversity and complexity of progenitor cells and their role in differentiation. This diversity is reflected in the nomenclature as well; progenitor cells are also called Transit-amplifying cells, Precursor cells, Progenitors, Lineage stem cells, and Tissue-determined stem cells.
The table below shows these complex stages:
|Early in development:
|Late in development: type 1
|Late in development: type 2
Table 2: Summarized from information in references 6 and 7.
The number of stem cells present in an adult is far fewer than the number seen in early development because most of the stem cells have differentiated and multiplied. This makes it extremely difficult to isolate stem cells from an adult organism, which is why scientists hope to use embryonic stem cells for therapy because embryonic stem cells are much easier to obtain.
B. The role of adult stem cells in tissue repair
During development, stem cells divide and produce more specialized cells. Stem cells are also present in the adult in far lesser numbers. The role of adult stem cells (also called somatic stem cells) is believed to be replacement of damaged and injured tissue. Observed in continually-replenished cells such as blood cells and skin cells, stem cells have recently been found in other tissue, such as neural tissue.
Organ regeneration has long been believed to be through
organ-specific and tissue-specific stem cells. Hematopoietic stem
cells were believed to replenish blood cells, stem cells of the
gut to replace cells of the gut and so on. Recently, using cell
lineage tracking, stem cells from one organ have been discovered
that divide to form cells of another organ. Hematopoietic stem
cells can give rise to liver, brain and kidney cells. This plasticity of adult stem
cells has been observed not only under experimental conditions,
but also in people who have received bone marrow transplants.4
Tissue regeneration is achieved by two mechanisms:
(1) Circulating stem cells divide and differentiate under appropriate signaling by cytokines and growth factors, e.g. blood cells; and (2) Differentiated cells which are capable of division can also self-repair, e.g. hepatocytes, endothelial cells, smooth muscle cells, keratinocytes and fibroblasts. These fully differentiated cells are limited to local repair. For more extensive repair, stem cells are maintained in the quiescent state, and can then be activated and mobilized to the required site.5
For wound healing in the skin, epidermal stem cells and bone-marrow
progenitor cells both contribute.6
Thus it is likely that organ-specific progenitors and hematopoietic
stem cells are involved in repair, even for other organ repair.
Fundamental remaining questions regarding adult stem cells include: Does one common type of stem cell migrate to different organs and repair tissue or are there multiple types of stem cells? Does every organ have stem cells (some of which have not yet been discovered)? Are the stem cells programmed to divide a finite number of times or do they have unlimited cell proliferation capacity?
C. Role of stem cells in cancer
Ontogeny (development of an organism) and oncology (cancer development) share many common features.
From the 1870s the connection between development and cancer has been reported for various types of cancers. Existence of "cancer stem cells" with aberrant cell division has also been reported more recently. The connection between cancer and development is clearly evident in teratocarcinomas.
As early as 1862, Virchow discovered that the germ cell tumor teratocarcinoma is made up of embryonic cells. In 1970, Stevens derived embryonal carcinoma cells from teratocarcinomas. A teratocarcinoma is a spontaneous tumor of germ cells that resembles development gone awry. This tumor may contain several types of epithelia: areas of bone, cartilage, muscle, fat, hair, yolk sac, and placenta. These specialized tissues are often adjacent to an area of rapidly dividing unspecialized cells. The teratocarcinomas are able to differentiate into normal mature cells when transplanted into another animal. This alternation between developmental and tumor cells status demonstrates how closely development and cancer are related.
McCulloch explored the connection between normal development
of blood cells and leukemia.7
According to him, normal hematopoietic development requires the
interaction of stem cell factor with
its receptor, c-kit. A hierarchy of stem and progenitor cells
differentiates and produces different sublineages of cells resulting
from response to varied growth factors. Malignancies of the hematopoietic
system originate from two sources: those with an increased growth
in an early stem cell produce acute leukemia, while those that
arise from a decreased response to death or differentiation in
a stem cell produce chronic leukemia.
The present-day challenge is to decode the common molecular mechanism and genes involved in self-renewal for cancer cells and stem cells.8
II. Stem cells used in research and clinical
Rao and colleagues postulate that all stem cells, regardless
of their origin, share common properties.9
These researchers have reviewed the literature for candidate "stemness"
genes. They conclude that there are a set of candidate genes that
are present in all stem cells and can serve as universal markers
for stem cells. These code for proteins are involved in self-renewal
and differentiation. In addition they predict some differences
in gene expression between different populations of stem cells.
A. Types and characteristics of stem cells for culture:
Embryonic stem (ES) cells are obtained from the inner cell mass and cultured as illustrated:
ES cells from mouse embryos have been cultured since the 1980s by
various groups of researchers working independently.10
These pioneers established murine embryonic stem cells lines that
could differentiate into several different cell types.11
ES cell lines have been established from other mammals (hamsters,
rats, pigs, and cows). Thompson and colleagues at the University
of Wisconsin reported isolation of primate ES cells in 1995 and
human ES cells in 1998.12
ES cells are the best characterized of all the cultured stem
cells. Properties of ES cells:13
ES cells are pleuripotent, i.e. they have the ability to differentiate
into cells derived from all three germ layers, but not the embryonic
(ii) ES cells are immortal i.e. cells proliferate in
culture and have been maintained in culture for several hundred
doublings. The advantage of maintaining stem cells in culture
is that they are a source of a large number of cells in the undifferentiated
state. So far other adult stem cells have not been maintained
(iii) ES cells maintain a normal karyotype (there are no
major structural changes in the chromosomes)
(iv) ES cells display
Oct-4 protein and other unique markers on the cell surface.
Generally, ES cells are maintained in culture on feeder cells (mouse fibroblast cells) There have been recent reports of ES cultured on feeder cell-free medium.14
ES cells can be induced to differentiate in vitro by culturing in suspension to form three-dimensional cell aggregates called embryoid bodies (EBs).15 The cells spontaneously differentiate into various cell types, e.g. neurons, cardiomyocytes, and pancreatic beta cells. The addition of growth factors to the culture directs differentiation to specific cell types. However, it is still challenging to isolate pure differentiated cell types.
Following injection of ES cells into immunodeficient mice, teratomas develop with derivatives of all three germ layers. This is a major disadvantage of using ES cells for cell therapy since any contaminating undifferentiated cells could give rise to cancer.
Embryonic germ cells
Gearhart and colleagues originally derived stem cells from primordial germ cells.16 Cells cultured from the genital ridge of the human embryo have been isolated and cultured. These cells have a lesser capacity of proliferation than ES cells but have an advantage in that they are not tumorigenic, unlike ES cells.17
Embryonal carcinoma cells
Embryonal carcinoma cell lines were first developed in 1967 by Ephrussi and colleagues from mouse teratomas, followed in 1975 by Fogh and Tempe from a human testicular teratocarcinoma. These cells are malignant relatives of ES and EG cells, which were used in many of the techniques to cultivate them. EC cells can differentiate under the right conditions and have a potential to be used for research and perhaps clinical applications.18 Once they differentiate they would not be expected to cause cancer, but these cells have not been studied as well as ES cells and are of limited use at present.
Adult or somatic stem cells The existence of hematopoietic
stem cells was discovered in the 1960s, followed by the discovery
of stromal cells (also called
mesenchymal cells). Only in the 1990s did scientists confirm the
reports of neural stem cells in mammalian brains. Since then stem
cells have been found in the epidermis, liver and several other
Figure 4: Hematopoietic and Stromal stem cell differentiation
Adult stem cells offer hope for cell therapy to treat diseases in the future because ethical issues do not impede their use. In addition, if the patient's own cells are used, immunological compatibility is not an issue. However, ES cells have been found to be superior for both differentiation potential and ability to divide in culture.
Two concepts are useful to describe characteristics of adult stem cells:
Plasticity is a newly recognized ability of stem cells to expand their potential beyond the tissue from which they are derived. For example, Dental pulp stem cells develop into tissue of the teeth but can also develop into neural tissue.20
Transdifferentiation is the direct conversion of one cell type to another,21 e.g. transdifferentiation of pancreatic cells into hepatic cells and vice versa has been reported in both animals and humans as has the transdifferentiation of blood cells into brain cells and vice versa.22
Cell fusion: ES cells can fuse in vitro with neuronal cells and with hematopoietic stem cells.17 This has started a new debate in adult stem cell plasticity, namely that some cells may have fused and the nucleus was reprogrammed instead of transdifferentiating.
Cord blood stem cells
Cord blood, from the umbilical cord, was believed to be an alternate source of hematopoietic stem cells; however, it is impossible to obtain sufficient numbers of stem cells from most cord blood collections to engraft an adult of average weight. Development continues on techniques to increase the number of these cells ex vivo. Cord blood contains both hematopoietic and non-hematopoietic stem cells.23
B. Research and Clinical Applications of Cultured Stem Cells
What are the uses of Cultured Stem cells? The most prominent is cell therapy for treating conditions such as spinal cord injuries and for curing disease. Stem cells are used to investigate questions to further basic and clinical research. Here are the major applications to date:
The identification of hematopoietic stem cells in mice by Till and McCulloch in 1961 heralded the use of stem cell therapy.29 By 1999, 50 diseases had been treated by bone marrow and stem cell therapy with varying degrees of success,30 among them leukemia, breast cancer, inflammatory bowel disease and osteogenesis imperfecta (a bone disease) in humans. ES and adult stem cells now offer hope for reversing the symptoms of many diseases and conditions including cancer, neurodegenerative diseases, spinal cord injuries, and heart disease.
- Functional Genomic studies
In 1986, Gossler et al. reported using mouse ES cells to produce transgenic animals.24 Soon after, two landmark papers in the field of mouse genetics demonstrated the ability to manipulate a specific gene of ES cells.25 Combining these techniques, a specific gene can be introduced into ES cells to produce transgenic mice. This gene can be transmitted to their offspring through the germline. Today these techniques enable the study of the function of mammalian genes and proteins in the mouse (through introducing human histocompatibility genes into mice).26
- Study of biological processes
Studies of biological processes, namely development of the organism and progress of cancer, are facilitated by the ability to trace stem cell fate. The spleen colony assay developed by Till and McCulloch is an example study of the development of blood cells. In this method single cells were injected into heavily irradiated mice so that all the hematopoietic cells in these mice originated from the original colony. Studies of this nature helped decipher the clonal origin of cancer,
- Drug discovery and development
The combination of isolation and purification of mouse ES cells and genetic engineering techniques has led to the use of murine ES cells in drug discovery. With the sequencing of the human genome many potential targets of new drugs have been identified. Studies using human ES may follow those of murine ES cells.27 Interest in using stem cells as models for toxicology has also grown recently.28
- Cell-based therapy
Cultured ES cells spontaneously form embryoid bodies containing different cell types from all three germ layers. The desired cells are isolated and cultured and the differentiated cells are then used for therapy. ES cells have been induced to differentiate into neurons, cardiomyocytes and endoderm cells.
The following stem cell characteristics make them good candidates for cell-based therapies:31
i. Potential to be harvested from patients
Following is a summary of three diseases in which stem cell-based therapy has been used.
ii. High capacity of cell proliferation in culture to obtain large number of cells from a limited source
iii. Ease of manipulation to replace existing non
functional genes via gene transfer methods
iv. Ability to migrate to host's target tissues, e.g.
v. Ability to integrate into host tissue and interact with surrounding tissue
a) Heart disease
Cardiovascular disease is a leading cause of death worldwide killing 17 million people each year,32 especially due to heart attack and stroke. In the United States, heart disease is the number one cause of death. The high rate of mortality associated with heart diseases is the inability to repair damaged tissue33 due to the full differentiation of heart tissue. Interruption of blood supply to the tissue causes infarction of the myocardium and death of myocardiocytes.
A recent report used a swine model of atrioventricular block and transplanted human ES cell-derived cardiomyocytes into the pig's heart to work as a pacemaker.34 The ES cells survived, functioned and integrated well with the host cells. The researchers used embryoid bodies to select spontaneously beating areas of culture (cultured myocytes will actually beat in synchrony just like a heartbeat). This study bodes well for future myocardial regeneration using human ES cells.
Adult stem cells have also been used in cell therapy for the heart.35 Skeletal muscle myoblast transfers showed contraction but did not differentiate into cardiomyocytes and did not integrate with the host myocardium. Ideally, both contraction and integration with host myocardium should have occurred in order for the therapy to be effective. Endothelial progenitor cells transplants halted the degenerative process but did not initiate regeneration. Early clinical studies may soon follow.
Another approach is cardiac tissue engineering.36 Cohen and Leor grew embryonal heart cells in vitro with an alginate scaffold (alginate is an algal polysaccharide) to provide 3D-support and organization for the cells. They transplanted the cells with the scaffold into the scar tissue of the rats with myocardial infarction and observed extensively. The vascularization shows that there was acceptance of the engineered tissue. This unique method of treating heart disease is promising and may be explored in other animal models in the future.
Elevated glucose levels in the blood are responsible for diabetes. Diabetes affects 16 million Americans (5.9 percent of the population) and is the seventh leading cause of death.37 Worldwide it afflicts 120 million people and the World Health Organization estimates that the number will reach 300 million by 2025.38
Type I diabetes, or juvenile onset diabetes, is an autoimmune disease that causes destruction of the insulin-producing beta cells in the pancreas. Insulin injections are given to diabetics but they cause surges in blood glucose levels followed by a drop in the glucose levels and lack fine tuning. Pancreas transplantation has been performed in diabetics as more recently has pancreatic islet cell transplantation. The latter has the advantages that it does not require whole organ transplantation. However, the need for immunosuppression to prevent rejection of allogeneic islet transplants and a serious shortage of organ donors are lingering problems.25 The Edmonton protocol, developed by Shapiro and colleagues, is promising. This procedure transplants a large amount of islet cells and uses a glucocorticoid-free type of immunosuppression regimen. In early clinical testing it reversed diabetes in all of the patients tested.
c) Stem cell therapy for diabetes
Cells need to be able to self-regenerate and differentiate. Also it has been observed that the presence of all the islet cell types is preferable to only beta cells since the former are better able to respond to changing levels of glucose in the blood. Growth must be balanced with ability to produce insulin. The insulin producing cells tend not to divide and those which divide actively do not produce insulin.
Adult stem cells from the pancreas have been elusive so far. However, a recent report of a clone from mouse pancreas that can generate both pancreatic and neural cell lines is exciting, as is a second report that adult small hepatocytes (liver cells) can be induced to produce insulin.39 Both reports offer hope for using adult stem cells as a treatment and cure for diabetes.
d) Parkinson's Disease
Parkinson's disease is the second
most common neurodegenerative disease following Alzheimer's.
Approximately 1.5 million people in the United States suffer
from Parkinson's disease,40
which is caused when 80% or more of dopamine producing-neurons
in the substantia nigra of the brain die. Normally, dopamine
is secreted from the substantia nigra and transmitted to another
part of the midbrain. This allows body movements to be smooth
Patients with Parkinson's disease are treated with the drug levodopa (or L-dopa), which is converted to dopamine in the body. Initially effective, the treatment's success is reduced over time and side effects increase, leaving the patient helpless.41
It has been recognized that dopamine-producing cells are required to reverse Parkinson's disease. Since the 1970s, many types of dopamine-producing cells have been used for transplantation. These include adrenal glands from the patient, human fetal tissue and fetal tissue from pigs.42 Limited success has been achieved with these cells. Rat and monkey models of Parkinson's were used to test fetal mesencephalic cells.41 Success with animal models led to clinical trials.
Fetal tissue transplantation has been performed in 350 patients, including trials using pig fetal tissue. So far, the success of reversing Parkinson's disease using fetal tissue has been limited at best. However, in the most successful cases, patients have been able to lead an independent life without L-dopa treatment.43 The limitations include (i) lack of sufficient tissue for the number of patients in need, (ii) variation in results between patients ranging from no benefit to reversal of symptoms, and
(iii) Occurrence of uncontrolled flailing movements (called dyskinesias).
The many criteria for the cells used in therapy include the ability to produce dopamine, to divide and survive in the brain and to integrate into the host brain. For these reasons, differentiated embryonic stem cells offer more promise. Mouse ES cells have been used in rat models of Parkinson's disease and recently human ES cells have been reported to differentiate into dopamine-producing neurons in culture.44
Another consideration is the immune problem. It was believed that the brain is an immunologically privileged site tolerating transplanted cells from a different individual (meaning that the immune system will not attack tissue transplanted into this location). However, a recent report challenges this view.45 For this reason autologous cells may offer a safer alternative. Neural stem cells and hematopoietic stem cells are both likely candidates.31 Also, dental pulp cells in both rats and humans produce neurotrophic factors and are a candidate for autologous transplantation in Parkinson's.20
5) Therapeutic cloning
Somatic cell nuclear transfer
was used to clone Dolly, the sheep.42
Since then, seven animal species have been cloned using this technique.44
A modified version for use in humans is as follows: The patient's
DNA is injected into an enucleated unfertilized egg and used to
generate ES cells which are then cultured and allowed to differentiate,
followed by transplantation into the patient. This technique is
called therapeutic cloning. The use of such cells may bypass the
ethical objections and immunological issues of using ES cells
and is the future of stem-cell clinical application.
|Figure 5: Stem Cell Transplant Using a Patient's Own Cells|
This review has summarized the role of stem cells in basic biological processes in vivo, namely in development, tissue repair and cancer in Part I. Part II focused on cultured stem cells and their uses, describing the different sources of stem cells, their properties and their research uses and clinical applications.
Remarkable progress has been achieved in studying stem cells. The most exciting use of cultured stem cells is the promise for curing many devastating diseases like Parkinson's and diabetes. However, more basic research remains before stem-cell based therapy is widely used.
Of the stem cells discussed, ES cells have the most capacity to differentiate into a variety of cells and their proliferation capacity is also unsurpassed by any other cell type. There are three major problems with ES cells; ethical issues, immunological rejection problems and the potential of developing teratomas.
In the future, ideally, somatic stem cells from the patient will be extracted and manipulated and then reintroduced into the same patient to cure debilitating diseases. This would preclude the use of embryonic stem cells for cell therapy, eliminate the ethical objections against stem cell research, and also resolve immunological rejection problems. However, at present the cell proliferation and differentiation potential of embryonic stem cells remains far more likely to produce a cure than do the somatic cells.
- For ethical issues and stem cell research refer to http://stemcells.nih.gov/info/ethics.asp; http://athome.harvard.edu/programs/psc/index.html; http://www.aaas.org/spp/sfrl/projects/stem/main.htm
Ethical Issues Associated with Pluripotent Stem Cells. Human Embryonic Stem Cells (2003) ed. by Chiu A.Y., Rao, M.S, 3-25.
- Sell, S. (2004) Stem cells. Stem Cell Handbook ed. by Sell, S. 1-18.
- Forbes, S.J., Vig, P., Poulsom, R., Wright, N.A., Alison, M.R. (2002) Adult Stem Cell Plasticity: New Pathways of Tissue Regeneration become Visible. Clin. Sci. 103, 355-369.
- Asahara T., Isner, J.M. (2004) Endothelial Progenitor Cells. Stem Cell Handbook ed. by Sell, S. 221-227.
- Lindblad, W.J. (2004) Stem cells in Dermal Wound Healing. Stem Cell Handbook ed. by Sell, S. 101-105.
- McCulloch, E.A. (2004) Normal and Leukemic Hematopietic Stem cells and Lineages. Stem Cell Handbook ed. by Sell, S. 119-131.
- Tsai, R.Y.L. (2004) A Molecular View of Stem Cell and Cancer Cell Self-renewal. Intl. J. Biochem. Cell Biol. 36, 684-694.
- Cai, J., Weiss M.L., Rao, M.S. (2004) In Search of "stemness". Exp. Hematol. 32, 585-598.
- Roach, M.L., McNeish, J.D. (2002) Methods for the Isolation and Maintenance of Murine Embryonic Stem Cells. Embryonic Stem Cells Methods and Protocols ed. by Turksen K. 1-16.
- Evans, M.J., Kaufman, M.H. (1981) Establishment in Culture of Pluripotenial Cells from Mouse Embryos. Nature 292, 154-156; Axelrod, H.R. (1984) Embryonic Stem Cell Lines Derived from Blastocysts by a Simplified Technique. Dev. Biol. 101, 225-228; Wobus, A.M., Holzhausen H., Jakel, P., Schneich, J. (1984) Characterization of a Pluripotent Stem Cell Line Derived from a Mouse Embryo. Exp. Cell Res. 152, 212-219; Doetschman, T.C. Eistattaer, H., Katz, M., Schmidt, W., and Kemler, R. (1985) The in vitro development of Blastocyst Derived Embryonic Stem Cell Lines: formation of Yolk Sac, Blood Islands and Myocardium. J. Embryol. Exp. Morphol. 87, 27-45.
- Thompson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A., Hearn, J.P. (1995) Isolation of a Primate Embryonic Stem Cell Line. Proc. Natl. Acad. Sci. USA 86, 7844-7848; Thomson, J.A, Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshal, V.S., Jones, J.M. (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282, 1145-1147.
- Amit, M., Segev, H., Manor, D., Itskovitz-Eldor, J. (2003) Subcloning and Alternative Methods for the Derivation and Culture of Human Embryonic Stem Cells. Human Embyronic Stem Cells ed. by Chiu, M., Rao, M.S. 127-141.
- Carpenter, M.K., Xu, C., Daigh, C.A., Antosiewicz, J.E., Thomson, J.A. (2003) Protocols for the Isolation and Maintenance of Human Embryonic Stem Cells. Human Embyronic Stem Cells ed. by Chiu, M., Rao, M.S.
- Drukker M., Benvenisty, N. (2003) Genetic Manipulation of Human Embryonic Stem Cells. Human Embryonic Stem Cells ed. by Chiu, A.Y., Rao, M.S. 265-284.
- Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G. R., Gearhart J.D., (1998) Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ CellS. Proc. Natl. Acad. Sci.USA 95, 13726-13731.
- Doyonnas, R., Blau, H.M. (2004) What is the Future of Stem Cell Research? Stem Cell Handbook ed. by Sell, S. 491-499.
- Draper, J.S. Moore, H., Andrews, P.W. (2003) Embryonal Carcinoma Cells. Human Embryonic Stem Cells ed. Chiu, A. Y., Rao, M.S. 63-87.
- Adult Stem Cells ed. Turksen, K. (2004)
For reviews of hematopoietic stem cells: http://stemcells.nih.gov/info/scireport/chapter5.asp; http://www.stemcell.com/technical/Hema%20SC%20MiniReview.pdf;
for mesenchymal stem cells: http://www.stemcell.com/technical/MSC%20MiniReview.pdf; for neural stem cells: http://www.stemcell.com/technical/Neurocult%20MiniReview.pdf
- Nosrat, I.V., Smith, C. A., Mullally, P., Olson, L., Nosrat C.A. (2004) Dental Pulp Cells Provide Neurotrophic Support for Dopaminergic Neurons and Differentiate into Neurons in vitro; implications for Tissue Engineering and Repair in the Nervous System. Eur. J. of Neurosci. 19, 2388-2398.
- Shen, C-N., Horb, M.E., Slack, J.M.W., Tosh,D. (2003) Transdifferentiation of Pancreas to Liver. Mech. Dev.120, 107-116.
- Priller, J. (2004) From Marrow to Brain. Adult Stem Cells ed. by Turksen, K. 215-233.
- de Wynter, E.A. (2003) What is the future of Cord blood stem cells? Cytotech. 41, 133-138.
- Gossler, A., Doetschman, T.C., Eistattaer, H., Katz, M., Schmidt, W., Kemler, R. (1986) Transgenesis by means of Blastocyst Derived Embryonic Stem Cell Lines. Proc. Natl. Acad. Sci. USA 83, 9065-9069.
- Thomas, K.R., Capecchi, M.R. (1987) Site-directed Mutagenesis by Gene Targeting in Mouse Embryo-derived Stem Cells. Cell 51, 503-512.; Koller, B.H., Hageman, L.J., Doetschman, T.C., Hagaman, J.R., Huang, S., Williams, P.J., et. al. (1989) Proc. Natl. Acad. Sci. USA 86, 8924-8931.
- For review: Floss,T., Wurst, W. (2002) Functional Genomics by Gene-trapping in ES cells. Embryonic Stem Cells Methods and Protocols ed. by Turksen, K. 347-379.
- McNeish, J. (2004) Embryonic Stem Cells in Drug Discovery Nat. Rev. Drug Discov. 3, 70-80.
- Davila, J.C., Cezar, G.G., Thiede, M., Strom, S., Miki, T., Trosko J. (2004) Use and Application of Stem Cells in Toxicology. Toxicol. Sci. 79, 214-223.
- Till, J.E., McCulloch, E.A. (1961) A Direct Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells. Radiat. Res. 14, 2213-222.
- Thomas, E.D. (1999) Bone Marrow Transplantation: a Review. Semin. Hematol. 36, 95-103.
- Barker, R.A., Jain, M., Armstrong, R.J.E., Caldwell, M.A. (2003) Stem Cells and Neurological Disease. J. Neurol. Neurosurg. Psychiat. 74, 553-557.
- Jackson, K.A., Goodell, M.A. (2004) Generation and Stem Cell Repair of Cardiac Tissue. Stem Cell Handbook, edited by Sell, S. 259-266.
- Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G., Huber, I., Satin, J., Itskovitz-Eldor, J., Gepstein, L. (2004) Electromechanical Integration of Cardiomyocytes Derived from Human Embryonic Stem Cells . Nature Biotechnol. 22, 1282-1289.
- Fraser, J.K., Schreiber, R.E., Zuk, P.A., Hedrick, M.H. (2004) Adult Stem Cell Therapy for the Heart. Intl. J. Biochem. Cell Biol. 36, 658-666.
- Cohen, S., Leor, J. (2004) Rebuilding Broken Hearts. Scientific American Nov. 2004, 45-51.
- http://stemcells.nih.gov/info/scireport/chapter7.asp; Street, C.N., Sipione, S., Helms, L., Binette, T., Rajotte, R.V., Bleackley, R.C., Korbutt, G.S. (2004) Stem Cell-based Approaches to Solving the Problem of Tissue Supply for Islet Transplantation in Type I Diabetes. Intl. J. Biochem. Cell Biol. 36, 667-683.
- Bouwens, L. (2004) Islet Cells. Stem Cell Handbook ed. by Sell, S. 429-438.
- Seaberg, R.M., Smukler, S.R., Kieeffer, T.J., Enikolopov, G., Asghar, Z., Wheeler M.B., Korbutt, G., van der Kooy, D. (2004) Clonal Identification of Multipotent Precursors from Adult Mouse Pancreas that Generate Neural and Pancreatic Lineages. Nat. Biotechnol. 22, 1115-1124.; SeNakajima-Nagata, N., Sakurai, T., Mitaka, T., Katakai, T., Yamaot, E., Miyazaki, J., Tabata, Y., Sugai, M., Shimzu, A.. (2004) In vitro Induction of Adult Hepatic Progenitor Cells into Insulin-producing Cells. Biochem. Biophys. Res. Commun. 318, 625-630.
- Baier, P.C., Schindehutte HJ., Thinane, K., Flugge G., Fuchs, E., Mansouri, A., Paulus, W., Gruss, P.,Trenwalder, C.(2004) Behavioral Changes in Unilaterally 6-Hydroxy-Dopamine Lesioned Rats after Transplantation of Differentiated Mouse Embryonic Stem Cells without Morphological Integration. Stem Cells 22, 396-404.
- Lindvall O., Bjorklund, A. (2004) Cell Therapy in Parkinson's Disease. NeuroRx. 1, 382-393.
- Zheng, X., Cai, J., Chen, J., Luo, Y., Zhi-Bing Y., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., Chen, G-J., Rao, M.S., Freed. W.J. (2004) Dopaminergic Differentiation of Human Embryonic Stem Cells. Stem Cells 22, 925-940; Wilmut, I., Paterson, L.A. (2004) Stem cells and Cloning. Stem Cell Handbook ed. by Sell, S. 75-80.
- Barker, R.A., Widner, H. (2004). Immune Problems in the Central Nervous System Cell Therapy. NeuroRx. 1, 472-481.