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The field of regenerative medicine was born with the historic
discoveries of human embryonic stem (ES) and embryonic germ
(EG) cells (Thomson et al, 1998; Shamblott et al., 1998),
and has ignited the public imagination about the potential
of stem cell-based therapies to treat human diseases. Responsible
evaluation of the utility and limitations of stem cells
in translational animal models (e.g., mice, rats, and nonhuman
primates) and humans is critical and has become an important
area of PDC research. PDC investigators are actively studying
cellular and molecular mechanisms controlling the functions
of embryonic and adult tissue stem cells. Research is also
focused on understanding stem cell potentials, stem cell
transplantation, and discovering the role of stem cells
in aging. Biological investigation of stem cell populations
from different tissues and species will provide a critical
scientific foundation for practical, ethical and political
debates on stem cell biomedicine.
The PDC has a comprehensive teaching mission that includes
an annual hands-on training course, FRONTIERS
IN HUMAN EMBRYONIC STEM CELLS(FrHESC) that is supported
by an NIH training grant awarded to Drs. G. Schatten and
R. Pedersen.
Stem Cell Characterization
Studies are underway to characterize new and existing mouse,
rat, nonhuman primate, and human ES and EG cell lines. These
lines are subjected to a rigorous battery of tests to establish
their developmental potential, including marker analysis,
functional transplantation assays, and chimera formation.
Any setbacks in the human ES field might be attributed to
the source of cell lines, i.e. discarded embryos obtained
from infertile patients after cryopreservation. These issues
can be addressed responsibly using translational animal
models, particularly non-human primates. The PDC now has
several offspring born for which the identical ES lines
have been derived and research is ongoing. Also, because
inner cell mass cell replacement or reaggregation chimerae
are unthinkable with human ES cells, nonhuman primate investigations
are urgent. Furthermore, if nonhuman primate ES cells behave
as in mice, then in vitro modification and selection with
in vivo propagation in transgenics could be performed swiftly
(i.e. < one-year generation time vs. > five-years
in vivo) and efficiently. Finally, nuclear transfer studies
are aimed at understanding the genetic and epigenetic factors
controlling the developmental potential of stem, germ, and
somatic cell nuclei, as well as the developmental potentials
of ES vs. SCNT ES cells.
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Cell Biology Studies of ESCs
Human embryonic stem cells have great potential for cellular
therapy because they are pluripotent. Research in Dr. Paul
Sammak’s lab has demonstrated that pluripotent ESCs
from mice (mESC), humans (hESC), and non-human primates
(nhpESC) display minimal nuclear architecture and surprising
elasticity, deformability, and chromatin dynamics. In contrast,
somatic cells have no large-scale movement within the nucleus.
We have compared nuclear architecture in pluripotent and
early differentiated hESCs and found that upon differentiation:
- Lamin A/C is expressed and nucleoporins bind to the
nuclear envelope.
- Centromeres and telomeres become associated with the
nuclear periphery.
- The core nuclear pore protein Nup133 becomes bound to
mitotic centromeres
- Histone H3K9 becomes methylated at centromeres and DNA
becomes methylated on cytosine at telomeres.
- there is no overlap between methylated DNA, histone
and condensed DNA. After 10 days, methylated histone,
DNA and condensed DNA coalesce into canonical heterochromatin
and do not appear to be associated with the nuclear periphery.
Therefore, we hypothesize that the unanticipated hyperdynamic
chromatin and nuclear elasticity found in Pluripotent hESC
nuclei is a consequence of a rudimentary nuclear structure
and that chromatin stability is a consequence of anchoring
to the nuclear envelope and laminae at the initiation of
development. We also suggest that embryonic stem cell pluripotency
is characterized by plasticity within the nuclei and chromatin
of undifferentiated embryonic stem cells. Our long-term
objective is to determine how global factors including chromatin
mobility, the genesis of heterochromatin, and nuclear architecture
contribute to the global control of gene expression in pluripotent
cells and at the initiation of differentiation.
Our aims include:
- What is the comparative movement of core histones in
the nuclei?
- Are the driving components of chromatin mobility in
the nucleus and the restraining components of chromatin
mobility come from the nuclear envelope and lamina?
We hypothesize that a defining characteristic of pluripotency
is as much a novel “soft-off state”, independent
of heterochromatin, which silences genes in the pluripotent
state. This soft-off state may be dependent on global mechanisms
involving nuclear architecture and chromatin plasticity.
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Understanding Stem Cell Potentials
Embryonic stem cell (ESC) potentials are extrapolated from
murine studies, and the relatively few HESC lines available
are all derived from leftover embryos that were clinically
discarded by anonymous infertile couples. Because of their
derivations, many basic questions cannot be answered. To
bridge these gaps, the PDC is joining an international consortium
to discover fundamental and translational aspects of embryonic
(ESC) versus adult stem cells. Roger Pedersen is differentiating
in vitro the NIH approved HESC lines into hematopoietic
stem cells (HSCs). Massimo Trucco and Tom Starzl’s
teams are transplanting these HSCs into fetal rhesus to
generate primates that are immunotolerant with approved
HESCs. Later these now tolerant primates are being investigated
with transgenic HESC carrying reporters detectable by MRI,
µPET and fluorescence imaging in vivo. Essential questions
to be answered are whether HESCs, even after differentiation
and cell sorting, are indeed free of contaminating pluripotent
HESCs that might developed into teratomas or teratocarcinomas
in vivo. Our objectives are to determine prior to 2010 whether
HESCs are safe in these primates, and also whether HESCs
differentiated into dopaminergic neurons or ß-islets
cells will also behave properly in vivo. With this information,
MPTP will be used to generate Parkinson’s models and
STZ for Type I diabetic primates. These animals, again immunotolerant
for the specific approved HESC line, will be investigated
to learn of the efficacy of these HESCs for stem cell therapy.
Peter Donovan is deriving embryonic germ cells (EGCs) from
our primates at two fetal stages – just prior to,
and just after, the migration of the primordial germ cells
(PGCs) into the fetal gonad to analyze the imprinting status.
Mouse EGCs have been carefully studied by Dr. Donovan and
others; the embryonic genomic imprint remains on the PGCs
until they enter the gonad. Then all genomic imprints are
erased, with the sex-specific imprint added as gametogenesis
begins. John Gearhart, our collaboration at JHU, has generated
several fascinating human EGC lines, but because they are
the result of abortions many aspects of their development
and genetics cannot, of course, be determined. In other
studies, neonatal males are surgically modified as half
cryptorchids (i.e. one undescended testicle). In mice, the
male germ cells in this testicle develop into embryonic
carcinomas (ECCs), the first pluripotent stem cell investigated.
Having primate ESCs, EGCs and ECCs will answer questions
about primate versus murine stem cell potentials.
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Regenerative Medicine: Stem cell transplantation
Practical application of embryonic or adult tissue stem
cells in regenerative medicine will depend on the development
of effective transplantation techniques. Several factors
will need to be considered, including controlling stem cell
development to produce only the desired lineage, conditioning
the host tissue to support robust donor cell engraftment,
circumventing host immune surveillance and rejection, and
restoring tissue function. Dr. Orwig has helped to pioneer
methods for transplanting spermatogonial stem cells, the
adult stem cells of the testis, into the testes of infertile
recipients to produce spermatogenesis and restore fertility.
The spermatogonial stem cell transplantation technique provides
a biological assay for evaluating stem cell engraftment,
niche interactions and tissue development. Results from
these studies have relevance for the development of transplantation
techniques for other stem cells and other tissues.
Recent studies from our group have demonstrated that hES
cells can be differentiated into cells with midbrain DA
neuron characteristics (see preliminary data). There are
concerns that the successful treatment of (PD) with hES
derived DA neuron transplants could be hampered by poor
cell survival, loss of DA phenotype, deregulated growth
of transplanted cells, and/or immunological incompatibilities
between the transplanted cells as has been shown in preliminary
data. We propose to test the fate and function of hES
and non-human primate ES (nhpES) derived DA neurons after
transplantation into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) lesioned monkeys. Short term analysis of cell survival
and fate cannot be easily carried out in primates (due to
obvious cost and animal use concerns), therefore we have
developed imaging strategies in primates based on MRI and
microPET technology that can be used to monitor the grafted
cells non-invasively and repeatedly in individual monkeys.
Donor-recipient compatibility is an important factor that
will likely impact many of the parameters critical for DA
neuron survival in vivo. Due to our unique expertise in somatic
cell nuclear transfer in primates and its application to
murine models of PD and human and primate ES cell differentiation,
we are in a strong position to evaluate whether DA neurons
generated after somatic cell nuclear transfer from primates
will improve the survival and function of DA neuron grafts
towards a first proof-of-principle application of therapeutic
cloning in primates.
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Stem Cells and Aging
Human life expectancy has increased dramatically, particularly
in Western societies, as a result of rapid advances in modern
medicine. Unfortunately, the quality of life in the later
years can be hampered by increased frailty and diseases
associated with aging. Aging processes are evident in all
tissues, including neurological, circulatory, musculoskeletal,
immune, skin, digestive and reproductive systems. Yet, despite
the pervasive nature of aging, the mechanisms of its progression
are poorly understood. A National Institute of Aging funded
project in Drs. K. Orwig and A. Csoka’s laboratories
is designed test the hypothesis that aging results from
uncorrected defects in stem cell and/or niche function,
which lead to system failure. Using spermatogenesis as a
model system, Dr. Orwig will study correlations between
age related declines in male fertility and spermatogonial
stem cell/niche function in mice and rats. Using the spermatogonial
stem cell transplantation technique young or aged stem cells
can be removed from their cognate niches and transplanted
into the testes of young or aged recipients, thus, allowing
the dissection of stem cell vs. niche effects in aging and
fertility. Dr. Csoka’s laboratory will evaluate the
accumulation of mutations in the lamin A gene and telomere
length to gain insights into the molecular mechanisms of
stem cell aging in this system.
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