Note: Descriptions are shown in the official language in which they were submitted.
CA 02320423 2000-08-10
WO 99/43785 PCT/US99/04188
DERIVl1TI01~ OF CEhhB 11~1D TISSQ88 pROl~
~HRYO~TIC pR8-BTEM C»8 ?OR TRlII~BiPIaIf~'lITION THBRlIPIEB
Background of the Invention
The present invention relates to the derivation
of cells and tissues from embryonic pre-stem cells for
transplantation therapies.
Summary of the Invention
This invention relates to the use of dispersed
morula cells in preference to inner cell mass (ICM) from
l0 blastocysts. The morula stage is the last pre-embryonic
stage without expression of any differentiation, making
these cells (pre-stem cells) all progenitors of embryonic
stem cells (ESCs) later present in blastocysts.
Conversely, the ICM from the blastocyst is already
differentiated from trophoblastic cells, which are by then
destined to become part of the placenta.
This invention also relates to the use of
chimeric introductions into pre-stem cell cultures and stem
cell propagations in culture. That is, "teacher-cells" or
spent media from them, that derived from other sources
(e.g. adults, cord blood, fetal tissues, etc. ) will "teach"
undifferentiated pre-stem cells how to convert to our
sought-after therapeutic cell population both more rapidly
and more preferentially.
This invention also relates to the identification
and use of certain early markers of stem cell
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differentiation, such as Fe++ sequestration, hemoglobin
accumulation, myeloid fibers, insulin synthesis, dopamine
loading, etc.
Other features and advantages of the present
invention will become apparent from the following
description of the invention.
Detailed Description of the Invention
The present invention provides for a method of
isolating and propagating a line of embryonic stem cells
that originates from either morulae (pre-stem) or
blastocyst (ICM stem cells). Therefore, Morula stage,
undifferentiated pre-stem cells will be used as progenitors
of stem cell populations. The propagated line of embryonic
stem cells are then used for the purpose of transplanting
cells, tissues or organs.
The propagation of stem cells can be initiated by
formation of chimeric inner cell mass cells. Chimeric ICMs
will be developed from blastocysts. From such ICMs,
superior stem cell cultures are derived. Preferably, the
formation of chimeric inner cell mass cells comprises
nuclear transplantation, mitochondrial substitution, or
cytoplasmic depletion.
Preferably, at least one regulatory factor is
used to propagate the line of embryonic stem cells. More
preferably, the regulatory factor is derived from "Teacher
cells" or "Teacher cells " spent culture medium. "Teacher
cells" will be introduced into less differentiated pre-stem
or early stage stem cells to accelerate propagation of
target stem cells. Alternatively, spent media from
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"teacher cells" can be used to accelerate the propagation
of the target stem cells.
In a preferred embodiment, the embryonic stem
cells are cultured in a medium in the presence of at least
one agent or cytokine in order to differentiate into target
specific cells or tissues. Preferably, the agent or
cytokine is selected from the group consisting of IL-1,
TNF-a, IL-6, PTH, PDGF, PGEZ, cAl~, estrogens, anti-
estrogens, progestins, anti-progestins, cortisol, GH,
androgens, I3/T3, VGEF and cyclosporin. Also preferably,
the concentration of the agent or cytokine in culture
medium is from about 1.0 pg/ml to about 10.0 ng/ml.
In another preferred embodiment, the target
specific cells are selected from the group consisting of
, nerve cells, bone cells, immune cells, and pancreatic beta
cells.
Techniques and parameters for the use of a broad
spectrum of early stage metabolic markers are developed.
Some such markers are, for example: Fe++ sequestration, Hg
accumulation, myeloid fibers, nerve growth factor,
apoptotic factors, insulin synthesis, dopamine loading,
hemoglobin loading, etc. Additionally, other early markers
of embryonic stem cells can be identified.
Specific techniques are developed to demonstrate
the foregoing. In one embodiment of the invention,
embryonic stem ( ES ) cells are derived from either morula or
blastocyst stage embryos by placing cells on fibroblast
feeder layers. The colonies are evaluated for
differentiation state using accepted markers. Further
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evaluation is done by injecting ES cells into surrogate
embryos to produce chimeras and evaluating the contribution
of the ES cells to the adult tissues. Finally, ideal
colonies of ES cells are used as nuclear donors for nuclear
transplantation.
Clonal properties of the propagated stem cells
can be achieved by adding apoptotic factors, cytokines or
other agents to the culture medium to eliminate
contaminating members of the stem cells that did not
l0 properly differentiate. Preferably, the cytokines or
agents are selected from the group consisting of IL-1, TNF-
a, IL-6, PTH, PDGF, PGE2, cAMP, estrogens, anti-estrogens,
progestins, anti-progestins, cortisol, GH, androgens, I3/T3,
vGEF and cyclosporin.
Alternatively, the propagation of the line of
embryonic stem cells is done in vivo by transplanting
"Teacher cells" inta an area sufficiently close to the
embryonic stem cells to allow for at least one regulatory
factor made by the teacher cells to contact the embryonic
cells.
The presence or absence of different
concentrations of calcium can be used to regulate the
propagation of the line of embryonic stem cells.
Preferably, the propagated line of embryonic stem cells is
grown in a three dimensional manner before being used for
transplantation.
This invention will allow for the efficient, safe
and commercially viable derivation of cells and tissues
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from embryonic pre-stem cells for transplantation
therapies. Specifically, growing-out of human blastocyats
at a rate greater than 50~ from the 2-cell stage of the
pre-embryo should be achieved. Also, efficient harvesting
of either morula stage pre-stem cells and/or stem cells
isolated from the inner cell mass of blastocysts should be
achieved. These embryonic pre-stem and stem cell
populations should preferably remain viable in culture for
more than one week.
~~ple 1
Clonal production of stem cells will be
undertaken. These clones will respond to the ambient
levels of glucose in their milieu, and in turn, insulin-
dependent diabetes would be treated by transplanting these
-stem cell lines to served by a peripheral blood supply.
the insulin secretory cells must accomplish renewal y
propagation through mitogenic proliferation.
Pluripotent stem cells will be isolated and
directed to differentiate into hemopoietic destinies.
Therefore, tissues derived from the blood cell group or
beta cells of the immune response system will be replaced
in deficient patients suffering from conditions such as HIV
infection, post-chemotherapy, or irradiation depletion.
Culture condition in vitro will direct the rate and degree
of differentiation manifested by these pluripotent stem
cells, such as the presence of "teacher cells" or certain
additives to the media, e.g. cytokines.
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The inherent capabilities of stem cells will be
modified by formation of chimeric cell lines that
incorporate "hybrid" metabolic functions that when
transplanted will provide the transplant recipient with
long-term relief from organ/tissue deficiencies. lPor
instance, the production of dopamine fn situ can modify
neurological treatments for patients manifesting muscular
rigidity and loss of motor control in disease states such
as Parkinson~s disease. Unlike pharmaceutical therapeutics
which are partially effective temporarily, transplantation
of chimeric stem cells that regulate the production
dopamine and serotonergic factors will offer these patients
superior outcomes.
The invention has been described in terms of
preferred embodiments thereof, but is more broadly
applicable as will be understood by those skilled in the
art. the scope of the invention is therefore limited only
by the following claims.
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