Note: Descriptions are shown in the official language in which they were submitted.
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SPORE-LIKE CELLS AND USES THEREOF
Field of the Invention
The invention relates to compositions and methods for tissue engineering
and cell therapies.
Background of the Invention
The relatively new field of tissue engineering has provided alternatives to
many previously tried methods for restoring tissue function. The field is an
interdisciplinary one that applies, primarily, engineering and life science
principles to
develop biological substitutes that maintain, improve, or restore tissue
function
(Tissue Engineering, R. Skalak and C.F. Fox, Eds., Alan R. Liss, New York, NY,
1988; Nerem, Ann. Biomed. Eng. 19:529, 1991).
Tissue engineers have used three general strategies to create new tissue.
The first strategy employs isolated cells or cell substitutes. This approach
avoids
many of the complications of surgery (e.g., complications that arise following
organ
transplantation), allows replacement of only those cells that supply the
needed
function, and permits manipulation of cells before they are administered to a
patient.
However, the cells used do not always maintain their function in the recipient
and can
evoke an immune response that results in their destruction.
The second strategy employs tissue-inducing substances. For this
approach to succeed, appropriate signal molecules, such as growth factors,
must be
purified and appropriately targeted to the affected tissue. While there is
some
understanding of how particular cells respond to particular growth factors, it
is not
currently possible to regulate, through extrinsic application of signaling
molecules,
the growth and differentiation of each cell type and to orchestrate the
formation of
three-dimensional organ structures.
The third strategy employs cells placed on or within matrices. In closed
systems, these cells are isolated from the body by a membrane that is
permeable to
nutrients and wastes, but impermeable to harmful agents such as antibodies and
immune cells. Closed systems can be implanted or used as extra-corporeal
devices.
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In contrast, in open systems, cell-containing matrices are implanted and
become
incorporated into the body. The matrices are fashioned from natural materials
such as
collagen or from synthetic polymers. The risk of immunological rejection is
lessened
by systemic administration of immunosuppressive drags or by the use of
autologous
cells. Of course, immune suppression places the patient at substantial risk
(e.g., the
patient is at risk of contracting infectious diseases), and it can be
extremely difficult to
obtain the requisite number of autologous cells.
Summary of the Invention
The present invention is based on the discovery of highly undifferentiated
cells called spore-like cells, which can be isolated from many different
tissues and
bodily fluids and used to treat a wide variety of disorders. For example,
spore-like
cells can be used to reengineer damaged or diseased tissue, to augment
existing tissue,
to create new tissue, or to otherwise improve the condition of a patient who
is
suffering from a disorder that is amenable to treatment by a cell- or gene-
based
therapy.
Accordingly, the invention features an isolated spore-like cell that, when
first isolated,
is multipotent, less than one to approximately seven microns in diameter, and
tolerant
of oxygen deprivation. A spore-like cell is "first isolated" when it is
separated from
substantially all of the differentiated cells with which it is naturally
associated in an
organism for a period of time less than about four hours. Of course, spore-
like cells
can retain some or all of their unique features for longer periods of time
depending on
the culturing conditions, which can be selected to induce or inhibit
differentiation
and/or proliferation. For example, spore-like cells can remain tolerant of
oxygen
deprivation for more than four hours in culture. The cells of the invention
can have
one or more areas of high contrast when viewed by transmission electron
microscopy
(i.e., areas of high contrast similar in appearance to the areas of contrast
in the spore-
like cell shown in Figs. 2C and 2D). Moreover, isolated spore-like cells can
remain
viable following exposure for more than ten minutes to an environment that is
at least
42 C or less than 0 C. Cells that are tolerant of such extreme conditions can
also have
one or more areas of high contrast when viewed by transmission electron
microscopy.
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Any of the spore-like cells of the invention can be less than one (e.g., one-
tenth to one-third of a micron) to approximately seven (e.g., one to seven or
one to
three) microns in diameter and isolated from a post-natal animal (e.g., an
adult animal
such as an adult human). The cells can also be isolated from a deceased animal
(e.g. a
deceased human). Spore-like cells are widely distributed within these sources
and can
be isolated from a tissue that develops from the endoderm, mesoderm, or
ectoderm.
At least about half the volume of the cell can be comprised of nucleic acids
and, when
first isolated, the cell can fail to express the protein nestin when analyzed
by
immunocytochemistry. A spore-like cell fails to express the protein nestin
when any
apparent binding of an anti-nestin antibody to a spore-like cell is not
significantly
greater than that observed in a negatively controlled experiment. For example,
a
spore-like cell fails to express nestin when the signal generated by an anti-
nestin
antibody is not statistically significantly greater than the signal generated
when no
primary antibody is included in the reaction or when the primary antibody has
been
inactivated. Of course, the reaction must be carried out under conditions in
which
nestin would be bound if it were present. In other words, there must be an
effective
positive control. For example, the reaction must be carried out under
conditions in
which known nestin-positive cells are bound by an anti-nestin antibody.
Any of the spore-like cells of the invention, when isolated from a post-natal
mammal and placed in a damaged, infected, or malfunctioning tissue, can
develop
into a cell having a phenotype substantially similar to the phenotype of a
healthy cell
normally found in the tissue. A spore-like cell develops into a cell that has
a
phenotype substantially similar to the phenotype of a healthy cell when the
spore-like
cell develops into a cell that expresses a protein that is deficient in the
animal or tissue
into which the spore-like cell is placed. For example, the phenotype is
"substantially
similar" when a cell that develops from a spore-like cell expresses alpha-
galactosidase A, estrogen, a chloride channel, or insulin in an animal or
tissue that is
deficient in alpha-galactosidase A, estrogen, that chloride channel, or
insulin. In the
event tissue is damaged (e.g., by trauma) or otherwise destroyed, the
phenotype is
"substantially similar" when a cell that develops from a spore-like cell
expresses a
protein once supplied by the damaged tissue. Given these examples, one of
ordinary
skill in the art can recognize many more "substantially similar" phenotypes.
For
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example, when isolated from the dermis of a post-natal mammal and placed in a
dermal wound, a spore-like cell can develop into a cell having a phenotype
substantially similar to that of a sympathetic or parasympathetic neuron, or a
cell
within a sweat gland, a sebaceous gland, or a hair follicle; when isolated
from the
epidermis of a post-natal mammal and placed in an epidermal wound, a spore-
like cell
can develop into a cell having a phenotype substantially similar to that of a
melanocyte, a keratinocyte, or a Merkel cell; when isolated from the retina of
a post-
natal mammal and placed in a damaged or malfunctioning retina, a spore-like
cell can
develop into a cell having a phenotype substantially similar to that of a
pigmented
epithelial cell, a photoreceptor cell, a bipolar cell, a horizontal cell, an
amacrine cell, a
ganglion cell, an interplexiform cell, or a radial cell of Muller; when
isolated from the
pancreas of a post-natal mammal and placed in a damaged or malfunctioning
pancreas, a spore-like cell can develop into a cell that produces glucagon,
somatostatin, pancreatic polypeptide, or insulin; when isolated from the lung
of a
post-natal mammal and placed in a damaged or malfunctioning lung, a spore-like
cell
can develop into a cell that exchanges oxygen or secretes a surfactant.
The invention also features a tissue construct comprising a spore-like cell. A
"tissue construct" is any medium, structure or device that contains or
supports
(physically or chemically) biological material, including material that
consists of or
includes the novel cells of the present invention. For example, a tissue
engineering
construct can include a biocompatible solution (e.g. a saline solution) that
includes
cytokines, growth factors, and antibiotics. The construct can include a
support
structure and, further, a hydrogel (the hydrogel and the spore-like cell form
a
hydrogel-spore-like cell composition).
In another aspect, the invention features methods for isolating a spore-like
cell by
dissociating a tissue sample and passing the dissociated tissue sample through
a first
device having an aperture no greater than about 15 microns. One can pass the
tissue
sample through a second device having an aperture greater than 15 microns
before the
sample is passed through the first device. One can also isolate spore-like
cells by
passing a tissue sample through a series of devices having progressively
smaller
apertures, the smallest aperture being about 15 microns. The first device, the
second
device, or one or more of the series of devices can be a pipette or filter.
The tissue
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can also be exposed to digestive enzymes, such as trypsin or collagenase. For
example, the tissue can be incubated with approximately 0.05% trypsin at 37 C
for
approximately five minutes.
Spore-like cells and their progeny (e.g., a skin, pancreatic, or retinal
progenitor cell) must originally be isolated from their natural environment
(i.e.,
removed from a place where they reside within an animal) to fall within the
present
invention. Accordingly, an "isolated" spore-like cell or a tissue-specific
(e.g. a skin,
pancreatic, or retinal progenitor cell) can be a cell that is placed in cell
culture, even
temporarily. The term covers single, isolated spore-like cells and their
progeny as
well as cultures of spore-like cells that have been significantly enriched
(i.e., cultures
in which less than about 10% of the cells are fully differentiated cells).
As used herein, the terms that are used to describe the progeny of a spore-
like cell (e.g., "progenitor" or "a spore-like cell and/or its progeny")
refers to a
descendent of a spore-like cell that differentiates into a cell having some
but not all of
the characteristics of a mature cell (e.g., a pigmented epithelial cell, a
photoreceptor
(Le., a rod or a cone), a bipolar cell, a horizontal cell, an amacrine cell, a
ganglion
cell, an interplexiform cell, or a neuroglial cell (the radial cell of
Muller)). Thus, the
progeny of spore-like cells are distinguishable from the differentiated cell
types
naturally found in tissues such as the retina. Accordingly, cells are excluded
from the
invention when they assume characteristics that render them indistinguishable
from
previously identified stem cells (e.g., rnesenchymal stem cells), precursor
cells (e.g.,
the islet cell precursors described by Cornelius et al. (Honn. Metab. Res.
29:271-277
(1997)), or the progenitors from central nervous tissue described by
Shihabuddin et al.
(Exp. Neurol. 148:577-586 (1997)) or Weiss et al. (J. Neurosci. 16:7599-7609
(1996))
or terminally differentiated cells. These characteristics can be assessed by
those of
ordinary skill in the art in numerous ways (e.g., by routine histological,
biochemical,
or, preferably, electron microscopic analysis).
A "hydrogel" is a substance formed when an organic polymer, which can
be natural or synthetic, is set or solidified to create a three-dimensional
open-lattice
structure that entraps molecules of water or other solutions to form a gel.
Solidification can occur by aggregation, coagulation, hydrophobic
interactions, cross-
linking, or similar means. Preferably, the hydrogels used in conjunction with
spore-
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like cells and their progeny solidify so rapidly that the majority of the
cells are
retained at the application site. This retention enhances new cell growth at
the
application site. The hydrogels are also biocompatible (e.g., they are not
toxic to
cells). The "hydrogel-cell composition" referred to herein is a suspension
that
includes a hydrogel and a spore-like cell or its progeny.
The invention has many advantages. The compositions and methods
described herein fill a therapeutic void. For example, there have been no
successful
attempts to repair damaged retinas by tissue engineering. Indeed, others have
postulated that progenitor cells do not exist in the retinas of adult mammals
(and no
one has described cells having the characteristics of the spore-like cells
described
herein). For example, Reh and Levine have stated that, "Where is currently no
evidence for a neural/glial stem cell at the ciliary margin in the adult
mammalian
retina, and the retina of the mature mammal does not show regenerative
capacity after
damage" (J. Neurobiol. 36:206.-220 (1998), at 217). The compositions and
methods of
the invention can also be used to generate cells, for example, islet cells,
that can not only
be maintained in culture, but also expanded in culture to yield a large number
of
biologically active, for example, insulin-producing, cells. Thus, the new
compositions
and methods described herein make it possible to use a single tissue sample to
produce
= enough cells to manufacture significant amounts of biological materials
(e.g. *proteins
such as enzymes and hormones) and to replace missing, damaged, infected, or
malfunctioning cells in a patient (e.g., a diabetic patient). This is true
even for spore-like
cells that are precursors of cells that, when fully differentiated, are
difficult to maintain
or expand in culture (e.g., neurons, hepatocytes, and islet cells). Other
features and
advantages of the invention will be apparent from the following detailed
description
and from the claims.
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In one aspect, the invention relates to an isolated spore-like cell, wherein
the
spore-like cell, when first isolated, is: multipotent; less than seven microns
in diameter; and is
viable following oxygen-deprivation for a period of twenty four hours, wherein
the cell has a
nuclear membrane containing one or more dark stripes when viewed by
transmission electron
microscopy, and wherein the cells do not express nestin when first isolated.
In another aspect, the invention relates to an isolated spore-like cell,
wherein
the spore-like cell remains viable following exposure for more than ten
minutes to an
environment that is at least 42 C or less than 0 C, and wherein the cell has a
nuclear
membrane containing one or more dark stripes when viewed by transmission
electron
microscopy.
In another aspect, the invention relates to use of the cell as described above
in
the preparation of a tissue construct.
In another aspect, the invention relates to a construct comprising a support
structure and the cell as described above.
In another aspect, the invention relates to a method for isolating a spore-
like
cell, the method comprising dissociating a tissue sample and passing the
dissociated tissue
sample through a first device having an aperture no greater than about 15
microns.
In another aspect, the invention relates to a method for isolating a spore-
like
cell, the method comprising passing a tissue sample through a series of
devices having
progressively smaller apertures, the smallest aperture being about 15 microns.
In another aspect, the invention relates to a method for isolating a spore-
like
cell from a biological sample, the method comprising exposing the sample to an
oxygen-
deficient environment for a time sufficient to kill substantially all of the
non-spore-like cells
in the sample and culturing the sample, thereby isolating spore-like cells
from the sample.
In another aspect, the invention relates to a method for isolating a spore-
like
cell from a biological sample, the method comprising exposing the sample to a
non-
physiological temperature for a time sufficient to kill substantially all of
the non-spore-like
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cells in the sample and culturing the sample, thereby isolating spore-like
cells from the
sample.
In another aspect, the invention relates to a method for generating an
artificial
tissue, the method comprising combining hydrogel with the spore-like cell as
described above.
In another aspect, the invention relates to use of the spore-like cell as
described
above for treating a patient who has cancer, wherein the cell is for use in an
area within the
patient from which a tumor has been removed or, where the cancer is a cancer
of a blood-
borne cell, in the bloodstream.
In another aspect, the invention relates to use of the spore-like cell as
described
above for treating a patient who has a damaged, infected, or malfunctioning
tissue, wherein
the cell is for use in the damaged, infected, or malfunctioning tissue.
In another aspect, the invention relates to use of the spore-like cell as
described
above for the manufacture of a medicament for treating a patient who has
cancer, the
medicament being suitable for application to an area within the patient from
which a tumor
has been removed or, where the cancer is a cancer of a blood-borne cell, in
the bloodstream.
In another aspect, the invention relates to use of the spore-like cell as
described
above for the manufacture of a medicament for treating a patient who has a
damaged,
infected, or malfunctioning tissue, the medicament being suitable for
application to the
damaged, infected, or malfunctioning tissue.
In another aspect, the invention relates to the spore-like cell as described
above, for use in treating a patient who has cancer, wherein the cell is for
use in an area within
the patient from which a tumor has been removed or, where the cancer is a
cancer of a blood-
borne cell, in the bloodstream.
In another aspect, the invention relates to the spore-like cell as described
above, for use in treating a patient who has a damaged, infected, or
malfunctioning tissue,
wherein the cell is for use in the damaged, infected, or malfunctioning
tissue.
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In another aspect, the invention relates to a cluster of spore-like cells,
comprising spore-like cells that are multipotent; are approximately one-tenth
to seven microns
in diameter; are viable following oxygen-deprivation for a period of twenty
four hours and
have an outer membrane containing one or more dark stripes when viewed by
transmission
electron microscopy, wherein the aggregate of spore-like cells is produced by
culturing
isolated spore-like cells.
In another aspect, the invention relates to an isolated spore-like cell
population
comprising spore-like cells that: are multipotent; are less than 1 micron in
diameter; are viable
following oxygen-deprivation for a period of twenty four hours, do not express
nestin when
first isolated; and have an outer membrane containing one or more dark stripes
when viewed
by transmission electron microscopy; wherein the population of spore-like
cells is produced
by culturing isolated spore-like cells.
Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those described
herein can be used in the practice or testing of the present invention, useful
methods and
materials are described below. All publications, patent applications, patents,
and other
references mentioned herein are incorporated by reference in their entirety.
In case of
conflicting subject matter, the present
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specification, including definitions, will control. The materials, methods,
and
examples presented herein are illustrative only and not intended to be
limiting.
Brief Description of the Drawings
Figs. 1A-1C are scanning electron micrographs of spore-like cells obtained
from the liver of an adult rat. The cells are magnified 5,000X in Figs. 1A and
1B, and
10,000X in Fig. 1C. The scale bars represent 1.0 tt.
Figs. 2A-2D are transmission electron micrographs of spore-like cells
obtained from the liver of an adult rat and placed in culture for 12 days. The
magnification in Figs. 2A-2D is 25,000X, 39,000X, 17,000X, and 90,000X
respectively.
Figs. 3A-3C are photographs of cells isolated fi-om an adult rat heart and
placed in culture. The newly isolated cells shown in Fig. 3A include
undifferentiated
spore-like cells (magnified 100X). After three days in culture, early
myocardial cells
can be seen (Fig. 3B). After two weeks in culture, Purkinje-like structures
can be
seen (Fig. 3C).
Figs. 4A-4C are photographs of cells isolated from the small intestine of
an adult rat. The newly isolated cells shown in Fig. 4A include
undifferentiated
spore-like cells. After three days in culture, clusters of small intestinal
cells (Fig. 4B)
and autonomic neurons (Fig. 4C) can be seen. Figs. 4A-4C are shown at a
magnification of 200X.
Figs. 5A and 5B are photographs of cells isolated from the bladder of an
adult rat. The newly isolated cells shown in Fig. 5A include undifferentiated
spore-
like cells (magnification at 100X). After two days in culture, the isolated
spore-like
cells, or their progeny, appear to be differentiating (Fig. 5B; magnification
at 200X).
Figs. 6A and 6B are photographs of cells isolated from the kidney of an
adult rat. The newly isolated cells shown in Fig. 6A include undifferentiated
spore-
like cells (magnification at 100X). After three days in culture, aggregates of
cells
resembling kidney structures can be (Fig. 6B; magnification at 200X).
Figs. 7A-7E are photographs of cells isolated from the liver of an adult rat.
The newly isolated cells shown in Figs. 7A and 7C include undifferentiated
spore-like
cells (magnification at 100X). After three days in culture, an aggregate of
cells
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resembling a differentiating liver structure can be seen (Fig. 7B;
magnification at
200X). After seven days in culture, cells resembling hepatocytes can be seen
(Fig. 7D). After 12 days in culture, many cells isolated from the liver
express bile, as
evidenced by a Hall's stain (Fig. 7E; 400X).
Figs. 8A-8C are photographs of cells isolated from the lung of an adult rat;
Fig. 8D is a photograph of cells in a culture initiated by spore-like cells
obtained from
an adult sheep lung; and Fig. 8E is a photograph of a semi-thin section of a
feline
lung. The newly isolated cells shown in Fig. 8A include undifferentiated spore-
like
cells. After six weeks in culture, alveolar-like cells can be seen (Figs. 8B
and 8C).
After 30 days in culture, spore-like cells have formed alveolar-like
structures
(Fig. 8D) similar to those seen in the lungs of adult mammals (Fig. 8E).
Figs. 9A-9D are photographs of cells isolated from the adrenal gland of an
adult rat. Undifferentiated spore-like cells can be seen at Day 0 (see the
arrows in
Figs. 9A (200X) and 9B (400X)). After two days in culture, primitive adrenal
cells
can be seen (Figs. 9C (200X) and 9D (400X)).
Figs. 10A-10C are photographs of islet-like structures. These structures
formed in cultures of spore-like cells that were isolated from pancreatic
tissue that
contained no islets (the islets were harvested prior to the isolation of spore-
like cells).
After six days in culture, more than 100 islet-like structures were present
per field (at
100X magnification; Figs. 10A and 10B). The islet-like structures were
immunostained, which revealed insulin expression (Fig. 10C).
Fig. 11 is a photograph of a culture that includes undifferentiated spore-
like cells isolated from adult human blood.
Figs. 12A and 12B are photographs of cultured cells. The cultures were
established seven days earlier and contained spore-like cells isolated from
adult
human blood. In Fig. 12A, the cells are viewed with phase contrast microscopy.
In
Fig. 12B, the cells are illuminated with fluorescent light following
immunohistochemistry for nestin.
Fig. 13 is a schematic of a permeable support structure filled with a
hydrogel-spore-like cell composition.
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Detailed Description
The present invention provides compositions and methods for repairing,
replacing, or generating tissue (which can include a single cell type or a
combination
of cell types) or another biologically useful substance (e.g., a hormone, an
enzyme, or
an anti-angiogenic factor). The compositions include spore-like cells (e.g.,
mammalian spore-like cells) and certain of their progeny, either or both of
which can
be administered to a patient by the methods described below or by way of
existing
tissue engineering or cell therapy procedures known to those of ordinary skill
in the
art.
, Spore-like cells, their novel progeny, and exemplary methods for their
isolation and use are described below.
A. Spore-like Cells
1. Source
Spore-like cells and their progeny can be obtained from a donor (e.g., a
member of an avian, reptilian, amphibian, or mammalian class). For example,
mammalian spore-like cells can be isolated from a rodent, a rabbit, a cow, a
pig, a
horse, a goat, a sheep, a dog, a cat, a non-human primate, or a human. Spore-
like
cells can be isolated not only from many different types of animals, but also
during
many different stages of the animal's life, including stages where the animals
are
quite mature (e.g. adolescence and adulthood). Notably, because spore-like
cells
tolerate oxygen deprivation and exposure to extreme temperatures better than
differentiated cells (this ability is discussed further below), viable spore-
like cells can
also be isolated from deceased animals, including animals that have been
deceased for
many days, if not weeks, months, or years (e.g., animals that have been
deceased for
1,000 years or more).
In addition, spore-like cells can be obtained from a variety of sources
within a given donor. For example, spore-like cells can be obtained from
bodily
fluids (e.g., blood, saliva, or urine), and most, if not all, functional
organs. While
spore-like cells have been isolated from body fluids and solid functional
organs, it is
not clear whether they originate exclusively in either of these places. It may
be that
tissues and organs are the primary sources for spore-like cells, which appear
in body
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fluids only secondarily, for example, when the cells are "washed out" of those
tissues.
However, it is also possible that spore-like cells originate in bodily fluids
or from the
same source as other cells that are present in bodily fluids (e.g., spore-like
cells may
originate only in the bone marrow). If so, spore-like cells could then be
subsequently
delivered from those fluids to specific tissues. Moreover, delivery can be
upregulated
when the tissue is affected by, for example, a disorder, a regenerative
process, or
wound healing.
The Examples below demonstrate that spore-like cells can be isolated
from, inter alia, adult mammalian liver, lung, heart, bladder, kidney, and
intestine,
and can differentiate into hepatocytes, alveolar cells, cardiac myocytes,
bladder cells,
renal cells, and autonomic neurons, respectively. Given the variety of known
sources
for spore-like cells, it is reasonable to expect that these cells can be found
in most, if
not all, tissues and bodily fluids.
The donor of spore-like cells can be the recipient (e.g., a human patient or
an animal) who will be subsequently treated with those cells, another person,
or an
animal of, e.g., the same or a different species from the recipient. In other
words,
autologous, allogenic, and xenogeneic spore-like cells can be obtained and
used to
treat human patients (methods of treatment are described further below).
Regardless of the source from which they are obtained, spore-like cells can
be placed in culture, and cell lines derived from spore-like cells can be
developed
using techniques routinely practiced by those of ordinary skill in the art.
Thus,
cultured spore-like cells and cell lines derived from spore-like cells can
also be used
to treat human patients and are within the scope of the present invention.
2. Features and Characteristics
Spore-like cells were so-named because they have characteristics
reminiscent of those of spores. Structurally, they have a primitive appearance
and,
functionally, they tolerate extreme conditions. Spore-like cells are typically
small and
generally spherical. Many cells in a culture of newly isolated spore-like
cells are
approximately 1 to 3 p, in diameter. Most spore-like cells have a diameter of
approximately one to seven microns (e.g., a diameter of one to two, two to
four, three
to five, about five, or five to ten microns). However, larger and smaller
spore-like
cells have been identified (e.g., using electron microscopy). Given that spore-
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cells can differentiate into a variety of mature cell types, and that
differentiation is a
gradual process, it is difficult to define the precise upper size limit of
spore-like cells.
=However, spore-like cells 4 to 5, and 7 to 10 in diameter have been
identified in
scanning electron micrographs. Occasionally, even larger cells (e.g., cells as
large as
12 to 18 u) have been observed. From the observations to date, it is difficult
to
determine with certainty whether the apparent large cells are in fact single
cells
(perhaps on the verge of cell division) or conglomerates of several spore-like
cells.
The lower size limit of the spore-like cells is more definite and is certainly
unique.
Spore-like cells that are only about one-third of a micron in diameter (e.g.,
one-tenth,
one-third, one-fourth, one-fifth, or one-half micron) have been observed in
scanning
electron micrographs and some cells are as small as about one-tenth of a
micron.
This extremely small size is in keeping with the unique composition of
spore-like cells, which contain a great deal of nuclear material and
relatively little
cytoplasm. In most differentiated cells, the nucleus comprises approximately
10-20%
of the cells' volume. However, approximately 50% and up to approximately 90%
of
the volume of a spore-like cell is comprised of nuclear material. Without
limiting the
invention to cells that arise by any particular mechanism, spore-like cells
may arise
when essential DNA fragments (which represent compressed DNA) are shed from
mature cells (e.g., those undergoing cell death by apoptosis or other means)
and re-
packaged and protected within, for example, a glycolipid-rich coat. Indeed,
the
concept of a minimal genome is beginning to emerge. This concept is
exemplified by
a mycoplasm that contains 517 genes but only requires 265 to 350 of these
genes to
survive (Hutchison et al. Science 286:2165-2169, 1999). If one considers the
exquisite simplicity of DNA and the genetic code, it seems plausible that the
complex
information stored in DNA could be compressed considerably. The unique size of
newly-isolated spore-like cell is perhaps best appreciated by viewing the
cells with an
electron microscope (e.g., see Figures 1A-1C and 2A-2D).
The nuclear material appears to be surrounded by a coat containing more
glycolipids and mucopolysaccharides than are normally found on the surface of
normal differentiated cells. There are a number of standard assays for
glycolipids,
which are carbohydrate and lipid compounds that contain 1 mole each of a fatty
acid,
sphingosine, and hexose. Common reactions for carbohydrates include the
periodic
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acid-Schiff (PAS) reaction, diastase, alcian blue staining, colloidal iron,
and
hyaluronidase. Spore-like cells isolated from adult liver are stained by PAS
and
mucicarmine stains, which indicates that these cells are coated with
mucopolysaccharids and glycolipids. In any event, the content of the coat is
such that
when the cells are viewed with a transmission electron microscope, they can
appear to
have a striped appearance (i.e. a plurality of areas of high contrast as shown
in
Figs. 2C and 2D). When the cells are exceedingly small (e.g., less than about
one
micron) the areas of contrast are not as obvious, but they can nevertheless be
seen
with a trained eye.
Functionally, spore-like cells are unique in at least three ways. First, even
though they can be isolated from a mature (e.g., a post-natal, juvenile,
adolescent, or
adult) animal, they can differentiate into a variety of different cell types
(i.e., they are
multipotent). For example, spore-like cells isolated from the lung develop not
only
into cells that mediate gas exchange, but also into cells that form the
connective tissue
and vasculature of the lung. Similarly, spore-like cells isolated from the
liver develop
not only into hepatocytes, but also into cells that form the connective tissue
and
vasculature of the liver. Thus, multipotent spore-like cells are those that
produce
some or all of the cell types found within complex tissue structures. At a
minimum,
spore-like cells must be shown to differentiate into two or more (e.g., 2, 3,
4, or 5) cell
types.
Second, spore-like cells tolerate exposure to extreme conditions (e.g.,
dessication, oxygen-deprivation, and exposure to temperatures that are both
much
higher and much lower than normal body temperature (which, for warm-blooded
mammals, is about 37 C)). As shown in the Examples below, spore-like cells can
survive in low-oxygen environments, such as those that exist within the
tissues of a
deceased animal, or within a capped container of phosphate-buffered saline
(PBS), for
many hours (e.g., four, six, ten, twelve, or 24 hours or more). While
differentiated
cells are able to survive oxygen deprivation for variable periods of time
(e.g., neurons
are particularly sensitive, surviving only about 4-15 minutes after oxygen
deprivation,
while cartilage can remain viable, with refrigeration, for a month or so),
spore-like
cells isolated from differentiated tissue outlive the differentiated cells of
that tissue
when the tissue is oxygen-deprived. Moreover, spore-like cells can survive
without
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special preservatives. For example, hepatocytes can survive ex vivo for
approximately two days if they are specially preserved, but spore-like cells
isolated
from the liver can survive for the same period (and much longer) without
special
preservatives.
Spore-like cells can also survive exposure to temperatures that are much
higher and much lower than body temperature. For example, spore-like cells
remain
viable within tissues that are stored at about 4 C for a prolonged period of
time (e.g.,
one, three, five, seven, or more days). They also remain viable at
temperatures that
vary even further from a physiological body temperature. For example,
substantially
pure populations of spore-like cells (e.g., spore-like cells isolated from a
mammal)
and spore-like cells within tissues (e.g., spore-like cells within mammalian
tissues)
can survive freezing or heating to more than 5 C in excess of a physiological
body
temperature. That is, viable spore-like cells survive within tissues that have
been
cooled to 0 C, or below, or heated to about 43 C or above. As with oxygen-
deprivation, spore-like cells can survive exposure to these conditions without
special
treatment (e.g., they can survive exposure to freezing temperatures even
without
treatment with a cryopreservative). Viable spore-like cells can also be
isolated from
tissues that have been thoroughly dried (e.g., by placement in a dessicator
for
approximately 24 hours). Given their ability to survive exposure to extreme
conditions, spore-like cells can be isolated from an animal (including a
human) that
has been dead for many hours, for several days or weeks, or even longer. While
most
differentiated cells, particularly those within oxygen-sensitive tissues such
as the
brain, will no longer be viable, spore-like cells will be. Thus, the methods
of the
present invention are useful in forensic science to, for example, identify a
deceased
person. Similarly, spore-like cells can be isolated from tissues found at a
crime scene,
such as blood, and used to identify the perpetrator. Further, since spore-like
cells can
be isolated from tissues that have been frozen without a cryopreservative,
they can be
isolated from animals that have died in the wild in frigid climates and, quite
probably,
from animals that have been frozen for many, many years. Similarly, because
spore-
like cells remain viable even after exposure to heat, they can also be
recovered from
animals that have died in fires, arid landscapes, or in warm springs. Some of
the
animals from which spore-like cells can be isolated may now be extinct.
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Third, under the conditions described in the examples below, spore-like
cells have a greater capacity to proliferate than terminally differentiated
cells isolated
from specialized tissues such as the islet cells of the pancreas. When
cultured under
the conditions described in the Examples below, skin progenitor cells appear
to
double approximately every 24-36 hours. Cell viability and proliferative
capacity can
be assessed using standard techniques, including visual observation with a
light or
scanning electron microscope and Trypan blue exclusion. Proliferative capacity
is an
important attribute because tissue engineering, cell therapies, and gene-based
therapies are often hampered by physicians' inability to obtain sufficient
numbers of
cells to administer to a patient.
The features and characteristics described herein can be used to distinguish
spore-like cells from previously identified cell types. For example, the spore-
like
cells of the invention can be identified by one or more of the following
attributes:
their ability to differentiate into a variety of terminally differentiated
cell types found
in mature animals (such as those illustrated in the Examples below) and
thereby give
rise to complex tissue structures; their typical spherical shape, small size
(as small as
0.1-0.3 pt, in diameter and generally 1.0 to 3.0 p, in diameter), and
cytoarchitecture
(which includes relatively large amounts of nuclear material and relatively
small
amounts of cytoplasm); and their ability to survive in environments having a
low or
even non-existent oxygen supply.
3. Methods of Obtaining Spore-like Cells
To obtain spore-like cells, a sample of biological material is harvested
from an animal (e.g. a human). Spore-like cells and their progeny can be
obtained
from the sample either immediately after collection (or soon after) or after
the sample
has been stored under either normal cell storage conditions or in an oxygen-
poor
environment or at a non-physiological temperature, for example, a temperature
that
varies from a normal body temperature by more than 5 C (e.g., a temperature
more
than 42 C or at or below freezing). One of the easiest samples to obtain from
an
animal is a sample of whole blood. Those of ordinary skill in the art will
appreciate
that the isolation method will vary slightly depending on the type of tissue
used as the
starting material. For example, in the event the sample is a blood sample, it
can be
placed in a tube containing an anti-coagulant. After collection, tissue
samples,
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whether they are samples of bodily fluids or cell suspensions obtained from
solid
organs, are centrifuged for a time and at a speed sufficient to pellet the
cells within the
sample at the bottom of the centrifuge tube. The resulting pellet is
resuspended in a
suitable medium (e.g., DMEM/F-12 medium supplemented with glucose,
transferrin,
insulin, putricine, selenium, progesterone, epidefinal growth factor (EGF) and
basic
fibroblast growth factor (bFGF; see the Examples, below).
The suspended cells are then transferred to a tissue culture vessel and
incubated at or near 37 C. Initially, when the sample is a blood sample, the
culture
flasks contain primarily hematopoietic cells. However, after several days in
culture,
the red blood cells lyse and degenerate so that the culture contains
primarily, if not
exclusively, spore-like cells. When spore-like cells are isolated from solid
tissues, the
differentiated cells can be lysed by triturating the sample with a series of
pipettes,
each having a smaller bore diameter than the one before. For example, the last
pipette
used can have a bore diameter of approximately 15[1. After several additional
days in
culture, the spore-like cells multiply and can coalesce to form clusters of
cells. Over
time, usually on the order of approximately 7 days, their number can increase
greatly.
Typically, more than 90% of the cells are viable according to Trypan blue
exclusion
studies when isolated as described above.
When spore-like cells are obtained from a solid organ, for example by an
excisional biopsy of the skin, the skin is swabbed with betadine and
infiltrated with
lidocaine. Under sterile conditions, a piece of skin can then be removed with
a
scalpel or punch. Once obtained, the dermis, the epidermis, or full-thickness
skin can
be placed in culture. If desired, spore-like cells or skin progenitor
cellsican be
isolated individually from both the dermis and epidermis.
To obtain spore-like cells or skin progenitor cells, the piece of skin is
placed in a buffered solution (e.g., phosphate buffered saline), which can
include one
or more antibiotics, and the tissue can be dissociated mechanically (e.g., by
macerating the tissue or by scraping it with a scalpel or similar instrument),
chemically (e.g., by exposure to one or more enzymes, such as trypsin or
collagenase,
that facilitate tissue degradation), or both. Generally, the more aggressive
the
dissociation, the more spore-like cells or skin progenitor cells one will
obtain. To
isolate individual spore-like cells or skin progenitor cells, the tissue can
be triturated,
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first with a normal bore Pasteur pipette and subsequently with a series of
fire polished
pipettes having bore sizes ultimately reduced to less than about fifteen
microns. This
procedure (which is described in further detail in the Examples below)
destroys large
mature cells, but allows the smaller spore-like cells and skin progenitor
cells (i.e.,
cells generally having a diameter smaller than that of the smallest bore
pipette) to
survive.
The surviving cells are placed in an incubator and can be grown under
conditions that either allow them to differentiate into the specialized cell
types found
in mature skin or that discourage differentiation. For example, spore-like
cells and
skin progenitor cells can be encouraged to differentiate by exposing them to
the
processes and basal nutrient media described in U.S. Patent No. 5,292,655.
Alternatively, growth factors that cause progenitor Cells to mitose (e.g.,
epidennal
growth factor (EGF), basic fibroblast growth factor (bFGF) and other
cytokines) can
be applied to help maintain the cells in an undifferentiated state. For
example, the
isolated cells can be cultured in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with a hormone mixture containing glucose, transferrin, insulin,
putricine, selenium, progesterone, EGF, and bFGF (see the Examples below). The
media can be changed approximately every three days, and cells can be passaged
approximately every 7-9 days.
Another way to promote proliferation is to expose spore-like cells or skin
progenitor cells to agonists of Notch function, as described in U.S. Patent
No. 5,780,300. Agonists of Notch include, but are not limited to, proteins
such as
Delta or Serrate or Jagged (Lindsell et al., Cell 80:909-917, 1995) or
biologically
active fragments thereof. These proteins or protein fragments mediate binding
to
Notch and thereby activate the Notch pathway. Spore-like cells or skin
progenitor
cells can be contacted in culture with agonists of Notch or can be transfected
with
genes that encode Notch agonists. Techniques for transfecting cells in culture
are
routinely practiced by those of ordinary skill in the art. Progenitors that
remain
undifferentiated in culture can differentiate when administered to a patient;
their
differentiation being orchestrated by the microenvironment they encounter
within the
patient (differentiation is discussed further below).
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Those of ordinary skill in the art will recognize that trituration through
reduced bore pipettes is not the only way to isolate spore-like cells from
larger,
differentiated cells. For example, a suspension containing spore-like cells
and
differentiated cells can be passed through a filter having pores of a
particular size.
The size of the pores within the filter (and, similarly, the diameter of the
pipette used
for trituration) can be varied, depending on how stringent one wishes the
isolation
procedure to be. Generally, the smaller the pores within the filter, or the
smaller the
diameter of the pipette used for trituration, the fewer the number of
differentiated cells
that will survive the isolation procedure.
4. Spore-like Cell Differentiation
When first isolated, spore-like cells may not express any known markers
(i e., proteins or other biological molecules associated with a given cell
type, e.g., a
teiminally differentiated cell type). After being placed in culture, some
spore-like
cells express nestin, a marker of neuroectodermally-derived cells. Those that
do
express nestin probably do so before expressing markers associated with
terminally
differentiated neural cells.
Spore-like cells or their progeny, when cultured, will eventually express
cellular markers associated with terminally differentiated cells (see the
Examples
below), or can be made to do so by transfection with a gene encoding the
marker or a
biologically active substance of interest. Those of ordinary skill in the art
can
identify, by techniques routinely practiced in the art (e.g.,
immunochemistry),
numerous markers associated with terminally differentiated cells. For the
purpose of
defining the cells of the present invention, these cells become terminally
differentiated
(and thus fall outside the scope of the invention) when they express
essentially the
same phenotype as a mature cell. More specifically, a cell of the present
invention is
terminally differentiated when it expresses at least half of the
distinguishing markers
presently known in the art to be expressed by a particular mature (i.e. fully
differentiated) cell. Antibodies to these markers are commercially available
or
otherwise readily attainable.
Alternatively, histological stains and microscopy can be used to identify
mature cells. For example, electron microscopy can be performed to reveal
melanosomes, and a Fontana stain can be performed to identify melanin granules
in
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melanocytes. Electron microscopy is also useful in identifying dense core
granules in
another epithelial cell type, the Merkel cell. Analysis of cellular morphology
can also
be used to identify sweat glands, which contain clear cells filled with
glycogen, and
sebaceous glands that are filled with lipids, triglycerides, cholesterol, and
wax-like
substances. To distinguish differentiated cells within the visual system, one
can
identify pigmented epithelial cells by the characteristic granules visible
under the
electron microscope; horizontal type I cells by the formation of connections
with rods;
horizontal type II cells by the formation of connections with cones; bipolar
cells by
their bipolar morphology and connections to rods or cones (they also send an
axon to
synapse with ganglion cells); amacrine cells by their numerous dendrites, but
lack of
an axon; interplexiforrn cells by their synapses with amacrine cells (they are
pre- and
post-synaptic to amacrine cells) and pre-synaptic connection with horizontal
cells and
bipolar cells; and ganglion cells as the terminal link in the neural network
of the retina
via their connection with the optic nerve.
One method of inducing differentiation is to allow spore-like cells or their
progeny to establish contact (e.g., physical contact) with a solid support.
For
example, spore-like cells can differentiate when they establish contact with a
glass or
plastic surface, a mesh, or other substrate suitable for use in tissue culture
or
administration to a patient. Contact can be facilitated by coating the solid
support
with one or more components of the extracellular matrix, such as laminin or
fibronectin. On the other hand, keeping the cells suspended tends to inhibit
differentiation.
Spore-like cells can also differentiate when they establish contact with a
tissue within a patient's body or when they are sufficiently close to a tissue
to be
influenced by substances (e.g., growth factors, enzymes, or hormones) released
from
the tissue. In other words, a spore-like cell can establish contact with a
tissue (e.g.,
the dermis or epidermis) by virtue of receiving signals from the tissue. Such
signalling would occur, for example, when a receptor on the surface of a spore-
like
cell, or on the surface of a cell descended from a spore-like cell (e.g., a
skin
progenitor cell), binds and transduces a signal from a molecule such as a
growth
factor, enzyme, or hormone that was released by a tissue within the patient.
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Alternatively, or in addition, spore-like cells and their progeny can be
induced to differentiate by adding a substance (e.g., a growth factor, enzyme,
hormone, or other signalling molecule) to the cell's environment. One of
ordinary
skill in the art can readily identify substances known to direct
differentiation along
various paths. For example, transforming growth factor alpha (TON is known to
stimulate amacrine cell differentiation (J. Neurobiol. 36:206-220, 1998) and
to inhibit
differentiation into rods (J. Neurobiol. 36:206-220, 1998). In contrast,
retinoic acid
stimulates rod differentiation and inhibits amacrine cell differentiation (J
Neurobiol.
36:206-220, 1998).
One or more substances that evoke differentiation can be added to a
culture dish containing spore-like cells, to a mesh or other substrate
suitable for
applying spore-like cells to a tissue, or to a tissue within a patient's body.
When a
substance that induces spore-like cells to differentiate is administered,
either
systemically or locally, it can be administered according to pharmaceutically
accepted
methods. For example, proteins, polypeptides, or oligonucleotides can be
administered in a physiologically compatible buffer, with or without a carrier
or
excipient. Of course, either the cells within a patient's body or the cells
being
administered (here, spore-like cells or their progeny) can be made to express
particular factors following genetic manipulation. Thus, spore-like cells or
their
progeny can differentiate either in culture or in a patient's body, and can do
so
following contact with a solid support or exposure to substances that are
either
naturally expressed, exogenously administered, or expressed as a result of
genetic
manipulation. Regardless of the stimulus for differentiation, spore-like cells
or their
progeny are useful so long as they will differentiate or have differentiated
sufficiently
to aid in the maintenance or repair of an injured or malfunctioning tissue.
For
example, cells that have differentiated sufficiently to repair the dermis or
epidermis
can be administered to a patient at the site of a burn or other traumatized
area of skin.
While spore-like cells or their progeny may eventually become fully
differentiated, and while this is desirable in some circumstances (e.g., where
the cells
are used to recreate a histologically mature and complete tissue), not all of
the cells
administered need to be fully differentiated to achieve successful treatment;
spore-like
cells or their progeny need only differentiate to a point sufficient to treat
the patient.
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That point can be reached either before or after the cells are administered to
the
patient. As described herein, the spore-like cell can also be genetically
modified
using routine techniques to express a beneficial enzyme or hormone (e.g.
insulin).
B. Use
In view of the preceeding discussion and given the number of
differentiated phenotypes already observed (see the Examples, below), it is
reasonable
to expect that spore-like cells can fully or partially differentiate into
most, if not all,
types of cells. Thus, spore-like cells can be used to study fundamental
aspects of
cellular differentiation. The rate and course of spore-like cell
differentiation is
influenced by the number and type of mature cells to which the spore-like
cells are
exposed. For example, when isolating spore-like cells from the liver, the more
mature
hepatocytes that remain in the culture of spore-like cells, the more quickly
the spore-
like cells will differentiate and the more likely it is that they will
differentiate into
hepatocytes. Thus, spore-like cells proliferate and differentiate in response
to agents
(e.g., growth factors or hormones) within tissue, including tissue that has
been injured
or that is otherwise malfunctioning due to a medical condition, disorder, or
disease.
Regardless of their origin -- blood, another body fluid, the bone marrow, or a
solid,
functional tissue or organ -- spore-like cells can be influenced by agents
that are either
exogenously applied or provided by naturally occurring cells to which they
have been
exposed.
Spore-like cells can be used to maintain the integrity and function of a
wide variety of tissues as well as to reengineer, repair, or otherwise improve
tissue
associated with a medical disorder. For example, spore-like cells can be used
to
maintain or reengineer tissue that develops from the endoderm, mesoderm, or
ectoderm. More specifically, spore-like cells can be used to maintain or
reengineer:
bone; bone marrow; muscle (e.g., smooth, skeletal, or cardiac muscle);
connective
tissue (e.g., cartilage, ligaments, tendons, pleura, or fibrous tissues); lung
tissue;
vascular tissue; nervous tissue (e.g., neurons and glial cells in the central
or peripheral
nervous systems), glandular tissue (e.g., tissue of the thyroid gland, adrenal
gland, or
sweat or sebaceous glands); epithelial cells, keratinocytes, or other
components of the
skin; lymph nodes; the immune system; reproductive organs; or any of the
internal
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organs (e.g., liver, kidney, pancreas, stomach, bladder, or any portion of the
alimentary canal).
The preceeding list is intended to illustrate, not limit, the types of cells
and
tissues that can benefit from administration of spore-like cells
(administration of
spore-like cells is described further below). Those of ordinary skill in the
art, given
the present disclosure, will understand the wide variety of uses for spore-
like cells.
For example, life-like artificial skin can be produced by culturing spore-like
cells and
allowing them, when applied to a living body or used in conjunction with
present skin
replacement methods, to differentiate into epidermal and dermal cells
(including
melanocytes) as well as into hair follicles, sweat glands, sebaceous glands,
ganglia,
and similar adnexal structures.
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Spore-like cells can also be used to treat diseases and disorders that affect
other sensory systems. For example, spore-like cells can be used to treat
blindness
caused by, for example, retinal detachment. Presently, without treatment,
retinal
detachment often becomes total within six months (see, e.g., Chapter 7 of
Current
Medical Diagnosis and Treatment, 37th Edition, Tierney et al., Eds., 1998).
Blindness can also result when the central retinal vein or its branches become
occluded, as occurs in Behcet's syndrome, and visual acuity is impaired by
systemic
diseases such as diabetes mellitus, essential hypertension, preeclampsia-
eclampsia of
pregnancy, blood dyscrasias, and AIDS. Spore-like cells and retinal progenitor
cells
can be used to repair the retina when it has been injured or when its ability
to function
is otherwise compromised (e.g., by macular degeneration, retinitis pigmentosa,
retinal
detachment, occlusion of a retinal artery or vein (or occlusion of one or more
of their
branches), or a retinopathy such as diabetic retinopathy, hypertensive
retinochoroidopathy, blood dyscrasias, or cytomegalovirus retinitis). Spore-
like cells
or retinal progenitors, when administered to a patient, will differentiate
into cells
having some or all of the beneficial features of the following cell types:
pigmented
epithelial cells, photoreceptors (i.e., rods or cones), bipolar cells,
horizontal cells,
amacrine cells, ganglion cells, interplexiform cells, and neuroglial cells
(the radial
cells of Milner). The cells of the invention can be placed in an area of the
eye
previously occupied by the retina (i.e., the cells can be administered to a
patient
whose retina has been wholly or partially removed as a consequence of surgery
or a
disease process).
Spore-like cells and their progeny can also be used to treat patients who
have a metabolic, enzymatic, or hormonal deficiency. For example, the
compositions
and methods of the invention can be used to treat a patient who has either
Type I or Type
II diabetes, who has suffered a traumatic injury to the pancreas, or who
required surgery
that adversely affected the pancreas (e.g., surgical removal of some or all of
the pancreas
due to chronic pain syndrome or cancer). When used to treat a metabolic,
enzymatic, or
hormonal deficiency, the cells of the invention can be administered to
numerous
locations including, but not limited to, the site where the damaged or
malfunctioning
cells normally reside. For example, cells used to treat diabetes need not be
delivered into
the recipient's pancreas but can be provided beneath the kidney capsule or
within an
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implantable device. When spore-like cells or their progenitors are
administered to the
pancreas, where they have access to the growth factors and other compounds
that
normally influence islet cells, it is less important that they be administered
in a
differentiated state. However, when the cells are administered elsewhere
(e.g., beneath
the kidney capsule or within an implantable device) it is more important that
they be
administered in a differentiated or committed state or adrninistered together
with factors
that will promote their differentiation.
When spore-like cells are used in cell therapies, they can be administered
just as more differentiated cells have been administered. For example, when
spore-
like cells are used to treat diabetes, they can be administered just as mature
insulin-
expressing cells have been administered (e.g., by implantation under the renal
capsule
or within various implantable or extracorporeal devices). In fact, spore-like
cells can
be placed within a containment device and implanted, for example, within a
patient's
abdomen to treat a variety of disorders. This method of administration is
particularly
well suited for treating systemic disorders, such as those caused by an
enzymatic
imbalance. Implantation by way of containment devices is also useful when
cells
require protection from the patient's immune system.
Alternatively, as described below, spore-like cells and their progeny can be
used in conjunction with tissue constructs (i.e., any medium, structure or
device that
contains or supports (physically or chemically) biological material, including
material
that consists of or includes the novel cells of the present invention). A
tissue
construct can include a biocompatible solution (e.g. a saline solution) that
includes
cytokines, growth factors, and antibiotics, and materials or devices useful in
reengineering damaged, diseased, or otherwise unhealthy tissue. The constructs
can
include support structures, such as a mesh, and/or a hydrogel. Together, the
hydrogel
and the spore-like cells of the invention form a hydrogel-spore-like cell
composition.
Similarly, a hydrogel combined with a progenitor cell forms a hydrogel-
progenitor cell
composition.
Spore-like cells can be combined with a liquid hydrogel that can be placed in
a
permeable, biocompatible support structure that is delivered to a patient
(either before
or after it is filled with the hydrogel-cell composition). As the hydrogel-
cell
composition fills the support structure, it assumes the structure's shape.
When spore-
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like cells proliferate and differentiate to such an extent that they form new
tissue, the
support structure guides the shape of the developing tissue. For example, the
support
structure can be formed in the shape of a bone (or a fragment thereof), a
meniscus
within a joint, an ear, an internal organ (or a portion thereof), or other
tissue (e.g., the
skin). However, the support structure need not be strictly fashioned after
naturally
occurring tissue in every case. For example, the support structure can be
shaped in a
way that simply facilitates delivery of spore-like cells to a patient. For
example, the
support structure can be shaped to fit under the renal capsule or within some
other
organ or cavity (e.g., the support structure can be shaped to lie within a
portion of the
gastrointestinal tract or to fill a space once occupied by tissue, such as the
spaces
created when a tumor is surgically removed or when a tissue has been destroyed
following trauma, ischemia, or an autoimmune response).
In some instances, including instances where spore-like cells are
administered in the course of cell or gene therapy, spore-like cells can be
administered
without containment devices, hydrogels, or support structures. It is well
within the
ability of one of ordinary skill in the art to determine when spore-like cells
should be
confined within a space dictated by a support structure and when they should
not. For
example, one of ordinary skill in the art would recognize that when treating
respiratory distress syndrome (RDS) with spore-like cells that are made to
secrete
surfactant, or that differentiate into cells that secrete surfactant, the
surfactant, which
reduces surface tension within the alveoli, must be supplied locally. Thus,
spore-like
cells obtained, e.g., from healthy human lung tissue, for example, taken from
a
healthy portion of a lung of the patient or from a donor, are delivered to the
diseased
portions of the patient's lungs, e.g., by inhalation in the form of an
aerosol.
Cells obtained from an immunologically distinct donor can illicit an
immune response, in which case, the patient (the recipient of the cells) can
be treated
with standard immunosuppressant therapy (e.g., with cyclosporine and/or
steroid
hormones). While immunosuppression is commonly required when transplanting
typical mature cells (e.g., when transplanting an organ such as the liver or
kidney), it
should not be required when administering the unique cells of the invention.
These
cells, particularly spore-like cells, are so undifferentiated that they may
not express
surface antigens and thus would not elicit an immune response, even if
isolated from a
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different species or a different individual of the same species as the patient
who will
receive the cells.
C. Administration of Cells via Hydrogel
The novel cell types described herein can be administered to a patient by
way of a composition that includes a hydrogel. For example, a hydrogel can be
combined with spore-like cells, their non-terminally differentiated progeny,
one or
more types of differentiated cells, or a combination of these cell types. This
cell-
hydrogel mixture can be applied directly to a tissue that has been damaged or
to a
device that is then implanted in, or attached to, a patient. For example, as
described in
U.S.S.N. 08/747,036, a hydrogel-cell mixture can simply be applied to a
desired
surface of a tissue (by, e.g., brushing, dripping, or spraying the mixture
onto the
surface) or poured into or otherwise made to fill a desired cavity or device.
The
hydrogel provides a thin matrix or scaffold within which the cells (e.g., the
spore-like
cells) adhere and function (by, e.g., differentiating, proliferating, or
secreting a
biologically active substance such as insulin). The methods of administration
described here are especially well suited when the tissue associated with a
patient's
disorder has an irregular shape or when the cells are applied at a distant
site (e.g.,
when spore-like cells that are made to express insulin or that differentiate
into cells
that express insulin are placed beneath the renal capsule, elsewhere in the
abdomen,
or within an extracorporeal device to treat diabetes).
Alternatively, the hydrogel-cell mixture can be introduced into a
permeable, biocompatible support structure so that the mixture essentially
fills all, or
a desired portion of, the support structure and, as it solidifies, assumes the
support
structure's shape. Thus, the support structure can guide the development and
shape of
tissues that mature from cells (e.g., spore-like cells and their progeny)
within it. As
described further below, the support structure can be provided to a patient
either
before or after being filled with the hydrogel-cell mixture. For example, the
support
structure can be placed within a tissue (e.g., a damaged area of the skin,
liver, lung, or
the skeletal system) and subsequently filled with the hydrogel-cell
composition using
a syringe, catheter, or other suitable device. When desirable, the shape of
the support
structure can be made to conform to the shape of the damaged tissue. In the
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subsections, suitable support structures, hydrogels, and delivery methods are
described (cells suitable for use are described above).
1. Hydrogels
The hydrogels used to practice this invention should be biocompatible,
capable of sustaining living cells (Le., nontoxic to the cells combined with
it), and,
preferably, should solidify rapidly in vivo (e.g., in about five minutes after
being
delivered to the support structure). Large numbers of cells (e.g., spore-like
cells) can
be distributed evenly within a hydrogel; a hydrogel can support approximately
5 x 106
cells/ml. The hydrogel should contain, but in most cases not isolate, the
cells within
it. Nutrients should be able to diffuse through the hydrogel to reach the
cells and
waste products or other secreted substances should be able to reach the
patient.
A variety of different hydrogels can be used to practice the invention.
These include, but are not limited to: (1) temperature dependent hydrogels
that
solidify or set around body temperature (e.g., PLURONICSTm); (2) hydrogels
cross-
linked by ions (e.g., sodium alginate); (3) hydrogels set by exposure to
either visible
or ultraviolet light (e.g., polyethylene glycol polylactic acid copolymers
with acrylate
end groups); and (4) hydrogels that are set or solidified upon a change in pH
(e.g.,
TETRONICSTm).
Materials that can be used to form these different hydrogels include, but
are not limited to, polysaccharides such as alginate, polyphosphazenes, and
polyacrylates, which are cross-linked ionically, block copolymers such as
PLURONICSTM (also known as POLOXAMERSTm), which are poly(oxyethylene)-
poly(oxypropylene) block polymers solidified by changes in temperature,
TETRONICSTM (also known as POLOXAMINESTm), which are poly(oxyethylene)-
poly(oxypropylene) block polymers of ethylene diamine solidified by changes in
pH.
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a. Ionic Hydrogels
Ionic polysaccharides, such as alginates or chitosan, can be used to
suspend living cells, including spore-like cells and their progeny. These
hydrogels
can be produced by cross-linking the anionic salt of alginic acid, a
carbohydrate
polymer isolated from seaweed, with ions, such as calcium cations. The
strength of
the hydrogel increases with either increasing concentrations of calcium ions
or
alginate. U.S. Patent No. 4,352,883 describes the ionic cross-linking of
alginate with
divalent cations, in water, at room temperature, to form a hydrogel matrix.
Spore-like cells, their progeny, or a mixture of cells including either of
these cell types, are mixed with an alginate solution, the solution is
delivered to an
already implanted support structure, which then solidifies in a short time due
to the
presence of physiological concentrations of calcium ions in vivo.
Alternatively, the
solution is delivered to the support structure prior to implantation and
solidified in an
external solution containing calcium ions.
In general, the polymers described here are at least partially soluble in
aqueous solutions (e.g., water, aqueous alcohol solutions that have charged
side
groups, or monovalent ionic salts thereof). There are many examples of
polymers
with acidic side groups that can be reacted with cations (e.g.,
poly(phosphazenes),
poly(acrylic acids), and poly(methacrylic acids)). Examples of acidic groups
include
carboxylic acid groups, sulfonic acid groups, and halogenated (preferably
fluorinated)
alcohol groups. Examples of polymers with basic side groups that can react
with
anions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl
imidazole).
Polyphosphazenes are polymers with backbones consisting of nitrogen and
phosphorous atoms separated by alternating single and double bonds. Each
phosphorous atom is covalently bonded to two side chains. Polyphosphazenes
that
can be used have a majority of side chains that are acidic and capable of
forming salt
bridges with di- or trivalent cations. Examples of acidic side chains are
carboxylic
acid groups and sulfonic acid groups.
Bioerodible polyphosphazenes have at least two different types of side
chains: acidic side chains that can form salt bridges with multivalent cations
and side
chains that hydrolyze in vivo (e.g., imidazole groups, amino acid esters,
glycerol, and
glucosyl). Bioerodible or biodegradable polymers (i.e., polymers that dissolve
or
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degrade within a period that is acceptable in the desired application (usually
in vivo
therapy)), will degrade in less than about five years and most preferably in
less than
about one year, once exposed to a physiological solution of pH 6-8 having a
temperature of between about 25 C and 38 C. Hydrolysis of the side chain
results in
erosion of the polymer. Examples of hydrolyzing side chains are unsubstituted
and
substituted imidizoles and amino acid esters in which the side chain is bonded
to the
phosphorous atom through an amino linkage.
Methods for synthesis and the analysis of various types of
polyphosphazenes are described in U.S. Patent Nos. 4,440,921, 4,495,174, and
4,880,622. Methods for synthesizing other polymers described above are known
to
those of ordinary skill in the art. See, for example Concise Encyclopedia of
Polymer
Science and Engineering, J.I. Kroschwitz, Ed., John Wiley and Sons, New York,
NY,
1990. Many polymers, such as poly(acrylic acid), alginates, and PLURONICSTm
are
commercially available.
Water soluble polymers with charged side groups are cross-linked by
reacting the polymer with an aqueous solution containing multivalent ions of
the
opposite charge, either multivalent cations if the polymer has acidic side
groups, or
multivalent anions if the polymer has basic side groups. Cations for cross-
linking the
polymers with acidic side groups to form a hydrogel include divalent and
trivalent
cations such as copper, calcium, aluminum, magnesium, and strontium. Aqueous
solutions of the salts of these cations are added to the polymers to form
soft, highly
swollen hydrogels.
Anions for cross-linking the polymers to form a hydrogel include divalent
and trivalent anions such as low molecular weight dicarboxylate ions,
terepthalate
ions, sulfate ions, and carbonate ions. Aqueous solutions of the salts of
these anions
are added to the polymers to form soft, highly swollen hydrogels, as described
with
respect to cations.
Those of ordinary skill in the art are well able to adjust the size of the
polymers formed to accommodate various uses and needs. For example, when the
polymer in the hydrogel ranges from 10 to 18.5 kDa, most antibodies will not
pass
into the hydrogel, but nutrients will. When desired, smaller polymers can be
made
that give rise to hydrogels of higher density (i.e., smaller pores).
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b. Temperature-Dependent Hydrogels
Temperature-dependent, or thermosensitive, hydrogels can also be used in
the methods of the invention. These hydrogels have so-called "reverse
gelation"
properties (i.e., they are liquids at or below room temperature and gel when
warmed
to higher temperatures (e.g., body temperature)). Thus, these hydrogels can be
easily
applied at or below room temperature as a liquid and automatically form a semi-
solid
gel when warmed to body temperature. As a result, these gels are especially
useful
when the support structure is first implanted into a patient, and then filled
with a
hydrogel-cell composition. Examples of such temperature-dependent hydrogels
are
PLLTRONICSTM (BASF-Wyandotte), such as polyoxyethylene-polyoxypropylene
F-108, F-68, and F-127, poly (N-isopropylacrylamide), and N-
isopropylacrylamide
copolymers.
These copolymers can be manipulated by standard techniques to affect
their physical properties such as porosity, rate of degradation, transition
temperature,
and degree of rigidity. For example, the addition of low molecular weight
saccharides
in the presence and absence of salts affects the lower critical solution
temperature
(LCST) of typical thermosensitive polymers. In addition, when these gels are
prepared at concentrations ranging between 5% and 25% (WN) by dispersion at 4
C,
the viscosity and the gel-sol transition temperature are affected, the gel-sol
transition
temperature being inversely related to the concentration. These gels have
diffusion
characteristics capable of allowing spore-like cells and their progeny to
survive and be
nourished.
U.S. Patent No. 4,188,373 describes using PLURONICTM polyols in
aqueous compositions to provide thermal gelling aqueous systems. U.S. Patent
Nos.
4,474,751, '752, '753, and 4,478,822 describe drug delivery systems that
utilize
thermosetting polyoxyalkylene gels. With these systems, any of which can be
used to
deliver the novel cells of the present invention to a patient, both the gel
transition
temperature and the rigidity of the gel can be modified by adjusting the pH or
the
ionic strength, as well as by the concentration of the polymer.
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c. pH-Dependent Hydrogels
Other hydrogels suitable for use in the methods of the invention are pH-
dependent. These hydrogels are liquids at, below, or above specific pH values,
and
gel when exposed to specific pHs, for example, 7.35 to 7.45, the normal pH
range of
extracellular fluids within the human body. Thus, these hydrogels can be
easily
delivered to an implanted support structure as a liquid and automatically form
a semi-
solid gel when exposed to physiological pH. Examples of such pH-dependent
hydrogels are TETRONICSTm (BASF-Wyandotte)
polyoxyethylene-polyoxypropylene polymers of ethylene diamine, poly(diethyl
aminoethyl methacrylate-g-ethylene glycol), and poly(2-hydroxymethyl
methacrylate). These copolymers can be manipulated by standard techniques to
affect
their physical properties.
d. Light Solidified Hydrogels
Other hydrogels that can be used to administer spore-like cells or their
progeny are solidified by either visible or ultraviolet light. These hydrogels
are made
of macromers including a water-soluble region, a biodegradable region, and at
least
two polymerizable regions (see, e.g., U.S. Patent No. 5,410,016). For example,
the
hydrogel can begin with a biodegradable, polymerizable macromer including a
core,
an extension on each end of the core, and an end cap on each extension. The
core is a
hydrophilic polymer, the extensions are biodegradable polymers, and the end
caps are
oligomers capable of cross-linking the macromers upon exposure to visible or
ultraviolet light, for example, long wavelength ultraviolet light.
Examples of such light solidified hydrogels include polyethylene oxide
block copolymers, polyethylene glycol polylactic acid copolymers with acrylate
end
groups, and 10K polyethylene glycol-glycolide copolymer capped by an acrylate
at
both ends. As with the PLURONICTM hydrogels, the copolymers comprising these
hydrogels can be manipulated by standard techniques to modify their physical
properties (such as rate of degradation, differences in crystallinity, and
degree of
rigidity).
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2. Preparation of Hydrogel-Cell Mixtures
Once a hydrogel of choice (e.g., a thermosensitive polymer at between
5% and 25% (WN), or an ionic hydrogel such as alginate dissolved in an aqueous
solution (e.g., a 0.1 M potassium phosphate solution, at physiological pH, to
a
concentration between 0.5% to 2% by weight) is prepared, isolated spore-like
cells or
their progeny (with or without differentiated cells) are suspended in the
polymer
solution. If desired, the concentration of the cells can mimic that of the
tissue to be
generated. For example, the concentration of cells can range between 10 and
100
million cells/m1 (e.g., between 20 and 50 million cells/ml or between 50 and
80
million cells/m1). Of course, the optimal concentration of cells to be
delivered into
the support structure can be determined on a case by case basis, and will vary
depending on cell type and the region of the patient's body into which the
support
structure is implanted or onto which it is applied. To optimize the procedure
(i.e., to
provide optimal viscosity and cell number), one need only vary the
concentrations of
the cells or the hydrogel.
3. Support Structures
The support structure is a permeable structure having pore-like cavities or
interstices that shape and support the hydrogel-cell mixture. For example, the
support
structure can be a porous polymer mesh, or a natural or synthetic sponge. The
porosity of the support structure should be such that nutrients can diffuse
into the
structure, thereby effectively reaching the cells inside, and products
produced by the
cells (either products that can harm the cells, such as waste products, or
products that
can aid the patient (e.g., insulin, somatostatin, alpha-galactosidase A)) can
diffuse out
of the structure.
The support structure can be shaped to conform to the space in which new
tissue is desired. For example, the support structure can be shaped to conform
to the
shape of an area of the skin that has been burned or the portion of cartilage
or bone
that has been lost. Depending on the material from which it is made, the
support
structure can be shaped by cutting, molding, casting, or any other method that
produces a desired shape (as described below, in some instances, the support
structure
can be shaped by hand). Moreover, the shaping process can occur either before
or
after the support structure is filled with the hydrogel-cell mixture. For
example, a
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support structure can be filled with a hydrogel-cell mixture and, as the
hydrogel
hardens, molded into a desired shape by hand.
As the hydrogel solidifies, it will adopt the flexibility and resiliency of
the
support structure, which can be important for accommodation of compressive and
tensile forces. Thus, for example, replaced skin will accommodate tensile
forces
associated with pulling and stretching, as well as compressive forces
associated with
weight bearing, as occurs, for example, on the soles of the feet. The
flexibility and
resiliency of the support structure also provides greater ease of
administration than do
many currently available skin replacement methods in which the tissue is
extremely
delicate and must therefore be handled with the utmost care.
Like the hydrogel itself, the support structure is biocompatible (i.e., it is
not toxic to the spore-like cells suspended therein) and can be biodegradable.
Thus,
the support structure can be formed from a synthetic polymer such as a
polyanhydride, polyorthoester, or polyglycolic acid. The polymer should
provide the
support structure with an adequate shape and promote cell survival and, when
required proliferation and differentiation, by allowing nutrients to reach the
cells.
Additional factors, such as growth factors, other factors that induce
differentiation or
dedifferentiation, secretion products, immunomodulators, anti-inflammatory
agents,
regression factors, biologically active compounds that promote innervation or
enhance
the lymphatic network, and drugs, can be incorporated into the polymer support
structure.
An example of a suitable polymer is polyglactin, which is a 90:10
copolymer of glycolide and lactide, and is manufactured as VICRYLTM braided
absorbable suture (Ethicon Co., Somerville, NJ). Polymer fibers (such as
VICRYLTM) can be woven or compressed into a felt-like polymer sheet, which can
then be cut into any desired shape. Alternatively, the polymer fibers can
be
compressed together in a mold that casts them into the shape desired for the
support
structure. In some cases, additional polymer can be added to the polymer
fibers as
they are molded to revise or impart additional structure to the fiber mesh.
For
example, a polylactic acid solution can be added to this sheet of polyglycolic
fiber
mesh, and the combination can be molded together to form a porous support
structure.
The polylactic acid binds the crosslinks of the polyglycolic acid fibers,
thereby
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coating these individual fibers and fixing the shape of the molded fibers. The
polylactic acid also fills in the spaces between the fibers. Thus, porosity
can be varied
according to the amount of polylactic acid introduced into the support. The
pressure
required to mold the fiber mesh into a desirable shape can be quite moderate.
All that
is required is that the fibers are held in place long enough for the binding
and coating
action of polylactic acid to take effect.
Alternatively, or in addition, the support structure can include other types
of polymer fibers or polymer structures produced by techniques known in the
art. For
example, thin polymer films can be obtained by evaporating solvent from a
polymer
solution. These films can be cast into a desired shape if the polymer solution
is
evaporated from a mold having the relief pattern of the desired shape. Polymer
gels
can also be molded into thin, permeable polymer structures using compression
molding techniques known in the art.
Many other types of support structures are also possible. For example, the
support structure can be formed from sponges, foams, corals, or biocompatible
inorganic structures having internal pores, or mesh sheets of interwoven
polymer
fibers. These support structures can be prepared using known methods.
4. Application of the Support Structure
Any of the liquid hydrogel-cell mixtures described above can be placed in
any of the permeable support structures (also described above). Fig. 13 is a
schematic
of a filled support structure in cross-section. This structure, as
illustrated, is suitable
for application of compositions containing spore-like cells, or their progeny,
to the
skin. The support structure 10 is formed from a bilayered mesh of interwoven
polymer fibers 12 having epidermal layer 12a and dermal layer 12b. The spaces
between the fibers form interconnected pores 14 that are filled with liquid
hydrogel-
cell mixture. Within a short time of placing the mixture in the support
structure (e.g.,
in approximately three to five minutes), hydrogel 16 solidifies, thereby
keeping
suspended cells 18 within pores 14 of support structure 10. The solidified
hydrogel
16 helps maintain the viability of the cells by allowing diffusion of
nutrients
(including growth and differentiation factors) and waste products through the
interconnected pores of the support structure. The ultimate result is the
growth of
new skin and its engraftinent to the patient's body.
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The liquid hydrogel-cell mixture can be delivered to the shaped support
structure either before or after the support structure is implanted in or
applied to a
patient. The specific method of delivery will depend on whether the support
structure
is sufficiently "sponge-like" for the given viscosity of the hydrogel-cell
composition,
i.e., whether the support structure easily retains the liquid hydrogel-cell
mixture
before it solidifies. Sponge-like support structures can be immersed within,
and
saturated with, the liquid hydrogel-cell mixture, and subsequently removed
from the
mixture. The hydrogel is then allowed to solidify within the support
structure. The
hydrogel-cell-containing support structure is then implanted in or otherwise
administered to the patient.
The support structure can also be applied to the patient before the hydrogel
completely solidifies. Alternatively, a sponge-like support structure can be
injected
with the liquid hydrogel-cell mixture, either before or after the support
structure is
implanted in or otherwise administered to the patient. The hydrogel-cell
mixture is
then allowed to solidify.
The volume of the liquid hydrogel-cell mixture injected into the support
structure is typically less thari, but somewhat comparable to, the volume of
the
support structure, i.e., the volume of the desired tissue to be grown.
Support structures that do not easily retain the liquid composition require
somewhat different methods. In those cases, for example, the support structure
is
immersed within and saturated with the liquid hydrogel-cell mixture, which is
then
allowed to partially solidify. Once the cell-containing hydrogel has
solidified to the
point where the support structure can retain the hydrogel, the support
structure is
removed from the partially solidified hydrogel, and, if necessary, partially
solidified
hydrogel that remains attached to the outside of the support structure is
removed (e.g.,
scraped off the structure).
Alternatively, the liquid hydrogel-cell mixture can be delivered into a
mold containing the support structure. For example, the liquid hydrogel-cell
mixture
can be injected into an otherwise fluid-tight mold that contains the support
structure
and matches its outer shape and size. The hydrogel is then solidified within
the mold,
for example, by heating, cooling, light-exposure, or pH adjustment, after
which, the
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hydrogel-cell-containing support structure can be removed from the mold in a
form
that is ready for administration to a patient.
In other embodiments, the support structure is implanted in or otherwise
administered to the patient (e.g., placed over the site of a burn or other
wound, placed
beneath the renal capsule, or within a region of the body damaged by
ischemia), and
the liquid hydrogel-cell mixture is then delivered to the support structure.
As noted
above, the hydrogel-cell mixture can be delivered to the support using any
simple
device, such as a syringe or catheter, or merely by pouring, dripping, or
brushing a
liquid gel onto a support structure (e.g., a sheet-like structure).
Here again, the volume of hydrogel-cell composition added to the support
structure should approximate the size of the support structure (i.e., the
volume
displaced by the desired tissue to be grown). The support structure provides
space
and a structural template for the injected liquid hydrogel-cell mixture. In
the event
some of the hydrogel-cell mixture leaks from the support structure prior to
solidifying, existing tissue beneath or around the support structure will
constrain the
liquid hydrogel-cell mixture until it gels.
In any of the above cases, the hydrogel is solidified using a method that
corresponds to the particular hydrogel used (e.g., gently heating a
composition
including a PLURONICTM temperature-sensitive hydrogel).
To apply or implant the support structure, the implantation site within the
patient can be prepared (e.g., where the support structure is applied to the
skin, the
area can be prepared by debridement), and the support structure can be
implanted or
otherwise applied directly at that site. If necessary, during implantation,
the site can
be cleared of bodily fluids such as blood (e.g., with a burst of air or
suction).
5. Administration of Cells with Specific Skin Replacement Therapies
Spore-like cells, their non-terminally differentiated progeny, one or more
types of differentiated cells, or a combination of these cell types (e.g.,
spore-like cells
and skin progenitor cells or spore-like cells and keratinocytes or
fibroblasts) can also
be administered by way of existing skin replacement therapies. Including the
novel
cells of the present invention improves existing methods because the present
cells can
differentiate into cell types and structures that would not otherwise be
present, and
thereby improve the structure, functions, and appearance of the replacement
skin.
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a. VICRYLTM Mesh
Spore-like cells and skin progenitor cells can be administered in
connection with dermal tissue replacements that are placed beneath meshed,
split-
thickness skin grafts. To construct the tissue replacement, human dermal
fibroblasts
are isolated from neonatal foreskin as follows. Epidermis and dermis are
separated by
incubation in 0.25% trypsin with 0.2% ethylenediaminetetraacetic acid for 1-2
hours
at 37 C. The dermis is minced and digested with collagenase B, and the tissue
digest
is filtered through sterile gauze to remove debris. Fibroblasts are maintained
in
Dulbecco's modified eagle's medium (DMEM) and passaged when cells reach 80-
90% confluence. Cells are removed from flasks and resuspended for seeding at a
concentration of 4 x 106 cells/ml.
Spore-like cells or skin progenitor cells are isolated and cultured as
described herein, and resuspended for seeding at a concentration of
approximately
4 x 106 cells/ml.
Dermal grafts are then prepared by seeding viable fibroblasts and spore-
like cells and/or skin progenitor cells in a minimum volume of DMEM onto each
4
cm2 area of VICRYLTm mesh (Ethicon Inc., Somerville, NJ). The cells readily
attach
in vitro to the mesh fibers, and they become confluent in 2 to 3 weeks (i.e.,
all mesh
openings are covered by cells and tissue matrix, according to assay by
inverted phase
microscopy).
The patient's wound can be excised to subcutaneous fat, to fascia, or to
deep dermis and fat. Hemostatis can be achieved with topical thrombin-
epinephrine
solution and electrocoagulation. The graft, prepared as described above, is
placed on
the prepared site and can be affixed to the wound margins with staples or
sutures. If
desired, a hydrogel can be used together with the VicrylTM mesh.
b. Collagen-based Skin Substitutes
Spore-like cells, skin progenitor cells, or compositions of cells containing
them can also be used in conjunction with numerous permanent dermal skin
replacements that include, for example, resorbable synthetic composites
containing
collagen and chondroitin-6-sulfate (e.g., glycosaminoglycan (GAG) dermal
membranes are described by Burke et al., Ann. Surg. 194:413-428, 1981; Yarmas
et
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al., Science 214:174-176, 1982; and Yannas et al., U.S. Patent No. 4,060,081).
The
present cells, alone or in combination with one another or with one or more
differentiated cell types can also be used in lieu of keratinocytes in the
dermal
collagen-chondroitin-6-sulfate membrane described in EP 0 363 400 B1 or in the
collagen matrices that include transforming growth factor-a (TGF-a) described
in
U.S. Patent No. 5,800,811. TGF-a impregnated collagen matrices may be
advantageous in that they are thought to inhibit inflammatory processes while
promoting angiogenesis and histogenesis.
c. Multilayer Skin Substitutes
Spore-like cells, skin progenitor cells, or compositions of cells containing
them are especially well suited for use with multilayer skin substitutes.
Spore-like
cells or progenitor cells isolated from the dermis can be incorporated in the
lower or
"dermal" layer, where they would subsequently differentiate into adnexal
structures,
and spore-like cells or progenitor cells isolated from the epidermis can be
incorporated in the upper or "epidermal" layer, where they would subsequently
differentiate into melanocytes cells, keratinocytes, and Merkel cells. Several
multilayer skin equivalents that can be used in conjunction with compositions
that
include the cells of the present invention are described in WO 97/41208.
d. In Vivo Testing
Numerous in vivo models of wound healing are available to evaluate and
optimize the performance of spore-like cells, skin progenitor cells, and
compositions
containing them. For example, the ability to repair a burn can be tested in
domestic
outbred swine. The swine are anaesthesized by ketamine hydrochloride, followed
by
inhalation of a mixture of halothane, nitrous oxide, and oxygen. A portion of
their
skin is shaved, washed twice with BETADINETm, and washed once with 70%
alcohol.
Wounds that are 1 min deep are then created with a Brown dermatome on the
lateral
side. Wounds of this depth correspond to deep second degree burns and remove
all of
the epidermis and most of the dennis. Of course, the depth of the injury can
be
increased to mimic a more severe burn or decreased to mimic a less severe
burn. The
size of the wounds can also be varied. Typically, a wound is approximately 2.5
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inches square. Following surgery, the pigs are treated with analgesics to
alleviate
their pain.
A second reliable skin injury model has been developed in pigs to study
the use of hyaluronidase in the treatment of intravenous extravasation
injuries (Raszka
et al., J. Perinatol. 10:146-149, 1990). In this model, the flanks of recently
weaned
Yorkshire pigs are shaved and then cleaned with alcohol and povidone-iodine
solutions. A subcutaneous injection of CaCl2 (300 mEq/L; 2.0 ml) is then
given,
which causes ulceration (an area of full thickness skin necrosis greater than
0.5 cm2).
Murine wound healing models are also available. For example, Moore et
al. (Br. J. Cancer 66:1037-1043, 1992) describe a model that can be used to
compare
patterns of damage to skin and its supporting vasculature following treatment
by
hyperthennia and photodynamic therapy. More straight-forward injury models are
also available. For example, rodents used in studies of hyperthennia have
received
burns by exposure to a heat source. For example, an area of the skin can be
injured by
exposure to hot water (98 C) for a given number of seconds (9 seconds; as in
Farriol
et al., Burns 20:496-498, 1994). As in the models above, the animals are
medicated
to alleviate their pain.
EXAMPLES
The present invention will be further understood by reference to the
following non-limiting examples.
Example 1
Spore-like cells were isolated from human blood as follows. Samples of
whole blood (approximately five ml each) were acquired from adult humans and
placed in a tube containing an anti-coagulant. The samples were then
centrifuged
at 1200 rpm for approximately five minutes. The supernatant was removed and
the
resulting pellet was resuspended in DMEM/F-12 medium (15 ml) supplemented with
a combination of the following hormones and nutrients: glucose (23 mM),
transferrin
(10 mg/ml), insulin (20 mg/ml), putricine (10 mM), selenium (100 nM),
progesterone
(10 nM) (Life Technologies, Baltimore, MD), EGF (20 ng/ml), and bFGF (20
ng/ml)
(Collaborative Biomedical Products, Chicago, IL). The resulting suspension was
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transferred to 75 cm2 tissue culture flasks and incubated in 5% CO2 at 37 C.
The
media were changed every 3-4 days. Cells were passaged every 7-9 days.
Initially,
these culture flasks appeared to contain many hematopoeitic cells (e.g., red
blood
cells), but over time (usually, a matter of several days), these cells
disappeared,
leaving only spore-like cells.
After several days in culture, the spore-like cells multiplied and coalesced
to form clusters of cells. Trypan blue exclusion revealed cell viability to be
greater
than 90%. Figs. 11 and 12A are photographs of cultures that include
undifferentiated
spore-like cells isolated from adult human blood. The cells shown in Fig. 12A
were
isolated seven days earlier and are viewed with phase contrast microscopy.
Immunofluorescent staining was then performed. At this time, some of the cells
expressed nestin (see Fig. 12B).
Example 2
Spore-like cells were isolated from the skin of an adult rodent as follows.
Excisional biopsies of the skin of adult Fisher rats were made under sterile
conditions.
The biopsied tissue, which included the dermis and epidermis, was placed in a
petri
dish containing cold (50 C) phosphate buffered saline (PBS) and antibiotics
(penicillin (50 MU/m1) and streptomycin (90 mg/ml)). The epidermis was scraped
with a #11 scalpel to disassociate epidermal cells, and the tissue was then
transferred
to a second petri dish (also containing cold PBS and antibiotics) where the
dermis was
scraped with a #11 scalpel. The cells that were dissociated were then
centrifuged at
1200 rpm (GLC-2B, Sorvall, Wilmington, DE) for five minutes and resuspended in
10 ml of 0.05% trypsin (Life Technologies, Baltimore, MD). Following
resuspension
in trypsin, the tissue was incubated at 37 C for five minutes. Ten ml of
Dulbecco's
Modified Eagle Medium (DMEM)/F-12 containing 10% heat inactivated fetal bovine
serum (FBS) (Life Technologies, Baltimore, MD) was added to deactivate the
trypsin.
The tissue was then triturated, first with a normal bore Pasteur pipette and
subsequently with a series of fire polished pipettes having bores reduced to
about
15 ..in. The number of pipettes required can vary, depending upon a number of
factors, including the initial size of the tissue fragments obtained by
scraping the
excised skin and how frequently the pipettes become clogged with tissue.
Trituration
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was carried out until the tissue was dispersed as a fine suspension. The
suspension
was then centrifuged at 1200 rpm (GLC-2B, Sorvall, Wilmington, DE) for five
minutes. The supernatant was removed and the pellet was resuspended in 15 ml
of
DMEM/F-12 medium supplemented with a hormone mixture containing glucose
(23 mM), transfeiTin (10 mg/ml), insulin (20 mg/ml), putricine (10 mM),
selenium
(100 nM), progesterone (10 nM) (Life Technologies, Baltimore, MD), EGF
(20 ng/ml) and bFGF (20 ng/ml) (Collaborative Biomedical Products, Chicago,
IL).
The suspension was transferred to 75 cm2 tissue culture flasks (Collaborative
Biomedical Products, Chicago, IL) and incubated at 37 C in 5% CO2. The media
was
changed every three days and cells were passaged every 7-9 days. The cells
that
attached to the tissue culture flask appeared to differentiate more readily.
Spore-like cells isolated from the skin will differentiate upon exposure to
the processes and basal nutrient media described in U.S. Patent No. 5,292,655.
Alternatively, growth factors that cause spore-like cells to mitose (e.g.,
epidermal
growth factor (EGF), basic fibroblast growth factor (bFGF) and other
cytokines) can
be applied to help maintain the cells in an undifferentiated state. For
example, the
isolated cells can be cultured in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with a hormone mixture containing glucose, transferrin, insulin,
putricine, selenium, progesterone, EGF, and bFGF.
Spore-like cells were also isolated from excisional biopsies of the skin of
adult pigs according to the sane protocol described here for the adult rat.
Spore-like cells and skin progenitor cells isolated and cultured as described
herein can differentiate in culture. The progenitor cells have been seen to
form an
unidentified adnexal skin structure, a primitive adnexal gland. Without
limiting the
invention to cells that differentiate by a particular mechanism, it is
believed that
progenitor cells may develop along various and committed lineages depending on
the
cues they receive from neighboring cells; it may be that a concerted and
highly
interactive process directs the progenitor cell down a particular
differentiation
pathway.
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A biocompatible, biodegradable, reverse-thermosensitive copolymer gel
can be obtained by preparing a 30% weight/volume solution of a PLURONICTM
F127, F68 block copolymer (available from BASF). The solution remains in a
liquid
state at less than 15 C, and solidifies within 5 to 10 minutes as the
temperature is
increased to over 15 C. Skin progenitor cells isolated as described above are
added to
the hydrogel mixture to generate a final cellular density of about 2 x 106 to
6 x 107
cells per ml. Such a mixture can be delivered into a permeable support
structure.
Example 3
Spore-like cells were isolated from adult rat heart according to the protocol
described in Example 2. The newly isolated cells, which are shown in Fig. 3A,
include undifferentiated spore-like cells. After culturing the spore-like
cells for three
days, early myocardial cells can be seen (Fig. 3B), and after two weeks in
culture,
Purkinje-like structures can be seen (Fig. 3C).
Example 4
Spore-like cells were isolated from adult rat intestine according to the
protocol described in Example 2. The newly isolated cells, as shown in Fig.
4A,
include undifferentiated spore-like cells. After culturing the cells for three
days,
clusters of small intestinal cells (Fig. 4B) and autonomic neurons (Fig. 4C)
can be
seen.
Example 5
Spore-like cells were isolated from an adult rat bladder according to the
protocol described in Example 2. The newly isolated cells, which are shown in
Fig. 5A, include undifferentiated spore-like cells. After culturing the spore-
like cells
for two days, the isolated spore-like cells, or their progeny, appear to be
differentiating into mature bladder cells (Fig. 5B).
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Example 6
Spore-like cells were isolated from an adult rat kidney according to the
protocol described in Example 2. Cells newly isolated from the kidney of an
adult rat,
which are shown in Fig. 6A, include undifferentiated spore-like cells. After
culturing
the spore-like cells for three days, aggregates of cells resembling kidney
structures
can be seen (Fig. 6B).
Example 7
Spore-like cells were isolated from an adult rat liver according to the
protocol described in Example 2. Because the liver is highly vascularized, the
intact
tissue was washed with PBS. Cells newly isolated from the liver of an adult
rat,
which are shown in Figs. 7A and 7C, include undifferentiated spore-like cells.
After
culturing the spore-like cells for three days, an aggregate of cells
resembling a
differentiating liver structure can be seen (Fig. 7B). After culturing the
spore-like
cells for seven days, cells resembling hepatocytes can be seen (Fig. 7D).
Example 8
Spore-like cells were isolated from adult mammalian lungs according to
the protocol described in Example 2. Spore-like cells were isolated from the
lungs of
adult rats (see Figs. 8A-8C) and sheep (see Fig. 8D). The newly isolated cells
shown
in Fig. 8A include undifferentiated spore-like cells. After six weeks in
culture,
alveolar-like cells can be seen (Figs. 8B and 8C). After culturing the spore-
like cells
for 30 days, spore-like cells isolated from an adult sheep have formed
alveolar-like
structures (Fig. 8D) similar to those seen in the lungs of adult cats (Fig.
8E; Histology,
F. Hammersen, Ed., Urban & Schwarzenberg, Baltimore-Munich, 1980, Fig. 321).
Example 9
Spore-like cells were isolated from adult rat adrenal glands according to
the protocol described in Example 2. Undifferentiated spore-like cells
isolated from
the adrenal gland of an adult rat can be seen at Day 0 in Figs. 9A and 9B (see
the
arrows). After culturing the spore-like cells for two days, primitive adrenal
cells can
be seen (Figs. 9C and 9D).
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Example 10
Spore-like cells were isolated from the pancreas of an adult human and
from the pancreas of an adult rat. The dissections were carried out in 10%
cold fetal
serum albumin according to the protocol described in Example 2. Significantly,
spore-like cells have been isolated from a portion of the rat pancreas that
remained
after the islets were removed by ductal injection of collagenase (as
described, for
example, by Sutton et al., Transplantation, 42:689-691, 1986).
Islet-like structures that formed in cultures of spore-like cells isolated
from
islet-free pancreatic tissue are shown in Figs. 10A-10C. After six days in
culture,
more than 100 islet-like structures were present per field (see Figs. 10A and
10B),
even though the spore-like cells first placed in culture were isolated from a
tissue
from which the islets had been removed. When the islet-like structures that
nevertheless developed were inununostained, insulin expression can be seen
(Fig.
10C).
Example 11
To isolate spore-like cells and retinal progenitor cells from the retina of an
adult fisher rat, the animal's eyes were removed under sterile conditions and
cut in
half using a #11 scalpel. The retinal tissue was then scraped and
disassociated with a
# 11 scalpel in a petri dish containing cold (50 C) phosphate buffered saline
with
antibiotics (penicillin (50 mU/m1) and streptomycin (90 mg/ml)). The
dissociated
tissue was then centrifuged at 1200 rpm (GLC-2B, Sorvall, Wilmington, DE) for
five
minutes and resuspended in 10 ml of 0.05% trypsin (Life Technologies,
Baltimore,
MD) for an additional five minutes at 37 C. Ten ml of Dulbecco's Modified
Eagle
Medium (DMEM)/F-12, containing 10% heat inactivated fetal bovine serum (FBS)
(Life Technologies, Baltimore, MD) was then added to deactivate the trypsin.
The
samples were triturated with a normal bore pasteur pipette, followed by
sequential
trituration with fire polished, reduced bore pasteur pipettes, the smallest
being about
15 microns. Trituration was carried out until the tissue was dispersed as a
fine
suspension, and the solution was then centrifuged at 1200 rpm (GLC-2B,
Sorvall,
Willmington, DE) for five minutes. The supernatant was removed, and the
resulting
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pellet was resuspended in 15 ml of DMEM/F-12 medium (Life Technologies,
Baltimore, MD) supplemented with the hormone mixture described in Example 2.
The suspension was transferred to 75 cm2 tissue culture flasks (Collaborative
Biomedical Products, Chicago, IL) and incubated at 37 C in 5% CO2. The media
was
changed every three days, and cells were passaged every 7-9 days.
The isolation procedure described above can also be carried out following
enucleation of the eye from a deceased human donor. This procedure is well
established and is carried out under sterile conditions. To remove retinal
tissue from
an intact eye, vitreous humor is removed (vitrectomy) and cells, generally
from the
periphery of the retina, are removed using standard retinal biopsy techniques.
Spore-like cells and retinal progenitors isolated and cultured as described
herein can differentiate in culture. These cells have been seen to assume
morphologies reminiscent of both rods and cones. As with spore-like cells
isolated
from other sources, spore-like cells isolated from the retina and their
progeny may
develop along various and committed lineages depending on the cues they
receive
from neighboring cells; a concerted and highly interactive process directs the
progenitor cell down a particular differentiation pathway.
Retinal repair can be assessed in vivo using techniques routinely practiced
by those of ordinary skill in the art in various animal models (e.g., rabbit).
For
example, following implantation of spore-like cells, retinal progenitor cells,
or
compositions containing both, one can record cortical evoked responses to
light using
standard electrophysiology techniques. Recordings made before implantation of
the
cells can also be made to provide a baseline measurement. Of course behavioral
tests
and standard tests for visual acuity can also be performed.
Example 12
Due in part to the unusual appearance of spore-like cells under the light
microscope, the cells were examined under an electron microscope. Scanning and
electron microscopy was performed according to standard protocols. The
electron
micrographs revealed several interesting features. For example, the range of
spore-
like cell sizes may be greater than first appreciated with the light
microscope. Some
of the spore-like cells shown in Fig. lA have a diameter of approximately 0.3
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microns. The unique cytoarchitecture of the spore-like cell is apparent when
viewed
with transmission electron microscopy (see Figs. 2A-2D) or following nuclear
staining (such as the 4'6-diamidino-2-phenylindole (DAPI) stain described in
Example 13). The interior of the spore-like cell is comprised largely of
diffuse
nuclear material and some cells are surrounded by a "zebra" coating, which is
associated with deposits of glycolipids (i.e., carbohydrate and fat). For
example,
zebra bodies (so-called because of their striped appearance) are associated
with
mucopolysaccharidoses, such as Hurler's syndrome or with Fabry's disease, in
which
glycolipids accumulate due to an enzyme deficiency. Spore-like cells thus
appear,
during at least one stage of their existence, to be unique packets of DNA.
Example 13
A massive accumulation of nuclear material is also apparent when spore-
like cells are stained for nucleic acids by methods known to those of ordinary
skill in
the art. For example, DNA can be stained with either 4'6,-diamidino-2-
phenylindole
(DAPI) for total DNA staining or with propidium iodide for staining of double-
stranded DNA and RNA. DAPI and propidium iodide can be added directly to
antifade mounting medium (e.g., 90% glycerol, 1X PBS, and 2.5% 1,4-
diazabicyclo[2,2,2]octane (DABCO) (Sigma Chemical Co., St. Louis, MO). Spore-
like cells stained with DAPI contained a great deal of nuclear material; the
ratio of
nuclear to cytoplasmic material was much higher in spore-like cells than one
would
expect in most fully differentiated cell types.
Example 14
Four tissues (lung, liver, fascia, and spinal cord) were obtained from three
animals (Fisher rats) and kept in cold storage for five days. More
specifically, each
tissue type was removed from an animal less than two hours after the animal
was
killed and placed in a 50 cc centrifuge tube (Fisher Scientific, Pittsburg,
PA) filled
with PBS. The tubes were stored at 4 C without supplemental oxygen for five
days.
Spore-like cells were then isolated as follows.
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After excision from the animal, and using sterile technique, the selected
tissue was placed in cold PBS containing penicillin (50 mU/m1) and
streptomycin
(90 mg/ml) (Gibco, Grand Island, NY). The tissue was then manually
disassociated
with a #11 scalpel, and the disassociated cells were collected by
centrifugation at
1200 rpm for five minutes. The tissue was then resuspended in ten ml of 0.05%
trypsin (w/v) for five minutes at 37 C. The trypsin was inactivated by adding
10 ml
of DMEM/F-12 medium (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (Gibco). The cells were then dispersed by trituration using
progressively narrower fire-polished, reduced-bore pasteur pipettes. While the
aperatures are not measured, the opening of the smallest-bore pipette was
approximately 15 gm. The dispersed cells were collected by centrifugation at
1200
rpm for five minutes. The resulting pellet was resuspended in 10 ml of DMEM/F-
12
medium containing 33 mM glucose (Sigma Chemical Co., St. Louis, MO), 10 mg/ml
transferrin (Sigma), 20 mg/ml insulin (Sigma), 10 mM putrescine (Sigma), 100
riM
selenium (Sigma), 10 nM progesterone (Sigma), 20 ng/ml EGF (Peprotech, Rocky
Hill, NJ), and 20 ng/ml bFGF (Collaborative Biomedical, Raynham, MA). The
primary cell suspension was incubated at 37 C in 5% CO2, and the media were
changed every 3 days. Cells were passaged every 7-9 days by collecting the
nonadherent cell aggregates, centrifuging them at 1200 rpm for five minutes
and
removing the media. Cells were resuspended in fresh media, triturated using
narrow
fire polished reduced bore pasteur pipettes. The cell suspension was then
divided into
two suspensions and placed into two new culture dishes.
The technique described above was slightly modified to isolate hepatic
tissue: hepatic tissue was washed with cold PBS prior to disassociation.
Standard hematoxalin and eosin (H&E) staining was performed on tissue
fixed with 10% formalin. A simple Hall's stain was performed on liver-derived
spore-like cells for the presence of bile. Standard stains for mucicannine and
periodic
acid-Schiff were also performed.
To assess cellular proliferation, the time that was required for a population
of cells to double its number was estimated using periodic phase microscopy
field
counts (10 fields counted and averaged at 100x) or viable cell counts using
Trypan
blue with a hemocytometer.
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Based on the exclusion of Trypan blue, approximately 50% of the spore-
like cells within each of the four tissues that were exposed to 4 C, without
supplemental oxygen, for five days, remained viable at the end of that period.
Moreover, the spore-like cells isolated from lung, liver, fascia, and spinal
cord
retained their ability to proliferate and differentiate into tissue-specific
structures.
Example 15
After being killed, whole animals (Fisher rats) were placed in plastic bags
and stored in a freezer at ¨86 C. After being frozen for either two or eight
weeks, the
animals were removed from the freezer and placed in a 37 C water bath until
their
tissues thawed. Four tissues (lung, liver, fascia, and spinal cord) were then
harvested,
and spore-like cells were isolated as described in Example 14 and assessed by
Trypan
blue exclusion for viability. Spore-like cells could be obtained from oxygen-
deprived
and deeply frozen tissue just as they were from oxygen-deprived and chilled
tissue.
Approximately 50% of the spore-like cells within each of the four tissues that
were
exposed to -86 C, without supplemental oxygen, for two or eight weeks,
remained
viable at the end of those periods. Moreover, the spore-like cells isolated
from lung,
liver, fascia, and spinal cord retained their ability to proliferate and
differentiate into
tissue-specific structures.
Example 16
Four tissues (lung, liver, fascia, and spinal cord) were obtained from three
animals (Fisher rats) and heated to 85 C for 30 minutes. More specifically,
each
tissue type was removed from an animal less than two hours after the animal
was
killed and placed in a 50 cc centrifuge tube (Fisher Scientific, Pittsburg,
PA) filled
with PBS. The tubes were then placed in a heated water bath as the temperature
of
the bath was raised to 85 C. The temperature was monitored with sterile
thermometers, which were placed within each tube. The tubes were left in the
water
bath for 45 minutes after the temperature reached 85 C. The tissue was then
allowed
to cool to room temperature, and spore-like cells were isolated as described
in
Example 14.
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Based on the exclusion of Trypan blue, approximately 50% of the spore-
like cells within each of the four tissues that were heated to 85 C for 30
minutes
remained viable at the end of that period. Moreover, the spore-like cells
isolated from
lung, liver, fascia, and spinal cord retained their ability to proliferate and
differentiate
into tissue-specific structures.
Other Embodiments
One of ordinary skill in the art will appreciate that the spore-like cells
described herein can be administered in connection with existing tissue
engineering
methods, in lieu of differentiated cells in cell-based therapies, and in lieu
of cells
presently administered following genetic manipulation.
It is to be understood that while the invention has been described in
conjunction with the detailed description thereof, that the foregoing
description is
intended to illustrate and not limit the scope of the invention, which is
defined by the
scope of the appended claims.
Other aspects, advantages, and modifications are within the scope of the
following claims.
What is claimed is:
48
