U.S. patent application number 10/020778 was filed with the patent office on 2002-10-17 for isolation of spore-like cells from tissues exposed to extreme conditions.
Invention is credited to Vacanti, Charles A., Vacanti, Martin P..
Application Number | 20020151050 10/020778 |
Document ID | / |
Family ID | 22922348 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151050 |
Kind Code |
A1 |
Vacanti, Charles A. ; et
al. |
October 17, 2002 |
Isolation of spore-like cells from tissues exposed to extreme
conditions
Abstract
Highly undifferentiated spore-like cells can be isolated from
many different tissues and bodily fluids after those tissues and
fluids have been exposed to extreme conditions. The spore-like
cells can be used to treat a wide variety of disorders.
Inventors: |
Vacanti, Charles A.;
(Lexington, MA) ; Vacanti, Martin P.;
(Westborough, MA) |
Correspondence
Address: |
LEE CREWS, PH.D.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
22922348 |
Appl. No.: |
10/020778 |
Filed: |
October 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60244347 |
Oct 30, 2000 |
|
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
C12N 5/0602 20130101;
A61K 35/12 20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 005/06 |
Claims
What is claimed is:
1. A method for isolating a spore-like cell from a biological
tissue or fluid, the method comprising (a) obtaining a tissue or
fluid that has been exposed to an environment in which
differentiated or partially differentiated cells in the tissue or
fluid die and (b) separating the spore-like cells from the
differentiated or partially differentiated cells that have
died.
2. The method of claim 1, further comprising disrupting the tissue
or fluid either before or after step (a).
3. The method of claim 2, wherein disrupting the tissue or fluid
comprises cutting the tissue into pieces, scraping the tissue with
a blunt instrument, or passing the tissue or fluid through a series
of devices having progressively smaller apertures.
4. The method of claim 3, wherein the devices are pipettes.
5. The method of claim 4, wherein the smallest pipette has an inner
bore diameter of approximately 15 microns.
6. The method of claim 3, wherein the devices are filters.
7. The method of claim 6, wherein the finest filter has a pore size
of approximately 15 microns.
8. The method of claim 1, wherein the biological tissue comprises a
tissue that originates from the endoderm.
9. The method of claim 1, wherein the biological tissue comprises a
tissue that originates from the mesoderm.
10. The method of claim 1, wherein the biological tissue comprises
a tissue that originates from the ectoderm.
11. The method of claim 1, wherein the biological fluid comprises
blood, urine, or saliva.
12. The method of claim 1, wherein the biological fluid is
cerebrospinal fluid.
13. The method of claim 1, wherein the environment is an
oxygen-poor environment.
14. The method of claim 1, wherein the environment is one in which
the temperature is above or below the range of temperatures in
which differentiated or partially differentiated cells can
survive.
15. The method of claim 1, wherein the environment contains a toxin
or infectious agent that kills differentiated or partially
differentiated cells.
16. A method for isolating a spore-like cell from a biological
tissue or fluid, the method comprising obtaining a tissue or fluid
that has been exposed to an environment that is either 42.degree.
C., or more, or 0.degree. C., or less, placing the tissue or fluid
in a tissue culture vessel, allowing the spore-like cells to adhere
to the vessel, and rinsing away non-spore-like cells, the tissue or
fluid having been exposed to the environment without first being
treated with a protective agent.
17. The method of claim 16, further comprising disrupting the
tissue or fluid either before or after exposure to the environment
or before or after placement in the tissue culture vessel.
18. The method of claim 17, wherein disrupting the tissue comprises
cutting the tissue into pieces, scraping the tissue with a blunt
instrument, or passing the tissue or fluid through a series of
devices having progressively smaller apertures.
19. The method of claim 18, wherein the devices are pipettes.
20. The method of claim 14, wherein the smallest pipette has an
inner bore diameter of approximately 15 microns.
21. The method of claim 18, wherein the devices are filters.
22. The method of claim 21, wherein the finest filter has a pore
size of approximately 15 microns.
23. The method of claim 16, wherein the biological tissue or fluid
originates from the endoderm.
24. The method of claim 16, wherein the biological tissue or fluid
originates from the mesoderm.
25. The method of claim 16, wherein the biological tissue is a
tissue that originates from the ectoderm.
26. The method of claim 16, wherein the biological fluid is blood,
urine, or saliva.
27. The method of claim 16, wherein the biological fluid is
cerebrospinal fluid.
28. The method of claim 16, wherein the environment is 0.degree. C.
or less and the protective agent is a cryopreservative.
29. The method of claim 16, wherein the tissue or fluid is
intentionally exposed to the environment.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Ser. No.
60/224,347, filed Oct.30, 2000.
TECHNICAL FIELD
[0002] The invention relates to compositions and methods for tissue
engineering and cell therapies.
BACKGROUND
[0003] Every year, millions of people suffer tissue loss or
end-stage organ failure (see, e.g., Langer and Vacanti, Science
260:920-926, 1993). When possible, physicians treat this loss or
failure by transplanting organs from one individual to another,
performing surgical reconstruction, or using mechanical devices
such as kidney dialyzers. Although these therapies have saved and
improved countless lives, they are imperfect solutions.
Transplantation is severely limited by critical donor shortages,
which worsen every year, and surgical reconstruction can cause
long-term problems. For example, colon cancers often develop after
surgical treatment of incontinence that directs urine into the
colon. Mechanical devices are inconvenient for the patient, and
their performance to date cannot match that of an intact organ.
Few, if any, of these treatments can restore the tissue lost or
prevent progression of the underlying disorder.
[0004] An alternative to the measures described above is tissue
engineering, an interdisciplinary science that applies engineering
and physiological principles to the development of biological
substitutes that maintain, improve, or restore tissue function
(Tissue Engineering, R. Skalak and C. F. Fox, Eds., Alan R. Liss,
New York, N.Y., 1988; Nerem, Ann. Biomed. Eng. 19:529, 1991). Three
general strategies have been adopted for the creation of new
tissue. The first employs isolated cells or cell substitutes. This
approach avoids the complications of surgery, 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 do not always maintain their function in the
recipient, and they can evoke an immune response that results in
their destruction. The second approach 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. The third approach
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. 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. Immunological rejection may be
prevented by immunosuppressive drugs or by the use of autologous
cells.
SUMMARY
[0005] The present invention is based, in part, on the discovery
that highly undifferentiated cells remain viable within, and can be
isolated from, tissue that has been exposed to extreme conditions.
Because these cells are highly undifferentiated and have other
unusual characteristics, some of which are reminiscent of spores,
the cells are called spore-like cells. Spore-like cells are
"undifferentiated" in that, when first isolated, they have the
characteristics described herein regardless of the tissue type from
which they were isolated. The cells can lie dormant in many tissues
(they have been found in all tissues examined to date and seem to
be ubiquitous), and they are also present in bodily fluids such as
the blood. In addition, they are small, have an outer membrane that
is rich in glycolipids (or glycogen) and/or mucopolysaccharides,
and an unusual cytoarchitecture. They are also multipotent, and
they can survive in extreme conditions (e.g., conditions in which
differentiated cells would die). Finally, spore-like cells fail to
demonstrate activity in a microtetrazolium assay (all known living
cells demonstrate redox activity in this assay). These
characteristics are discussed in more detail below.
[0006] Some of the methods of the invention are based on spore-like
cells' remarkable ability to survive under conditions that would
kill differentiated cells or known multipotent stem cells (such as
those found within the bone marrow, nervous system, and pancreas).
For example, spore-like cells are tolerant of oxygen-deprivation.
As shown in the Examples below, spore-like cells can survive in
oxygen-poor (including essentially oxygen-free) environments, such
as those that exist within the tissues of a deceased animal
(including tissues that have been frozen for an extended period of
time), or within a capped container of phosphate-buffered saline
(PBS) for many hours (e.g., four, six, ten, twelve, or 24 hours or
more). Differentiated or partially differentiated cells (e.g., stem
cells) are able to survive oxygen deprivation for variable, but
much more limited, periods of time than spore-like cells can
survive the same deprivation. Differentiated neurons are
particularly sensitive, surviving in an animal for only about 4-15
minutes after oxygen deprivation (caused by, for example, heart
failure, cardiac or respiratory arrest, or a cerebrovascular
accident). Cartilage withstands oxygen deprivation better than most
other tissues. With refrigeration, cartilage can remain viable for
a month or so. But neither differentiated cells nor stem cells can
withstand oxygen deprivation to the same extent spore-like cells
can. The spore-like cells within a tissue (or within the blood) can
outlive the differentiated or partially differentiated cells of
that tissue (or within that blood sample) when the tissue (or blood
sample) is oxygen-deprived or exposed to another of the extreme
conditions described herein. Accordingly, one method of obtaining
(or isolating) spore-like cells includes exposing a tissue to
conditions that kill the differentiated or partially differentiated
cells, but do not kill the spore-like cells therein. The tissue can
be any biological tissue, including an intact tissue, a tissue that
has been disrupted in some way (by, for example, physical
dissociation), or a tissue remnant (e.g., a remnant of skin left at
the scene of an accident or epithelial cells that have been
naturally shed).
[0007] Moreover, spore-like cells can survive (i.e., remain alive
after) oxygen deprivation or other harsh conditions (i.e.,
conditions that would kill a differentiated or partially
differentiated cell) without special preservatives. For example,
hepatocytes can survive ex vivo for approximately two days if they
are specially preserved, but spore-like cells from the liver can
survive for the same period (and much longer) without special
preservatives.
[0008] Spore-like cells can also survive exposure to temperatures
higher or lower than temperatures in which differentiated or
partially differentiated cells can survive. For example, spore-like
cells can survive exposure to temperatures higher or lower than
body temperature (e.g., average body temperature, elevated body
temperature (as occurs, for example, with fever), or depressed body
temperature (as occurs, for example, with hypothermia)). For
example, spore-like cells remain viable within (and can be isolated
from) tissues that are stored at about 4.degree. 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.degree. C. in excess of a physiological body
temperature. That is, viable spore-like cells survive (e.g., within
tissues) exposure to temperatures of 0.degree. C., or below, or
43.degree. C. or above (e.g., 45, 50, 55, 58, 60, 75, 90, 95, or
100.degree. C.). 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 4, 8, 12, or
24 hours or more).
[0009] Because spore-like cells can survive exposure to extreme
conditions, they can be isolated from tissues that have been
exposed to any one of those conditions (the differentiated or
partially differentiated cells having been destroyed by the
condition). For example, spore-like cells can be isolated from an
animal (including a human) that has been dead for many hours, for
several days, or longer. The precise time is not critical. What is
important is that the conditions be such that spore-like cells in
the tissue of interest survive after the differentiated or
partially differentiated cells have died (that is not to say that
every spore-like cell that was in the living animal or tissue
sample need survive; any number of spore-like cells can survive
where no differentiated or partially differentiated cells survive).
As indicated above, differentiated cells within oxygen-sensitive
tissues, such as the brain, are not viable after only short periods
of oxygen deprivation. Thus, the precise conditions required vary
depending on the tissue type used for the isolation procedure; the
extent of the treatment (e.g., the time the tissue must be
oxygen-deprived) need only be sufficient to kill the differentiated
or partially differentiated cells in the tissue used.
[0010] Spore-like cells can also be isolated from tissues that have
been frozen without a cryopreservative. Thus, spore-like cells can
be isolated from any tissue (be it intact or processed or
manipulated in some way) that has been placed in a freezer without
first being treated with a cryopreservative (as noted above, the
time period of exposure need only be sufficient to kill the
differentiated or partially differentiated cells in the animal,
tissue, or bodily fluid). Similarly, spore-like cells 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.
[0011] Although spore-like cells can be isolated from tissue that
has been frozen (or boiled), methods of isolating spore-like cells
can be carried out at temperatures above freezing (or below
boiling). Here again, the temperature must only be low enough or
high enough to kill differentiated or partially differentiated
cells.
[0012] Spore-like cells can be used to analyze fundamental aspects
of cellular differentiation and to treat many types 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. For example, spore-like cells that
differentiate into various types of skin cells can be used to
repair skin damaged by physical, thermal, or chemical trauma.
Similarly, spore-like cells that differentiate into
insulin-secreting cells can be used to treat diabetes; spore-like
cells that differentiate into .alpha.-galactosidase A-expressing
cells can be used to treat Fabry disease; and spore-like cells that
differentiate into cells that express angiogenesis inhibiting
factors, such as an endostatin, or other anti-tumor agents (e.g.,
tumor necrosis factor), can be used to treat cancer. These are
merely examples of the ways in which spore-like cells can be used.
Alternatively, or in addition, one can use spore-like cells that
are engineered to secrete substances such as those described above.
The cells can be made to express a wide variety of substances by
genetic manipulation or exposure to factors that alter their course
of differentiation. Spore-like cells can also be used to treat
patients who have an infection. It is believed that spore-like
cells are so primitive that they remain unaltered by exposure to
agents that infect differentiated cells. For example, a patient who
has hepatitis can be treated by harvesting a portion of the liver,
isolating spore-like cells from that tissue sample, and using the
spore-like cells to reengineer mature liver cells and tissues. All
or a portion of the infected liver can be ablated before the
reengineering process. Similarly, one can treat patients who have
cancer using spore-like cells. For example, spore-like cells can be
isolated from a patient who has a type of leukemia before the
patient undergoes chemotherapy or any other therapy for the cancer.
Following therapy, spore-like cells, which may have been induced to
differentiate ex vivo, can be administered to the patient to
reconstitute a healthy cadre of blood cells. There is no evidence
that spore-like cells are susceptible to the processes that result
in malignancy, and the therapy administered can be very aggressive
(and thereby more likely to kill malignant blood cells and thus,
succeed in eradicating the cancer). The methods of the present
invention may also be useful in forensic science. For example, one
can isolate spore-like cells from a deceased person or from a
sample (e.g., a blood sample) found at a crime scene and use the
cells to identify the person that was their source. For example,
one can place the spore-like cells in culture, allow them to
differentiate, and analyze their genetic material by standard
techniques.
[0013] As noted above, spore-like cells can be isolated from a
tissue, including a tissue that has been exposed to any condition
that is extreme enough to kill the differentiated or partially
differentiated cells within the tissue. For example, the tissue can
be exposed to an oxygen deficient environment, a non-physiological
temperature, an insufficiently moist environment (as in, for
example, a dessicator), or a toxin or any other substance (e.g., a
salt or improperly buffered solution) or event (e.g., radiation)
that kills differentiated or partially differentiated cells. In
particular embodiments, spore-like cells can be isolated from a
tissue that was harvested from an animal whose heart ceased beating
at least four minutes ago (i.e., an animal that has been dead for
at least 4, 10, 20, or 30 minutes; at least 1, 2, 4, 10 or 24
hours; at least 2, 4, 7, 10 or 30 days; at least 5, 10, 20 or 40
weeks; or at least 1, 2, 4, 10 or 100 years). Generally,
differentiated cells will not survive unless they are within 200
.mu.m of an oxygen supply (such as a blood vessel carrying
oxygenated blood), but spore-like cells can survive even if they
are more than 200 .mu.m away from such an oxygen supply.
[0014] In other embodiments, spore-like cells can be isolated from
a tissue that was (or has been) exposed (e.g., placed in a bath of
hot or cold water, a cold room, freezer, or the like) to a
temperature that is more than 42.degree. C. or less than 0.degree.
C. without first being treated with a protective agent (e.g. a
cryopreservative such as glycerol). The isolation procedure
following exposure to any extreme condition can be carried out very
simply. For example, a biological sample that contains cells such
as tissue cells or a bodily fluid that has been exposed to a
condition extreme enough to kill differentiated or partially
differentiated cells can simply be placed in a tissue culture
vessel (e.g., a plate or flask). The dead cells can then be washed
away after the spore-like cells adhere to the vessel. If desired,
the tissue can be disrupted (by, e.g., cutting, shredding, or
scraping it with a blunt instrument) either before or after it is
placed in culture or before or after exposure to an extreme
condition. Given spore-like cells' ability to survive in extreme
conditions, there is no reason to expect that they would not
survive in culture under most, if not all, of the conditions used
to culture differentiated cells. Alternatively, or in addition,
spore-like cells can be isolated by passing a tissue, bodily fluid,
or cell culture medium that contains them through a series of
devices (e.g., size-exclusion devices such as pipettes or filters)
having progressively smaller apertures (the smallest of which can
be approximately 15 .mu.). Smaller diameters (i.e., diameters
smaller than 15 .mu.) can also be used when more aggressive
isolation is desired (i.e., when one desires fewer differentiated
cells in the resulting culture). More aggressive isolation may be
desired when one wishes to maintain the spore-like cells in their
highly undifferentiated state. As described below, the conditions
in which the cells are cultured can be such that their
proliferation is encouraged and their differentiation is
discouraged.
[0015] The ability to survive after being exposed to a condition
that kills differentiated or partially differentiated cells is only
one of the unusual characteristics that can be exploited to
identify and isolate spore-like cells. They can also be identified
and isolated on the basis of their size alone. Although there is
some variation in the size of spore-like cells (see below), it is
clear that many spore-like cells are smaller than any other known
biological cell. Accordingly, one can isolate spore-like cells by
isolating the smallest cells in a tissue or bodily fluid. The
isolation can be carried out by any method known in the art (e.g.,
flow cytometry). Alternatively, the method can be carried out using
a size-exclusion device, such as the pipettes and filters described
above.
[0016] In another aspect, the invention features isolated cells
that are non-terminally differentiated progeny of spore-like cells
that were isolated from non-neural and non-pancreatic tissues and
that develop into mature non-neural and non-pancreatic cells.
[0017] One way to distinguish the cells of the present invention
from previously described cell types is to isolate the present
cells from tissues where no stem cells are known to exist. For
example, conventional wisdom dictates that there are no stem cells
in the liver or the heart. Therefore, any cell isolated from the
liver or heart that is a dividing, non-mature cell is a spore-like
cell of the present invention or a non-terminally differentiated
progeny thereof.
[0018] Spore-like cells can be administered alone, with other cell
types, or in conjunction with tissue engineering constructs
(i.e.,tissue that includes materials or devices used to reengineer
damaged, diseased, or otherwise unhealthy tissue). These constructs
can include support structures, such as a mesh, 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. Thus, the invention features methods for generating an
artificial tissue by, for example, combining hydrogel with a
spore-like cell or the progeny of a spore-like cell. The
hydrogel-cell compositions can also be delivered into a permeable,
biocompatible support structure and used to treat damaged tissue
(e.g., a hydrogel-spore-like cell composition can be applied to the
damaged tissue).
[0019] In addition to methods for generating and repairing tissue,
the invention features methods of treating patients who have a
disorder, such as a skin disorder, a tumor, or a disease, such as
diabetes. These methods are carried out, for example, by
administering a spore-like cell or its progeny to the damaged
region (e.g., the damaged region of the patient's skin, the area
from which the tumor was ablated, or the pancreas). Systemic
administration is also possible. The methods of the invention can
be used to treat a patient who has a deficiency of functional cells
in any of a wide variety of tissues or systems, including the
retina (or other structures associated with vision), auditory
system, nasal epithelium, alimentary canal, pancreas, gallbladder,
bladder, kidney, liver, heart, lung (including respiratory support
structures such as the trachea and smaller airways), nervous
system, reproductive system, endocrine system, immune system, bone,
muscle, tooth, nail, or skin (including the hair follicles).
[0020] Spore-like cells and their progeny 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. Spore-like cells can be isolated from tissues that are
derived from the endoderm, mesoderm, or ectoderm. Similarly,
spore-like cells can differentiate into tissues that derive from
the endoerm, mesoderm, or ectoderm. The germ layers, and the
tissues they give rise to, are well known in the art. An "isolated"
spore-like cell can be one 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 (and their
progeny) that have been significantly enriched (i.e., cultures in
which less than about 10% of the cells are differentiated or
partially differentiated cells).
[0021] The term "disorder" encompasses medical disorders,
conditions, syndromes, illnesses, and diseases, regardless of their
etiology. For example, a disorder amenable to treatment with the
compositions and methods described herein can be caused by trauma,
a genetic defect, an infection, substance abuse, uncontrolled
cellular proliferation, or a degenerative process (e.g., neural
degeneration or muscular atrophy). A given disorder is treated when
the symptoms of the disorder are alleviated or the underlying cause
is eliminated or counteracted, either completely or partially.
[0022] 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-like cells solidify so rapidly that the majority of the
spore-like cells are retained at the application site. This
retention enhances new cell growth at the application site.
However, those of ordinary skill in the art will recognize that
cellular retention is not always necessary. For example, retention
is not necessary when treating a systemic disorder. 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.
[0023] There are many advantages to using spore-like cells. For
example, they can be used to produce sufficient biological material
for tissue engineering. This is not always possible when fully
differentiated cells are used as the starting material. In
addition, spore-like cells can differentiate into a greater variety
of cell types than previously identified progenitor cells isolated
from adult mammals. Thus, spore-like cells can be used to maintain
or repair many, if not all, tissues and organs, including those
(such as the retina) that have not been considered likely
candidates for tissue engineering. The pluripotent nature of
spore-like cells also allows more histologically complete
development of any given tissue. For example, spore-like cells can
be used to engineer skin that is pigmented and that contains
adnexal structures (i.e., accessory structures or appendages such
as hair follicles, sweat glands, sebaceous glands, nail beds, and
specialized sensory receptors that allow us to sense pain,
pressure, temperature, position, etc). The pigmentation and adnexal
structures render the skin replacement a more visually appealing
and functional replacement for natural, undamaged skin. Of course,
disorders affecting the skin are only one of the many types of
disorders that can be treated with spore-like cells. Analogous
benefits will be apparent when systemic disorders or disorders
affecting other organs (e.g., the pancreas, liver, heart or lung)
are treated. The use of spore-like cells may also obviate the need
to obtain cells or tissues from embryonic or fetal tissue and may
therefore diffuse the emotional and political debate that currently
surrounds the research and treatments that rely on embryonic or
fetal tissue.
[0024] 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 specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0025] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1C are scanning electron micrographs of spore-like
cells obtained from the liver of an adult rat. The cells are
magnified 5,000.times. in FIGS. 1A and 1B, and 10,000.times. in
FIG. 1C. The scale bars represent 1.0 .mu..
[0027] 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
25,000.times., 39,000.times., 17,000.times., and 90,000.times.
respectively.
[0028] FIGS. 3A-3C are photographs of cells isolated from an adult
rat heart and placed in culture. The newly isolated cells shown in
FIG. 3A include undifferentiated spore-like cells magnified
100.times.). 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).
[0029] 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 200.times..
[0030] 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
100.times.). After two days in culture, the isolated spore-like
cells, or their progeny, appear to be differentiating (FIG. 5B;
magnification at 200.times.).
[0031] 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
100.times.). After three days in culture, aggregates of cells
resembling kidney structures can be (FIG. 6B; magnification at
200.times.).
[0032] 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
100.times.). After three days in culture, an aggregate of cells
resembling a differentiating liver structure can be seen (FIG. 7B;
magnification at 200.times.). 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; 400.times.).
[0033] 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).
[0034] 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 (200.times.) and
9B (400.times.)). After two days in culture, primitive adrenal
cells can be seen (FIGS. 9C (200.times.) and 9D (400.times.)).
[0035] 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 100.times. magnification; FIGS. 10A and 10B).
The islet-like structures were immunostained, which revealed
insulin expression (FIG. 10C).
[0036] FIG. 11 is a photograph of a culture that includes
undifferentiated spore-like cells isolated from adult human
blood.
[0037] FIG. 12A is a photograph of cultured cells. The cultures
were established seven days earlier and, as shown by phase contrast
microscopy, contained spore-like cells isolated from adult human
blood.
[0038] FIG. 13 is a schematic of a permeable support structure
filled with a hydrogel-spore-like cell composition.
DETAILED DESCRIPTION
[0039] The present invention provides compositions and methods for
repairing, replacing, or generating tissue or another biologically
useful substance (e.g., a hormone, an enzyme, an anti-angiogenic
factor, a cytokine, a growth factor, or other biologically active
molecules, including chimeric molecules (e.g., a polypeptide that
contains a detectable marker)). The compositions include spore-like
cells (e.g., mammalian spore-like cells), and can be used to study
cellular differentiation, to detect a compound that has an adverse
effect on differentiation (and therefore a possible adverse effect
on development) or administered to a patient either 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. For
example, to screen compounds for possible adverse effects on
development, one can expose spore-like cells to the compound(s) and
follow their course of differentiation. If the compound inhibits or
alters cellular differentiation (relative to an appropriate
control, such as a spore-like cell or population of spore-like
cells that have been similarly treated but not exposed to the
compound), then the compound may have an adverse effect on
development (e.g., fetal development) and should be tested
further.
[0040] 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). Spore-like cells can also 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 (however,
a particular advantage of administering spore-like cells is that
they do not require such protection--they may be harvested from the
patient to whom they are subsequently administered or they may be
so primitive that they fail to evoke an immune response).
[0041] Alternatively, as described below, spore-like cells can be
combined with a liquid hydrogel that can be placed into 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-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 shaped as 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).
[0042] 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 substance that reduces surface tension within the
alveoli, they must be supplied locally.
[0043] Spore-like cells, and their progeny, and exemplary methods
for their isolation and administration, are described below.
Spore-Like Cells
[0044] As noted above, spore-like cells were so-named because they
have characteristics reminiscent of those of spores. More
specifically, spore-like cells have one or more of the following
characteristics. First, they are widely distributed throughout the
body. They have been isolated from every tissue examined to date,
and they have been isolated from bodily fluids as well. Thus, they
can be isolated from a wide variety of tissues (e.g., cardiac,
smooth and skeletal muscle, intestine, bladder, kidney, liver,
lung, adrenal gland, skin, retina, nasal epithelium, brain, spinal
cord, periosteum, perichondrium, fascia, and pancreas) and bodily
fluids (e.g., blood, cerebrospinal fluid, urine, and saliva).
Stated more generally, spore-like cells can be isolated from
tissues that are generated from the endoderm, mesoderm, or
ectoderm.
[0045] Second, spore-like cells are multipotent, i.e., they can
differentiate into two or more (e.g., two, three, four, five, or
more) cell types. For example, multipotent spore-like cells can
differentiate into epithelial cell, keratinocytes, and melanocytes.
They can also self-replicate. That is, a spore-like cell can divide
to produce either one or two new spore-like cells. Spore-like cells
can differentiate into the cell types of the tissue from which they
were isolated (e.g., spore-like cells isolated from the pancreas
can differentiate into insulin-producing islet cells or
glucagon-producing islet cells) or into the cell types of another
tissue. For example, spore-like cells isolated from the blood can
differentiate into insulin-producing islet cells, and spore-like
cells isolated from cartilage or periosteum can differentiate into
neurons.
[0046] Third, spore-like cells are small. 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, spore-like cells having a diameter
of less than approximately one micron in diameter (e.g., one-tenth,
one-fifth, one-third, or one-half of a micron) have also been
observed). The diameter of the spore-like cells may increase
somewhat as they adhere to a tissue culture vessel and "sit down."
Cell size is discussed further below.
[0047] Fourth, spore-like cells appear to be membrane-bound, as
biological cells typically are, but within the membrane or
associated with the membrane, spore-like cells have an unusually
high content of glycolipids (or glycogen) and/or
mucopolysaccharides. In fact, there are sufficient glycolipids (or
glycogen) and/or mucopolysaccharides that the cells appear to have
one or more dark stripes when viewed under a powerful microscope
(by, e.g., transmission electron microscopy). When the cells are
exceedingly small (e.g., less than about one or two microns), the
stripes are not as obvious, but they can, even in some of the
exceedingly small cells, be seen with a trained eye. While it is
not presently known, glycolipids (or glycogen) and/or
mucopolysaccharides may help protect spore-like cells and thus
better equip them to survive harsh conditions, such as those
described herein.
[0048] Fifth, spore-like cells have an unusual cytoarchitecture.
Electron micrographs and histological stains for nucleic acids
reveal that a large portion (e.g., at least about 50% and up to
about 90% or more) of the volume of a spore-like cell is comprised
of nucleic acids. Mitochondria have also been observed.
[0049] Sixth, spore-like cells can survive in extreme conditions
(i.e., conditions in which differentiated or partially
differentiated cells would die). For example, spore-like cells are
tolerant of oxygen-deprivation. 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, for many
hours (e.g., four, six, ten, twelve, or 24 hours or more).
[0050] Spore-like cells can be obtained by the methods of the
present invention from many different types of donors (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, preferably, a human. Spore-like cells can be
obtained from an animal even after it has reached adulthood.
[0051] Because spore-like cells tolerate oxygen deprivation and
exposure to extreme temperatures better than differentiated cells,
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).
[0052] 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, cerebrospinal
fluid, or urine), and most, if not all, functional organs and
mucous membranes. Moreover, spore-like cells can be obtained from
the patient who will be subsequently treated with those cells, from
another person, or from an animal of a different species. In other
words, autologous, allogenic, and xenogeneic spore-like cells can
be obtained and used to treat human patients.
[0053] 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. Given the unique
characteristics of spore-like cells, it is entirely reasonable to
think they are immortal (i.e., they can be cultured indefinitely).
However, if this is not the case, they can be immortalized 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.
[0054] Spore-like cells can differentiate into many different cell
types. For example, as shown below, spore-like cells can be
isolated from 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. Spore-like cells obtained from the retina
can differentiate into cells having morphologies similar to that of
rods and cones. Spore-like cells obtained from the spinal cord can
differentiate into cells having morphologies similar to that of
neurons, astrocytes and oligodendrocytes.
[0055] Spore-like cells can also be isolated from readily
obtainable bodily fluids, such as the blood. 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. Similarly, given the number of differentiated phenotypes
already observed, it is reasonable to expect that spore-like cells
can differentiate into most, if not all, types of cells.
[0056] As noted above, spore-like cells are generally spherical,
especially when first isolated, and are typically small. Many cells
in a culture of newly isolated spore-like cells are approximately 1
to 3 .mu. in diameter. However, larger and smaller spore-like cells
have been identified (e.g., using electron microscopy; see Example
2). Given that spore-like 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, as well as 7 to
10, .mu. in diameter have been identified in scanning electron
micrographs. Occasionally, even larger cells (e.g., cells as large
as 12 to 18 .mu. or more) have been observed. The larger cells may
have in fact been cells on the verge of cell division (structures
resembling mitotic clefts are often visible). Alternatively, the
larger cells may really be conglomerates of several spore-like
cells.
[0057] 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 have been observed in
scanning electron micrographs and some cells may be as small as
one-tenth of a micron. This extremely small size may reflect the
unique composition of spore-like cells. Newly isolated spore-like
cells contain a great deal of nuclear material and relatively
little cytoplasm. In most differentiated cells, the nucleus
consumes approximately 10-20% of the cells' volume. However,
approximately 50% and up to approximately 90% of the volume of a
spore-like cell is consumed with nuclear material. The nuclear
material appears to be surrounded by a coat containing glycolipids
and/or mucopolysaccharides.
[0058] Without limiting the invention to spore-like cells that
arise by any particular mechanism, it is believed that spore-like
cells may arise when essential DNA fragments (which may represent
compressed DNA) are shed from mature cells (e.g., those undergoing
cell death by apoptosis or other means) and re-packaged in a
glycolipid-rich coat. Indeed, the concept of a minimal genome is
beginning to emerge. This concept is exemplified by a mycoplasma
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.
[0059] The unique size and composition of newly-isolated spore-like
cell is perhaps best appreciated by viewing the cells with an
electron microscope (e.g., see FIGS. 1A-1C and 2A-2D).
[0060] Functionally, spore-like cells are unique in at least three
ways. First, even though they are present in mature animals (e.g.,
post-natal, adolescent, or adult animals), they can differentiate
into a wide variety of different cell types. Second, spore-like
cells tolerate (i.e., survive following exposure to) conditions
that kill differentiated or partially differentiated cells (e.g.,
oxygen-deprivation and exposure to temperatures that are either
much higher or much lower than normal body temperature (which, for
warm-blooded mammals, is 37.degree. C.)). Experiments have
demonstrated that spore-like cells can tolerate essentially
complete oxygen deprivation for at least five days (cells were
viable despite oxygen deprivation for either four hours, 24 hours,
or five days). Thus, spore-like cells can tolerate prolonged oxygen
deprivation for at least five days or longer. In addition,
spore-like cells have a greater capacity to proliferate than
terminally differentiated cells isolated from specialized tissues.
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.
[0061] To obtain spore-like cells, a sample is obtained from an
animal, such as a human. One of the easiest samples to obtain is a
sample of whole blood. Those of ordinary skill in the art will
appreciate that the isolation method may 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,
whether they are samples of bodily fluids, organs, tissues, or cell
suspensions thereof, can be stored. Moreover, spore-like cells can
be recovered from bodily fluids or other samples that have been
stored under conditions in which differentiated cells or known stem
cells (e.g., hematopoietic stem cells) cannot survive (e.g., frozen
storage without a cryopreservative).
[0062] Either immediately after collection or after storage (e.g.,
under normal cell storage conditions, but also in an oxygen-poor
environment or at a temperature more than 42.degree. C. or at or
below freezing), the cells can be centrifuged for a time and at a
speed sufficient to pellet them in 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, epidermal growth factor
(EGF) and basic fibroblast growth factor (bFGF; see the Examples,
below. Other media have been used and have worked as well as that
just described for the growth, proliferation, and differentiation
of spore-like cells. After collection, the tissue sample can be
intentionally exposed to harsh conditions, for example, those
described herein, to kill differentiated cells.
[0063] The suspended cells are then transferred to a tissue culture
vessel and incubated (obviously, the incubation temperature is not
critical, but most incubators are kept at or near 37.degree. 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 .mu. (methods in which spore-like
cells are isolated after exposure to an extreme condition and
methods in which spore-like cells are isolated based on another
characteristic, such as size, or their ability to withstand
infectious agents, can be carried out separately or in
combination). 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
spore-like cells are viable according to Trypan blue exclusion
studies when isolated as described above.
[0064] 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, flow cytometry can also be used. Alternatively, 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.
[0065] At the time of isolation, spore-like cells may not express
the receptors or other cellular components that make differentiated
cells susceptible to attack by infectious agents. This is another
characteristic that can be exploited to identify and isolate
spore-like cells. For example, one can infect a tissue or cell
culture with an infectious agent and then separate the live
spore-like cells from the differentiated cells that were killed by
the infectious agent.
[0066] The features and characteristics described above 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 their ability to differentiate into a variety of
terminally differentiated cell types found in mature animals (such
as those illustrated in the Examples below), their typical
spherical shape, small size (as small as 0.1-0.3 .mu. in diameter
and generally 1.0 to 3.0 .mu. in diameter), and cytoarchitecture
(which includes relatively large amounts of nuclear material,
relatively small amounts of cytoplasm, and a glycolipid- or
mucopolysaccharide-rich coat), their ability to survive in
environments in which other cell types would die (e.g.,
environments in which there is a low, or even non-existent, oxygen
supply, environments in which the temperature is higher or lower
than other cells can tolerate (without special protective
measures), environments containing toxins, non-physiological salt
concentrations, acids, bases, or radioenergy that other cells
cannot tolerate).
[0067] When cultured as described in the Examples below, spore-like
cells proliferate more rapidly and into more types of
differentiated cells than do terminally differentiated cells or
mesenchymal stem cells. Cell viability can be assessed using
standard techniques, including visual observation with light or
scanning electron microscopes and Trypan blue exclusion.
[0068] Spore-like cells have been isolated from body fluids (e.g.,
the blood) as well as from solid functional organs such as the
liver, but it is not clear that 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 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 in the bone marrow). If so, spore-like cells could then
be subsequently delivered from those fluids to specific tissues.
Moreover, delivery may be upregulated when the tissue is affected
by, for example, a disorder, a regenerative process, or wound
healing.
[0069] Without limiting the invention to spore-like cells that
differentiate by a particular mechanism, it is believed that the
rate and nature of spore-like cell differentiation can be
influenced by altering the number and type of mature cells that
come into contact (physical or functional contact) with spore-like
cells. A mature cell is in functional contact with a spore-like
cell when the mature cell emits a chemical signal (e.g. a growth
factor, cytokine, or neurotransmitter) that is detected by the
spore-like cell. 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, it is believed that
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 associated with a
medical disorder. These agents guide differentiation so that the
spore-like cells or their progeny come to express some or all of
the same phenotypic markers expressed by mature cells normally
present in the tissue in which they have been placed. Spore-like
cells can be influenced by agents within tissues regardless of
their origin (i.e., regardless of whether the spore-like cells
originate in the blood, another body fluid, the bone marrow, or a
solid, functional tissue or organ).
[0070] 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: bone; bone marrow; muscle (e.g., smooth, skeletal, or
cardiac muscle); connective tissue (e.g., cartilage, ligaments,
tendons, pleura, or fibrous tissues); epithelial and mucous
membranes; 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 organs
(e.g., liver, kidney, pancreas, stomach, bladder, or any portion of
the alimentary canal). This list is intended to illustrate, not
limit, the types of cells and tissues that can benefit from
administration of 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. Those of ordinary skill in the art will
recognize many other therapeutic uses for spore-like cells.
Spore-like Cell Differentiation
[0071] Spore-like cells or their progeny can differentiate into a
number of different cell types. For example, spore-like cells can
differentiate into epithelial cells, keratinocytes, melanocytes,
adipocytes, myocytes, chondrocytes, osteocytes, alveolar cells,
hepatocytes, renal cells, adrenal cells, endothelial cells, islet
cells (e.g., alpha cells, delta cells, PP cells, and beta cells),
blood cells (e.g., leukocytes, erythrocytes, macrophages, and
lymphocytes) retinal cells (and other cells involved in sensory
perception, such as those that form hair cells in the ear or taste
buds on the tongue), and fibroblasts or other cell types present in
organs and connective tissues.
[0072] Spore-like cells and their progeny can be induced to
differentiate in a variety of ways and may or may not be committed
to a particular differentiation pathway. 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 (e.g., adhere to) a glass or plastic surface, a mesh,
or other substrate suitable for use in tissue culture or
administration to a patient.
[0073] Spore-like cells can also differentiate when they establish
contact with a tissue within a patient's body or are sufficiently
close to a tissue to be influenced by substances (e.g., growth
factors, enzymes, or hormones) released from the tissue. Thus,
differentiation of a spore-like cell can be influenced by virtue of
signals the cell receives from the surrounding tissue. Such
signaling 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, bound and transduced a signal from a molecule such
as a growth factor, enzyme, neurotransmitter, or hormone that was
released by a tissue within the patient.
[0074] Alternatively, or in addition, spore-like cells can be
induced to differentiate by adding a substance (e.g., a growth
factor, enzyme, hormone, or other signaling molecule) to the cell's
environment. For example, a substance 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. For
example, spore-like cells can be made to express hormones, such as
insulin, by transfecting them with gene constructs that include
sequences that encode these factors. Thus, spore-like cells or
their progeny can differentiate either in culture or in a patient's
body, and may 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 that have differentiated, or that will do so,
sufficiently to aid in the maintenance or repair of tissue, can be
administered to a patient (e.g., at the site of a burn or other
traumatized area of skin, a bone fracture, a torn ligament, an
atrophied muscle, a malfunctioning gland, or an area adversely
affected by a neurodegenerative process (or trauma or ischemia, as
occurs with a cerebrovascular accident) or autoimmune response).
Based on simple observation, it appears that when spore-like cells
come into contact with each other, they orchestrate their own
development. It appears that once contact is initiated, a
developmental pattern is set into motion.
[0075] Another way to promote proliferation without differentiation
is to expose the spore-like cells, particularly those isolated from
the skin, to agonists of Notch function, as described in U.S. Pat.
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 isolated from the skin
can be contacted in culture with agonists of Notch or can be
transfected with genes that encode Notch agonists. As described
above, the techniques required to transfect cells in culture are
routinely practiced by those of ordinary skill in the art.
Spore-like cells that remain undifferentiated in culture can
differentiate when administered to a patient; their differentiation
being orchestrated by the microenvironment they encounter within
the patient.
[0076] As described in Example 7, below, many cells isolated as
spore-like cells from the liver express bile after 12 days in
culture. Bile expression can be seen following staining by Hall's
technique using Fouchet's reagent (FIG. 7E). Bile pigments can also
be identified by at least two other standard histological stains,
the Gmelin test, and Stein's method. Similarly, 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 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.
[0077] 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 essentially
recreate a histologically mature and complete tissue), fully
differentiated cells are not always necessary for successful
treatment; spore-like cells or their progeny need only
differentiate to a point sufficient to treat the patient. For
example, spore-like cells used to treat diabetes need not ever
differentiate into cells that are indistinguishable from fully
differentiated .beta. cells within the islets of Langerhans. To the
contrary, spore-like cells or their progeny need only differentiate
to the point where they express sufficient insulin to treat the
diabetic patient.
[0078] Excluded from the invention are cells having characteristics
that render them indistinguishable from previously identified stem
cells (e.g., mesenchymal stem cells), precursor cells (e.g., the
islet cell precursors described by Cornelius et al. (Horm. 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 histological, biochemical, or, preferably, electron
microscopic analysis).
Methods of Treatment
[0079] A. Administration of Spore-like Cells and their Progeny via
Hydrogel
[0080] The novel cell types described herein can be administered to
a patient by way of a composition that includes spore-like cells,
or their progeny, and a liquid hydrogel. This cell-hydrogel mixture
can be applied directly to a tissue that has been damaged. For
example, as described in U.S. Ser. No. 08/747,036, a hydrogel-cell
mixture can simply be brushed, dripped, or sprayed onto a desired
surface or poured or otherwise made to fill a desired cavity or
device. The hydrogel provides a thin matrix or scaffold within
which the spore-like cells adhere and grow. These methods of
administration may be 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 are placed beneath the renal capsule to treat diabetes).
[0081] Alternatively, the hydrogel-cell mixture can be introduced
into a permeable, biocompatible support structure so that the
mixture essentially fills the support structure and, as it
solidifies, assumes the support structure's shape. Thus, the
support structure can guide the development and shape of the tissue
that matures from spore-like cells, or their progeny, that are
placed 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,
the liver, 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
following subsections, suitable support structures, hydrogels, and
delivery methods are described (cells suitable for use are
described above).
[0082] 1. Hydrogels
[0083] The hydrogels used to practice this invention should be
biocompatible, biodegradable, capable of sustaining living cells,
and, preferably, capable of solidifying rapidly in vivo (e.g., in
about five minutes after being delivered to the support structure).
Large numbers of spore-like cells can be distributed evenly within
a hydrogel; a hydrogel can support approximately 5.times.10.sup.6
cells/ml. Hydrogels also enable diffusion so that nutrients reach
the cells and waste products can be carried away.
[0084] 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 at body temperature (e.g.,
PLURONICS.TM.); (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., TETRONICS.TM.).
[0085] 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 PLURONICS.TM. (also known as
POLOXAMERS.TM.), which are poly(oxyethylene)-poly(oxypropylene)
block polymers solidified by changes in temperature, TETRONICS.TM.
(also known as POLOXAMINES.TM.), which are
poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene
diamine solidified by changes in pH.
Ionic Hydrogels
[0086] Ionic polysaccharides, such as alginates or chitosan, can
also 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. Pat. No. 4,352,883 describes the
ionic cross-linking of alginate with divalent cations, in water, at
room temperature, to form a hydrogel matrix.
[0087] Spore-like cells 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.
[0088] In general, these polymers 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).
[0089] 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.
[0090] Bioerodible polyphosphazenes have at least two different
types of side chains: acidic side chains capable of forming 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 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.degree. C. and 38.degree. 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.
[0091] Methods for synthesis and the analysis of various types of
polyphosphazenes are described in U.S. Pat. Nos. 4,440,921,
4,495,174, and 4,880,622. Methods for the synthesis of the 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, N.Y., 1990. Many polymers, such as poly(acrylic acid),
alginates, and PLURONICS.TM. are commercially available.
[0092] 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.
[0093] 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.
[0094] For purposes of preventing the passage of antibodies into
the hydrogel, but allowing the entry of nutrients, a useful polymer
size in the hydrogel is in the range of between 10 and 18.5 kDa.
Smaller polymers result in gels of higher density with smaller
pores.
Temperature-Dependent Hydrogels
[0095] 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 the hydrogel-cell composition. Examples of such
temperature-dependent hydrogels are PLURONICS.TM. (BASF-Wyandotte),
such as polyoxyethylene-polyoxypropylene F-108, F-68, and F-127,
poly (N-isopropylacrylamide), and N-isopropylacrylamide
copolymers.
[0096] 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% (WNV) by dispersion at 4.degree. 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.
[0097] U.S. Pat. No. 4,188,373 describes using PLURONIC.TM. polyols
in aqueous compositions to provide thermal gelling aqueous systems.
U.S. Pat. Nos. 4,474,751, '752, '753, and 4,478,822 describe drug
delivery systems that utilize thermosetting polyoxyalkylene gels.
With these systems, both the gel transition temperature and/or the
rigidity of the gel can be modified by adjustment of the pH and/or
the ionic strength, as well as by the concentration of the
polymer.
pH-Dependent Hydrogels
[0098] 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 body pH.
Examples of such pH-dependent hydrogels are TETRONICS.TM.
(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.
Light Solidified Hydrogels
[0099] 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. Pat. 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.
[0100] 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 PLURONIC.TM. 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.
[0101] Thus, a variety of hydrogels can be used to practice the
present invention. They include, but are not limited to: (1)
temperature dependent hydrogels that solidify or set at body
temperature, e.g., PLURONICS.TM.; (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.,
TETRONICS.TM..
[0102] The materials that can be used to form these various
hydrogels include polysaccharides such as alginate,
polyphosphazenes, and polyacrylates, which are cross-linked
ionically, or block copolymers such as PLURONICS.TM. (also known as
POLOXAMERS.TM.), which are poly(oxyethylene)-poly(oxypropylene)
block polymers solidified by changes in temperature, or
TETRONMCS.TM. (also known as POLOXAMINES.TM.), which are
poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene
diamine solidified by changes in pH.
[0103] 2. Preparation of Hydrogel-Cell Mixtures
[0104] Once a hydrogel of choice (e.g., a thermosensitive polymer
at between 5 and 25% (W/V), 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 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/ml (e.g., between 20 and 50
million cells/ml or between 50 and 80 million cells/ml). Of course,
the optimal concentration of cells to be delivered into the support
structure may be determined on a case by case basis, and may 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.
[0105] 3. Support Structures
[0106] 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 waste products produced by the cells can diffuse out of the
structure.
[0107] 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
support structure can be filled with a hydrogel-cell mixture and,
as the hydrogel hardens, molded into a desired shape by hand.
[0108] As the hydrogel solidifies, it will adopt the flexibility
and resiliency of the support structure, which is important for
accommodation of compressive and tensile forces. Thus, for example,
replaced skin could 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. For example, in many
currently available skin replacement methods, the tissue is
extremely delicate and must be handled with the utmost care.
[0109] The support structure is also 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 growth and proliferation by
allowing nutrients to reach the cells by diffusion. 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.
[0110] An example of a suitable polymer is polyglactin, which is a
90:10 copolymer of glycolide and lactide, and is manufactured as
VICRYL.TM. braided absorbable suture (Ethicon Co., Somerville,
N.J.). Polymer fibers (such as VICRYL.TM.), 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.
[0111] 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
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.
[0112] 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 shaped 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.
[0113] 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.
[0114] 4. Application of the Support Structure
[0115] 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 is suitable for
application of 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 the suspended cells 18 within the 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 being the growth of new skin and its engraftment to
the patient's body.
[0116] 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.
[0117] 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.
[0118] The volume of the liquid hydrogel-cell mixture injected into
the support structure is typically less than, but somewhat
comparable to, the volume of the support structure, i.e., the
volume of the desired tissue to be grown.
[0119] 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).
[0120] 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 hydrogel-cell-containing support
structure can be removed from the mold in a form that is ready for
administration to a patient.
[0121] 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.
The hydrogel-cell mixture can be delivered to the support using any
simple device, such as a syringe or catheter, or merely by pouring
or brushing a liquid gel onto a support structure (e.g., a
sheet-like structure).
[0122] 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. As
described above, some of the hydrogel-cell mixture may leak from
the support structure prior to solidifying. However, in this event,
existing tissue beneath or surrounding the support structure would
sufficiently constrain the liquid hydrogel-cell mixture until it
gels.
[0123] 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 PLURONIC.TM.
temperature-sensitive hydrogel).
[0124] To apply or implant the support structure, the implantation
site within the patient can be prepared (e.g., in the event 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).
EXAMPLES
[0125] The present invention will be further understood by
reference to the following non-limiting examples.
Example 1
[0126] Spore-like cells were isolated from human blood as follows.
Five cc's of whole blood were acquired from an adult human and
placed in a tube containing an anti-coagulant. The blood sample was
then centrifuged at 1200 rpm for approximately five minutes. The
supernatant was removed, and the resulting pellet was resuspended
in 15 cc's of DMEMIF-12 medium 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, Ill.). The resulting suspension was transferred to 75
cm.sup.2 tissue culture flasks and incubated in 5% CO.sub.2 at
37.degree. 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
[0127] 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 phosphate buffered saline (PBS) and antibiotics
(penicillin (50 mU/ml) 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, Del.) 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.degree. 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.
[0128] 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 .mu.m. The number of
pipettes required can vary depending upon how frequently they
become clogged with tissue. Trituration was carried out until the
tissue was dispersed as a fine suspension. The suspension was then
centrifuged at 1200 rpm (GLC-2B, Sorvall, Wilmington, Del.) 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), 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, Ill.). The suspension was transferred to 75 cm.sup.2
tissue culture flasks (Collaborative Biomedical Products, Chicago,
Ill.) and incubated at 37.degree. C. in 5% CO.sub.2. The media was
changed every three days, and the cells were passaged every 7-9
days. The cells that attached to the tissue culture flask appeared
to differentiate more readily.
[0129] Spore-like cells isolated from the skin will differentiate
upon exposure to the processes and basal nutrient media described
in U.S. Pat. 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.
[0130] Spore-like cells were also isolated from excisional biopsies
of the skin of adult pigs according to the same protocol described
here for the adult rat.
Example 3
[0131] 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 three days in culture,
early myocardial cells can be seen (FIG. 3B), and after two weeks
in culture, Purkinje-like structures can be seen (FIG. 3C).
Example 4
[0132] 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 three days in culture, clusters of small
intestinal cells (FIG. 4B) and autonomic neurons (FIG. 4C) can be
seen.
Example 5
[0133] 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 two days in culture, the
isolated spore-like cells, or their progeny, appear to be
differentiating into mature bladder cells (FIG. 5B).
Example 6
[0134] 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 three days in
culture, aggregates of cells resembling kidney structures can be
seen (FIG. 6B).
Example 7
[0135] 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 three days in culture, an aggregate of cells
resembling a differentiating liver structure can be seen (FIG. 7B).
After seven days in culture, cells resembling hepatocytes can be
seen (FIG. 7D).
Example 8
[0136] 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
30 days in culture, 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
[0137] 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 two
days in culture, primitive adrenal cells can be seen (FIGS. 9C and
9D).
Example 10
[0138] 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).
[0139] 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
immunostained, insulin expression can be seen (FIG. 10C).
Example 11
[0140] 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. 1A have a
diameter of approximately 0.3 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 12). The interior of the cell is consumed largely with
diffuse nuclear material and the cell is 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 12
[0141] 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 anti-fade mounting medium (e.g., 90% glycerol, 1.times.
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 13
[0142] 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 animals was killed and
placed in a 50 cc centrifuge tube (Fisher Scientific, Pittsburg,
Pa.) filled with PBS. The tubes were stored at 4.degree. C. without
supplemental oxygen for five days. Spore-like cells were then
isolated as follows.After excision from the animal, and using
sterile technique, the selected tissue was placed in cold PBS
containing penicillin (50 mU/ml) and streptomycin (90 mg/ml)
(Gibco, Grand Island, N.Y.). 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.degree. C. The trypsin was inactivated by adding
10 ml of DMEMIF-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 .mu.m. 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 nM
selenium (Sigma), 10 nM progesterone (Sigma), 20 ng/ml EGF
(Peprotech, Rocky Hill, N.J.), and 20 ng/ml bFGF (Collaborative
Biomedical, Raynham, Mass.). The primary cell suspension was
incubated at 37.degree. C. in 5% CO.sub.2, 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.
[0143] The technique described above was slightly modified to
isolate hepatic tissue: hepatic tissue was washed with cold PBS
prior to disassociation.
[0144] 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 mucicarmine and periodic acid-Schiff were
also performed.
[0145] 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 100.times.) or viable cell counts using trypan blue
with a hemocytometer.
[0146] 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.degree. 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 14
[0147] After being killed, whole animals (Fisher rats) were placed
in plastic bags and stored in a freezer at -86.degree. C. After
being frozen for either two or eight weeks, the animals were
removed from the freezer and placed in a 37.degree. 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 13 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.degree. 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 15
[0148] Four tissues (lung, liver, fascia, and spinal cord) were
obtained from three animals (Fisher rats) and heated to 85.degree.
C. for 30 minutes. More specifically, each tissue type was removed
from an animal less than two hours after the animals 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.degree. 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.degree. C. The tissue
was then allowed to cool to room temperature, and spore-like cells
were isolated as described in Example 13.
[0149] 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.degree. 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.
Example 16
[0150] Spore-like cells isolated from the central nervous system
fail to show activity in a microculture tetrazolium assay
(conducted as described in Marshall et al. (Growth Regulation
5:69-84, 1995)), but do so when they differentiate into cells with
more usual and recognizable characteristics. This assay tests for
redox activity and, when negative, indicates that no cells are
present. This assay therefore provides further evidence that
spore-like cells are unique from known cell types and that they
exist in a dormant state without apparent metabolic activity.
Example 17
[0151] Blood from a patient who developed type I diabetes eight
years earlier was collected in a standard tube with anticoagulant
and subsequently frozen at -85.degree. C. without preparation
(i.e., without treatment with a cryopreservative or other
substance) for up to 26 weeks. Spore-like cells were successfully
isolated from this blood sample after up to four freeze-thaw
cycles. No other intact cells were observed (as was expected as no
known cell types are known to survive these conditions).
[0152] More specifically, after each thaw cycle, one cc of blood
was added to 20 ccs of complete medium (DMEM/F12 with progesterone,
b-FGF, and EGF) After the cells were added, the medium was
triturated with a pasteur pipette and reduced-bore pasteur pipettes
and filtered through, first, a 100 micron filter and then a 40
micron filter. The cells were then placed in a 175 cm.sup.2 flask
and incubated at 37.degree. C. with 5% CO.sub.2. Initially, a few
intact spore-like cells were suspended in the media with gel-like
properties, with 3-5 cc of fresh media added every 3 to 5 days. The
media then assumed a liquid quality, with rapid spore-like cell
replication (ten doublings in 5 days). At about day 12, the
spore-like cells formed small clusters. At this point, 0.5 cc of
30% glucose was added to the culture flask, raising the
concentration of glucose from about 100 mg/dl to 500 mg/dl (a
hyperglycemic condition). In response, the clusters enlarged, and
frequent nucleated cells could be seen within 5 days. Insulin
production was demonstrated by both standard immunofluorescent
assays and by RT-PCR for insulin mRNA.
OTHER EMBODIMENTS
[0153] 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.
[0154] 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.
[0155] Other aspects, advantages, and modifications are within the
scope of the following claims.
* * * * *