U.S. patent application number 11/329547 was filed with the patent office on 2006-05-18 for pharmaceuticals containing retinal stem cells.
Invention is credited to Bernard Chiasson, Derek van der Kooy, Roderick McInnes, Vincenzo Tropepe.
Application Number | 20060104960 11/329547 |
Document ID | / |
Family ID | 21833312 |
Filed Date | 2006-05-18 |
United States Patent
Application |
20060104960 |
Kind Code |
A1 |
Kooy; Derek van der ; et
al. |
May 18, 2006 |
Pharmaceuticals containing retinal stem cells
Abstract
The invention relates to stem cells isolated from the retina of
mammals and retinal cells differentiated from these stem cells. The
invention also relates to a method of isolating retinal stem cells
and inducing retinal stem cells to produce retinal cells. Retinal
stem cells may also be induced in vivo to produce retinal cells.
The invention also includes pharmaceuticals made with retinal stem
cells or retinal cells which may be used to restore vision lost due
to diseases, disorders or abnormal physical states of the retina.
The invention includes retinal stem cell and retinal cell culture
systems for toxicological assays, for isolating genes involved in
retinal differentiation or for developing tumour cell lines.
Inventors: |
Kooy; Derek van der;
(Toronto, CA) ; McInnes; Roderick; (Ontario,
CA) ; Chiasson; Bernard; (City of York, CA) ;
Tropepe; Vincenzo; (Toronto, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
21833312 |
Appl. No.: |
11/329547 |
Filed: |
January 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10846012 |
May 14, 2004 |
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11329547 |
Jan 10, 2006 |
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09333248 |
Jun 15, 1999 |
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10846012 |
May 14, 2004 |
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08937967 |
Sep 25, 1997 |
6117675 |
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09333248 |
Jun 15, 1999 |
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60026698 |
Sep 25, 1996 |
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Current U.S.
Class: |
424/93.7 ;
435/368 |
Current CPC
Class: |
A61K 35/44 20130101;
A61K 35/12 20130101; C12N 5/0621 20130101 |
Class at
Publication: |
424/093.7 ;
435/368 |
International
Class: |
C12N 5/08 20060101
C12N005/08; A61K 35/44 20060101 A61K035/44 |
Claims
1. A method of transplanting retinal cells comprising implanting
(i) isolated retinal stem cells from the retina of a mammal and/or
(ii) retinal cells differentiated from said retinal cells, into the
retina of an individual.
2. The method of claim 1, wherein said mammal is a human, and said
individual is a human, and said individual is suffering from a
retinal disease or disorder, or an abnormal physical state of the
retina of the eye.
3. The method of claim 2, wherein said retinal disease or disorder,
or abnormal physical state of the retina of the eye is selected
from the group consisting of blindness due to disease or damage to
the retina of the eye, cytomegalovirus retinitis, uveitis,
glaucoma, macular degeneration, retinitis pigmentosa, retinal
degeneration, retinal detachment, and cancers of the retina.
4. The method of claim 1, wherein said retinal cells are
differentiated from said retinal stem cells in vitro.
5. The method of claim 1, wherein the retinal stem cells comprise
cells isolated from a retinal pigment epithelial layer of the
retina and/or cells derived therefrom.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
10/846,012, filed May 14, 2004, pending, which is a continuation of
U.S. Ser. No. 09/333,248, filed Jun. 15, 1999, abandoned, which is
a continuation of U.S. Ser. No. 08/937,967, filed Sep. 25, 1997,
now U.S. Pat. No. 6,117,675, which claims priority from U.S.
Provisional Application No. 60/026,698 filed Sep. 25, 1996, each of
which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to stem cells isolated from the retina
of mammals. The invention includes a method for stimulating
proliferation of endogenous retinal stem cells in vivo and
pharmaceutical compounds that stimulate proliferation of retinal
stem cells. The invention also relates to a method for isolating
retinal stem cells, uses for the stem cells and pharmaceutical
compositions containing the stem cells or their progeny. The
invention can be used to treat individuals having retinal diseases,
disorders or abnormal physical states. The invention includes
retinal stem cells and retinal cell culture systems for
toxicological assays, drug development, isolating genes involved in
retinal differentiation or for developing tumour cell lines.
BACKGROUND OF THE INVENTION
[0003] Vision loss may be caused by disease or damage to the retina
of the eye. The retina consists of a specialized layer of cells at
the back of the eye where light entering the eye is sensed as an
image. These cells normally respond to all aspects of the light
emitted from an object and allow perception of colour, shape and
intensity. The types of cells located in the retina include retinal
pigment epithelial ("RPE") cells, rod cells, cone cells, bipolar
cells, amacrine cells, horizontal cells, Mueller cells, glial
cells, and retinal ganglion cells.
[0004] When normal retinal function is impaired, it may lead to a
loss of colour perception, blind spots, reduced peripheral vision,
night blindness, photophobia, decreased visual acuity or blindness.
For example, acquired immunodeficiency virus ("AIDS") patients may
suffer cytomegalovirus retinitis which is caused by spread of the
cytomegalovirus to the retina (Bloom et al., Medicine,
109(12):963-968 (1988)). This and other infectious processes can
lead to loss of visual field, decreased visual acuity, and
blindness.
[0005] Uveitis is an inflammation of the eye which can affect the
retina and can lead to decreased visual acuity. Its effects on the
retina include inflamed or leaking vasculature which may appear as
perivascular exudation or haemorrhage, oedema of the retina,
chorioretinal lesions, neovascularization or inflammatory changes
in the peripheral retina. (Anglade et al., Drugs, 49(2):213-223
(1995)).
[0006] Cancers of the retina also impair vision. One example is
retinoblastoma, which is a childhood type of cancer. Other diseases
may occur through age-related macular degeneration.
[0007] Many different genetic diseases lead to retinal damage and
blindness. A relatively common example is retinitis pigmentosa
("RP"), which affects one person in four thousand worldwide.
Patients with RP have normal vision for one or more decades, and
then experience progressive loss of vision due to the premature
death of rod or cone cells. Blindness may result. Other types of
retinal degenerations (retinal dystrophies) may result from the
programmed death of other retinal cell types.
[0008] Physical damage to retinal cells may also occur through
retinal detachment which leads to retinal degeneration and
blindness.
[0009] The therapeutic strategies for treating loss of vision
caused by retinal cell damage vary, but they are all directed to
controlling the illness causing the damage, rather than reversing
the damage caused by an illness by restoring or regenerating
retinal cells. As one example, the treatments of uveitis are drawn
from the knowledge of changes in the retinal environment when
inflammation occurs. Corticosteroids, such as prednisone, are the
preferred drug of treatment. However, these drugs are
immunosuppressants with numerous side effects. As well, the
systemic immunosuppression may have significant negative effects on
the development of children as well as on adults in poor health
such as the elderly and patients with chronic disease. These
patients must try alternate drugs such as alkylating agents or
antimetabolites which also have side effects. Clearly, patients
with eye diseases remain vulnerable to sustaining permanent damage
to the retinal cells, even if drug treatments are available.
[0010] There are no known successful treatments for RP and other
retinal dystrophies. There are also no treatments which regenerate
new cells endogenously or which transplant healthy tissue to the
retina. Even if it were possible to develop some form of
transplantation, it would be subject to the same problems that
accompany transplants in other organ systems. These include: [0011]
in many cases, implants provide only temporary relief as the
symptoms associated with the disease often return after a number of
years, [0012] rejection by the patient of foreign tissue, [0013]
adverse reactions associated with immunosuppression
(immunosuppression is needed to try to help the patient accept the
foreign tissue), [0014] the inability of a sufficient number of
cells in the tissue being implanted to survive during and after
implantation, [0015] transmitting other diseases or disorders may
be transmitted to the patient via the implant, and [0016] the
results may not justify the costs and efforts of a complex
procedure.
[0017] Thus, there is currently no way to reverse permanent damage
to the retina and restore vision. Drug treatments focus on treating
the illness and its symptoms to prevent further damage to the
retina. There is a need to reverse damage to the retina and restore
vision by endogenously generating new retinal cells or
transplanting retinal cells.
[0018] In tissues other than the eye, stem cells are used as a
source for alternative treatments of disease or injury to tissues.
Stem cells are undifferentiated cells that exist in many tissues of
embryos and adult mammals. In embryos, blastocyst stem cells are
the source of cells which differentiate to form the specialised
tissues and organs of the developing fetus. In adults, specialised
stem cells in individual tissues are the source of new cells which
replace cells lost through cell death due to natural attrition,
disease or injury. No stem cell is common to all tissues in adults.
Rather, the term "stem cell" in adults describes different groups
of cells in different tissues and organs with common
characteristics.
[0019] Stem cells are capable of producing either new stem cells or
cells called progenitor cells that differentiate to produce the
specialised cells found in mammalian organs. Symmetric division
occurs where one stem cell divides into two daughter stem cells.
Asymmetric division occurs where one stem cell forms one new stem
cell and one progenitor cell.
[0020] A progenitor cell differentiates to produce the mature
specialized cells of mammalian organs. In contrast, stem cells
never terminally differentiate (i.e. they never differentiate into
a specialized tissue cells). Progenitor cells and stem cells are
referred to collectively as "precursor cells". This term is used
when it is unclear whether a researcher is dealing with stem cells
or progenitor cells or both.
[0021] Progenitor cells may differentiate in a manner which is
unipotential or multipotential. A unipotential progenitor cell is
one which can form only one particular type of cell when it is
terminally differentiated. A multipotential progenitor cell has the
potential to differentiate to form more than one type of tissue
cell. Which type of cell it ultimately becomes depends on
conditions in the local environment as such as the presence or
absence of particular peptide growth factors, cell-cell
communication, amino acids and steroids. For example, it has been
determined that the hematopoietic stem cells of the bone marrow
produce all of the mature lymphocytes and erythrocytes present in
fetuses and adult mammals. There are several well-studied
progenitor cells produced by these stem cells, including three
unipotential and one multipotential tissue cell. The multipotential
progenitor cell may divide to form one of several types of
differentiated cells depending on which hormones act upon it.
[0022] Weiss et al., Review, 1-13 (1996) summarises the five
defining characteristics of stem cells as the ability to: [0023]
Proliferate: Stem cells are capable of dividing to produce daughter
cells. [0024] Exhibit self-maintenance or renewal over the lifetime
of the organism: Stem cells are capable of reproducing by dividing
symmetrically or asymmetrically. Symmetric division is a source of
renewal of stem cells. Symmetric division leads to increases in the
number of stem cells. Asymmetric division maintains a consistent
level of stem cells in an embryo or adult mammal. [0025] Generate
large number of progeny: Stem cells may produce a large number of
progeny through the transient amplification of a population of
progenitor cells. [0026] Retain their multilineage potential over
time: Stem cells are the ultimate source of differentiated tissue
cells, so it is a characteristic that they retain their ability to
produce multiple types of progenitor cells, which will in turn
develop into specialized tissue cells. [0027] Generate new cells in
response to injury or disease: This is essential in tissues which
have a high turnover rate or which are more likely to be subject to
injury or disease, such as the epithelium or blood cells.
[0028] Thus, the key features of stem cells are that they are
multipotential cells which are capable of long-term self-renewal
over the lifetime of a mammal.
[0029] There is great potential for the use of stem cells as
substrates for producing healthy tissue where pathological
conditions have destroyed or damaged normal tissue. For example,
stem cells may be used as a target for in vivo stimulation with
growth factors or they may be used as a source of cells for
transplantation.
[0030] There has been much effort to isolate stem cells and
determine which peptide growth factors, hormones and other
metabolites influence stem cell renewal and production of
progenitor cells, which conditions control and influence the
differentiation of progenitor cells into specialized tissue cells,
and which conditions cause a multipotent progenitor cell to develop
into a particular type of cell.
[0031] In several tissues, stem cells have been isolated and
characterised to develop new therapies to repair or replace damaged
tissues. For example, stem cells have been isolated from the
mammalian brain (Reynolds et al., Science 255:107 (1992)). WO
93/01275, WO 94/16718, WO 94/10292 and WO 94/09119 describe uses
for these cells. WO 95/13364 reports that delivery of growth
factors to the ventricles of the central nervous system ("CNS")
stimulates neural stem cells to proliferate and produce progenitor
cells which will develop into neurons, oligodendrocytes or
astrocytes. All of these publications restrict the isolation or use
of adult stem cells to the brain (in particular, the tissue around
the brain ventricles, the subependyma, which is the remnant of the
embryonic brain germinal zone). There is no reported isolation of a
retinal stem cell from the adult peripheral nervous system ("PNS")
of a mammal. There is no evidence for production of new neurons
within the adult eye, so those knowledgeable about stem cells would
not suspect that stem cells would be produced in the retina.
[0032] Step cell cultures also provide useful assay cultures for
toxicity testing or for drug development testing. Toxicity testing
is done by culturing stem cells or cells differentiated from stem
cells in a suitable medium and introducing a substance, such as a
pharmaceutical or chemical, to the culture. The stem cells or
differentiated cells are examined to determine if the substance has
had an adverse effect on the culture. Drug development testing may
be done by developing derivative cell lines, for example a
pathogenic retinal cell line, which may be used to test the
efficacy of new drugs. Affinity assays for new drugs may also be
developed from the stem cells, differentiated cells or cell lines
derived from the stem cells or differentiated cells.
[0033] The stem cells also provide a culture system from which
genes, proteins and other metabolites involved in cell development
can be isolated and identified. The composition of stem cells may
be compared with that of progenitor cells and differentiated cells
in order to determine the mechanisms and compounds which stimulate
production of stem cells, progenitor cells or mature cells.
[0034] It would be useful if stem cells could be identified and
isolated in areas of the CNS and PNS outside the adult brain, such
as the retina. Medical treatments could then be developed using
those stem cells. To date, no person has suggested that a retinal
precursor cell in a mammal even exists beyond the embryonic stage
of development, (a retinal stem cell would self-renew to exist from
embryonic development to adulthood). There are retinal precursor
cells in the embryonic eye which exhibit some of the
characteristics of stem cells. However, since it is believed that
these cells do not persist into the adult eye, this would indicate,
by definition, that the embryonic precursors could only be retinal
progenitor cells and not real retinal stem cells. The prior art
teaches that it is highly unlikely that there is a retinal stem
cell. Most of the prior art involves studies of embryonic precursor
cells isolated from the mammalian eye (see e.g. Anchan et al.,
Neuron 6:923-936, (1991), Lillien et al., Development 115:253-266
(1992); Cepko et al., PNAS 93:589-595 (1996)). Precursor cells can
be multipotential, however they usually have more restricted
phenotype potential than stem cells. They also have only limited
self-renewal capability. Anchan et al., Neuron 6:923-936, (1991),
and Lillien et al., Development 115:253-266 (1992) both isolated
embryonic retinal precursors in culture, however, they did not
discuss retinal stem cells nor establish that retinal stem cells
exist and can be isolated and purified. In summary, before this
invention, no one expected that stem cells even existed in the
retina or that the cardinal features of stem cells, self renewal
and multipotentiality, could be found in those cells.
[0035] Current medical and surgical drug treatments are inadequate
for restoring vision lost when retinal cells are damaged, so the
potential clinical applications of pharmaceutical compounds
containing retinal stem cells or to stimulate endogenous
proliferation of retinal stem cells are tremendous. Retinal stem
cells would have the potential to act as in vivo targets for
stimulation by growth factors in order to produce healthy tissue.
This may be done, for example, by injecting growth factors or
genetically engineered cells which secrete growth factors into the
eye. Some very preliminary work in this area was done by Park et
al., Developmental Biology 148:322-333 (1991). They stimulated
retinal pigment epithelial cells in embryonic birds with FGF2 in
vivo to regenerate a neural retina. However, this was in birds, not
mammals, and only in embryonic birds. Moreover, the cells of the
regenerated neuroretina formed in these chicks were not in their
normal location. Thus, the photoreceptors, normally closest to the
brain, were located farthest from it, and ganglion cells were
closest to the brain. Thus, there is a clear need to develop
techniques to safely and effectively target stem cells in vivo in
mammals with growth factors in order to regenerate healthy eye
tissue. The eye is easily accessible surgically or by injection,
and it would be helpful if this accessibility could be exploited by
targeting retinal stem cells in areas of damage.
[0036] It would also be useful if stem cells were discovered that
could proliferate in the absence of growth factors.
[0037] A need also exists for a pharmaceutical composition
containing retinal cells for transplantation in which (1) the
composition is accepted by the patient, thus avoiding the
difficulties associated with immunosuppression, (2) the composition
is safe and effective, thus justifying the cost and effort
associated with treatment, (3) the composition provides long term
relief of the symptoms associated with the disease, (4) the
composition is efficacious during and after transplantation. There
is a clear need to develop retinal stem cell cultures which can act
as a source of cells that are transplantable in vivo in order to
replace damaged tissue.
[0038] There is also a need for retinal stem cell cultures or
retinal cell cultures which may be used in toxicity testing, drug
development and to isolate new genes and metabolites involved in
cell differentiation. There is also a need for retinal cell
cultures which may be used to develop derivative cell lines, such
as retinoblastoma cell culture lines, for studying cancer or other
diseases, disorders or abnormal states.
SUMMARY OF THE INVENTION
[0039] The invention provides for stem cells isolated from the
mammalian retina and retinal cells differentiated from these stem
cells.
[0040] This invention overcomes the needs outlined above in that it
provides a method for stimulating stem cells of the retina to
proliferate in vivo to produce differentiated retinal cells.
Proliferation is induced by administering one or more growth
factors to the retina. Proliferation is also induced by
administering genetically engineered cells which secrete growth
factors into the eye.
[0041] The retinal stem cells may also be used as sources of
transplantable tissue, as they can be removed from the donor and
transplanted into a recipient either before or after
differentiation into retinal cells. This invention also satisfies
the needs outlined above in that the retinal stem cells of this
invention (1) are accepted by the patient because they can be taken
from the patient's own retina, (2) are safe in that the patient is
not receiving cells or tissue from another source, (3) are
effective in that the retinal stem cells can be differentiated into
retinal cells for implantation and survive during and after
implantation, and (4) offer the potential to provide long term
relief of the symptoms of conditions associated with loss of one or
more retinal cell types.
[0042] The invention also provides cell cultures which may be used
in toxicity testing, drug development and the isolation of new
genes and metabolites involved in cell differentiation.
[0043] Accordingly, it is an object of the invention to provide
retinal stem cells which are isolated and purified from the retina
of a mammal. Retinal cells are then differentiate from the retinal
stem cells. Retinal cells which may be produced from the stem cells
are rod cells, cone cells, bipolar cells, amacrine cells, retinal
ganglion cells, retinal pigment epithelial cells, Mueller cells,
horizontal cells or glial cells.
[0044] The retinal stem cells are characterized by the presence of
Chx 10 protein, which is a marker for precursor cells.
[0045] The retinal stem cells may be transformed or transfected
with a heterologous gene. The growth or differentiation retinal
stem cells may be stimulated by a trophic factor.
[0046] The retinal stem cells and the retinal cells are useful in
toxicity testing, drug development testing, developing derivative
cell lines, and isolating genes or proteins involved in cell
differentiation.
[0047] It is another object of the invention to provide a
pharmaceutical composition for use in implant therapy consisting of
the retinal stem cells and retinal cells in a pharmaceutically
acceptable carrier, auxiliary or excipient. The invention also
relates to a method of treating a disease, disorder or abnormal
state of the retina by stimulating proliferation of retinal stem
cells. According to one embodiment of this invention, a growth
factor is introduced to retinal pigment epithelial cells. In the
method, the disease may be one of blindness; cytomegalovirus
retinitis, uveitis, glaucoma, macular degeneration, retinitis
pigmentosa, retinal degeneration, retinal detachment and cancers of
the retina. An individual suffering from a degenerative disease,
disorder or abnormal physical state of the retina may also be
treated by implanting the retinal stem cells or retinal cells into
the eye of the individual.
[0048] Another object of the invention is to provide a method for
isolating and purifying retinal stem cells from the retina of a
mammal by taking a sample of the retina from the mammal,
dissociating the sample into single cells, placing the cells in
culture, isolating the cells which survive in culture and
differentiating the cells which survive in culture into retinal
cells.
[0049] In another embodiment of the invention, where the mammal is
a human and is suffering from a disease, disorder or abnormal
physical state of the eye, the method includes implanting the
retinal stem cells or retinal cells differentiated from the retinal
stem cells, into the eye of the human. Where the mammal is a human
and is not suffering from a disease, disorder or abnormal physical
state of the eye, the method includes implanting the retinal stem
cells or retinal cells differentiated from the retinal stem cells
into a second human who is suffering from the disease, disorder or
abnormal physical state. The disease, disorder or abnormal physical
state which may be treated may be one of the group consisting of
blindness, cytomegalovirus retinitis, uveitis, glaucoma, macular
degeneration, retinitis pigmentosa, retinal degeneration, retinal
detachment and cancers of the retina.
[0050] Another object of the invention is to provide a kit,
containing at least one type of cells selected from a group
consisting of the retinal stem cells and the retinal cells. The kit
may be used for the treatment of a disease, disorder or abnormal
physical state of the eye.
[0051] The cells of the invention may also be used in a method for
identifying a substance which is toxic to retinal stem cells and
retinal cells, by introducing the substance to a retinal stem cell
culture or a retinal cell culture differentiated from a retinal
stem cell culture, and determining whether the cell culture is
adversely affected by the presence of the substance, is
employed.
[0052] The cells of the invention may also be used in a method for
identifying a pharmaceutical which may be used to treat a disease,
disorder or abnormal state of the eye, by introducing the
pharmaceutical to a retinal stem cell culture or a retinal cell
culture differentiated from a retinal stem cell culture, and
determining whether the culture is affected by the presence of the
pharmaceutical.
[0053] The invention also includes a method of stimulating
proliferation of retinal stem cells, comprising introducing a
growth factor to retinal pigment epithelial cells. The growth
factor is selected from a group consisting of EGF, FGF2, NGF, CNTF,
BDNF, FGF4, FGF8 and heparin. Accordingly, another aspect of the
invention is a method of treating a disease, disorder or abnormal
state of the retina, by stimulating proliferation of retinal stem
cells. This is done by introducing a growth factor to retinal
pigment epithelial cells. The method of treatment may be used in
treating a disease, disorder or abnormal physical state selected
from a group consisting of blindness, cytomegalovirus retinitis,
uveitis, glaucoma, macular degeneration, retinitis pigmentosa,
retinal degeneration, retinal detachment and cancers of the
retina.
[0054] Pigment granules in individual sphere cells are used to mark
the lineage of a retinal cell and the differentiation state of a
retinal cell. Retinal stem cells have black pigment granules,
progenitor cells have less black pigment granules than retinal stem
cells, and neural retinal cells have no black pigment granules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The invention will now be described in relation to the
figures in which:
[0056] FIG. 1: Spheres formed from self-renewal of single retinal
stem cells from the retinal pigment epithelial layer of the adult
mouse eye. When grown in culture, only black spheres formed
initially which indicated that the retinal stem cells came from the
retinal pigment epithelial layer.
[0057] FIG. 2: After 2-3 days, the spheres depicted in FIG. 1 both
contained black and white cells, indicating that the black stem
cells produced white progenitor cells of the neural retina.
[0058] FIG. 3: Retinal stem cells proliferate in the absence of
growth factors. EGF & FGF2 facilitate proliferation.
DETAILED DESCRIPTION OF THE INVENTION
[0059] This invention discloses the isolation of a stem cell from
both embryonic and adult mouse retina as well as adult human
retina. This is the first indication that a retinal stem cell is
present in the adult mammalian retina. There are embryonic
precursor cells but an embryonic precursor cell is not a stem cell
as defined in this application because a characteristic of a stem
cell is its long term self-renewal (it self-renews throughout the
embryonic and adult stage). The prior art teaches that the
existence of a retinal stem cell in adult mammals is unlikely.
During embryonic and early postnatal development there is a
germinal zone (also called a ventricular zone) in the developing
eye. There is no obvious remnant of the embryonic retinal germinal
(ventricular) zone in the adult mammalian retina and the prior art
teaches that the germinal zone disappears postnatally. The ependyma
and the retinal pigment epithelium both arise from their respective
ventricular zones and both are generally considered to be
post-mitotic epithelial tissue in adult animals. However, the
neural retina and subependyma also descend from the ventricular
zones of the eye and forebrain, respectively, and differ
significantly as the neural retina is generally considered to be
largely post-mitotic neuronal tissue whereas the subependyma is a
complex of undifferentiated proliferating cells. There is no
morphological ventricular tissue to be found in the adult
retina.
[0060] The stem cells of this invention possess the two key
characterising features of stem cells: they are mutipotential and
self-renew. These cells can be stimulated in vivo to generate new
retinal cells. The presence of the stem cell in the mature retina
suggests that there is no inherent inability to repair any injury
or disease to the retina.
[0061] It may simply be a matter of administering the right
combination of exogenous growth factors to stimulate the adult
retinal stem cells to proliferate and differentiate to achieve and
replace the compromised parts of the retina. As a result of this
discovery, the stem cells may also be cultured in vitro to generate
large numbers of new stem cells. The stem cells may also be
differentiated by adding growth factors to the culture medium,
which provides a source of healthy differentiated retinal tissue
cells. The cells of this invention may be used in transplants,
toxicity testing, drug development testing, or studies of genes and
proteins.
[0062] The retinal tissue was dissected using the method of Opas et
al., Development Biology, 161:440-454, (1993), as described in
Example 1. Single cells were isolated from the embryonic day 14
retina and cultured in the presence of the growth factors,
epidermal growth factor ("EGF"), basic fibroblast growth factor
("FGF2") and heparin. The cells were also cultured in the presence
of EGF alone. This methodology is described in Example 2.
[0063] It is now clear that cells do proliferate in the absence of
growth factors and that for the most part EGF and FGF2 facilitate
proliferation but not extensively (FIG. 3). Our data suggest that
FGF2 can cause growth of individual spheres but not so greatly
increase the number of spheres generated. It was discovered that
these growth factors caused single cells to proliferate to form
floating spheres even in serum-free medium. We also isolate and
culture a human retinal stem cell (Example 10).
[0064] When these spheres were dissociated into single cells, the
retinal stem cells self-renewed and proliferated to form new
spheres. The cells also exhibited properties that indicated that
they were multipotential stem cells.
[0065] We identified pan-neuronal and glial markers for retinal
sphere derived cells. We identify specific neuronal cell types.
Cells which migrate out of plated spheres and begin to
differentiate do not express Chx 10 immunoreactivity as would be
expected for all cell types except perhaps bipolar and amacrine
cells. When these cells were plated, they differentiated into
various neural retinal cell types i.e. they showed retinal specific
patterns of differentiation. Furthermore, the proportions of cells
produced by differentiation were consistent with these stem cells
originating in the retina.
[0066] There is clear evidence that the cells of the invention are
precursor cells of retinal origin. Some of the cells in the retinal
spheres exhibited neural-specific markers, such as the Chx 10
marker (Example 3). The Chx 10 gene was cloned and reagents to it
made by Liu et al., Neuron, Vol. 13:377-393 (1994). Chx 10 is a
murine polypeptide which is a regulatory protein involved in
vertebrate retinal development. It is a marker of neural precursor
cells, although it is not clear if it is found in retinal
progenitor cells or stem cells. This protein is expressed in early
retinal cells in vivo but generally not by telencephalon cells or
their progeny. In the mature retina, bipolar and amacrine cells
express Chx 10. Some cells in the spheres also tested positively
for nestin which is a filamentous protein present only in
undifferentiated cells. We also locate other markers which identify
retinal precursor cells (Example 9). These markers clearly
distinguish retinal stem cells from forebrain stem cells.
[0067] Our experiments with mouse retinal cells further indicate
that these retinal stem cells come from the ciliary margin of the
adult retinal pigment epithelial layer and not the adult neural
retinal layer (Example 4).
[0068] The stem cells are stimulated to produce differentiated
retinal cells in vitro in the presence of growth factors (Example
5). The growth factors are introduced into the site of the stem
cells (retinal pigment epithelial cells see--Example 4) to grow the
cells in an attempt to repair diseased or damaged retina. The
differentiated retinal cells are characterized (Example 8).
Production of certain retinal cell types is biased by particular
growth factors.
[0069] We also administer exogenous growth factor in vivo to
stimulate retinal stem cell proliferation (Example 11). The retinal
stem cells interface with biomaterials to provide therapies that
stimulate axonal cell growth (Example 12).
[0070] The pharmaceutical compositions of this invention used to
treat patients having degenerative diseases, disorders or abnormal
physical states of the eye could include an acceptable carrier,
auxiliary or excipient. The compositions can be for topical,
parenteral, local, intraocular or intraretinal use.
[0071] The pharmaceutical composition can be administered to humans
or animals. Dosages to be administered depend on patient needs, on
the desired effect and on the chosen route of administration.
[0072] The pharmaceutical compositions can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and such that
an effective quantity of the cells is combined in a mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985).
[0073] On this basis, the pharmaceutical compositions could include
an active compound or substance, such as growth factors,
genetically engineered stem cells or retinal cells which secrete
growth factor or other substances, in association with one or more
pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable pH and isoosmotic with the
physiological fluids. The methods of combining growth factor or
cells with the vehicles or combining them with diluents is well
known to those skilled in the art. The composition could include a
targeting agent for the transport of the active compound or cells
to specified sites within the eye, such as specific cells, tissues
or organs.
[0074] The invention also relates to the use of the stem cells and
progenitor cells of this invention to introduce recombinant
proteins into the diseased or damaged retina. The cells act as a
vector to transport a recombinant molecule, for example, or to
transport a sense or antisense sequence of a nucleic acid molecule.
In the case of a recombinant molecule, the molecule would contain
suitable transcriptional or translational regulatory elements.
[0075] Suitable regulatory elements may be derived from a variety
of sources, and they may be readily selected by one of ordinary
skill in the art. Examples of regulatory elements include: a
transcriptional promoter and enhancer or RNA polymerase binding
sequence, a ribosomal binding sequence, including a translation
initiation signal. Additionally, depending on the vector employed,
other genetic elements, such as selectable markers, may be
incorporated into the recombinant molecule.
[0076] The recombinant molecule may be introduced into stem cells
or retinal cells differentiated from stem cells of a patient using
in vitro delivery vehicles such as retroviral vectors, adenoviral
vectors, DNA virus vectors, amplicons and liposomes. They may also
be introduced into these cells using physical techniques such as
microinjection and electroporation or chemical methods such as
coprecipitation and incorporation of DNA into liposomes.
[0077] Suitable regulatory elements may be derived from a variety
of sources, and they may be readily selected by one of ordinary
skill in the art. If one were to upregulate the expression of the
gene, one would insert the sense sequence and the appropriate
promoter into the vehicle. If one were to downregulate the
expression of the gene, one would insert the antisense sequence and
the appropriate promoter into the vehicle. These techniques are
known to those skilled in the art.
[0078] The pharmaceutical compositions could also include the
active compound or substance, such as the stem cells of this
invention or retinal progenitor cells or differentiated cells
derived from those stem cells, in association with one or more
pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable pH and iso-osmotic with the
physiological fluids. The methods of combining cells with the
vehicles or combining them with diluents is well known to those
skilled in the art. The composition could include a targeting agent
for the transport of the active compound to specified sites within
the eye, such as specific cells, tissues or organs.
EXAMPLE 1
Dissection of Retina and Retinal Pigment Epithelium (RPE)
[0079] We considerably narrowed the area from which the retinal
stem cell originates in the adult eye. The pigmented cells
associated with the ciliary margin is the only area from which
retinal spheres can be isolated in the adult animal. Dissections
done on younger animals (embryonic or early post-natal) typically
include the entire RPE. The cells of this invention may be used
with biomaterials in a method of medical treatment of ocular
dysfunction or disease. The dissection of the adult and embryonic
neural retinal and RPE layers was done similarly to the method
described for dissecting chick embryo retina in Opas et al.,
Development Biology, 161:440-454, (1993) in artificial
cerebrospinal fluid (aCSF) containing 124 mM NaCl, 5 mM KCl, 1.3 mM
MgCl, 2 mM CaCl.sub.2, 26 mM NaHCO.sub.3, and 10 mM D-glucose (pH
7.4) previously aerated (15 min) with 95% 0.sub.2-5% CO.sub.2 at
room temperature. RPE cultures were prepared by enucleating mouse
embryos or adults, removing anterior portions of the eye (including
the lenticular and corneal tissue) and by making an incision
through the sclera to facilitate the removal of the vitreous,
retina and associated vasculature. The retinal cups, with exposed
RPE, were incubated in a sterile dispase solution (Collaborative
Research) at 30-32.degree. C. for 10 min, and then in a sterile
high Mg.sup.2+, low Ca.sup.2+ aCSF solution at 37.degree. C. for 10
min in the presence of trypsin (1.3 mg/mL), hyaluronidase (0.66
mg/mL), and kyneurinic acid (0.1 mg/mL). In the case of embryonic
tissue, the retinal cups were taken from dispase and separated from
the basement membrane. The high Mg.sup.2+, low Ca.sup.2+ aCSF
solution, in the presence of trypsin, hyaluronidase and kyneurinic
acid was not used for embryonic tissue (only adult tissue). The RPE
was separated from its basement membrane (and associated choroidal
tissue and vasculature) and placed in serum-free media (described
below). The neural retinal tissue was prepared in a similar
manner.
EXAMPLE 2
Isolation and Culturing of Retinal Stem Cells in the Presence of
Growth Factors
[0080] Retinal cells were dissociated and cultured in the presence
of growth factors according to the methods described in Morshead et
al., Neuron, Vol. 13:1071-1082 (1994) and Reynolds et al., 1993.
After dissection and enzymatic treatment, neural retina and RPE
tissues were cut into 1 mm sections and transferred into serum-free
culture medium (described below) containing 0.7 mg/mL trypsin
inhibitor (Boehringer-Mannheim) to stop the enzymatic reaction and
mechanically dissociated (trituration) with a fire-polished Pasteur
pipette. The cell suspension was then centrifuged at 150.times.g
for 5 min, the media was aspirated and the pellet was resuspended
in fresh serum-free media only.
[0081] The dissociated cells were plated in noncoated 35 mm culture
dishes (NUNC 96 well plates) at desired densities (determined by
Trypan blue exclusion) with serum-free medium containing 20 ng/mL
EGF (UBI; purified from mouse sub-maxillary gland), or 10 ng/mL
FGF2 (UBI; human recombinant) with heparin (2 ug/mL; UBI). The
serum-free medium was composed of DMEM/F12 (1:1) (Gibco) and
contained a salt and hormone mix of insulin (25 ug/mL), transferrin
(100 ug/mL), progesterone (20 nM), Putrescine (60 uM), selenium
chloride (30 nM), glucose (0.6%), glutamine (2 mM), sodium
bicarbonate (3 mM), and HEPES buffer (5 mM). Single cells
proliferated in response to the defined growth factors in
serum-free media to form floating spheres. These retinal stem cells
were passaged by mechanical dissociation of spheres into single
cells. The number of spheres were counted in each 96 well culture
dish after a 5-10 day incubation period.
[0082] This procedure was used to obtain retinal cell cultures from
mouse embryo and adult mouse. We use similar procedures to obtain
retinal stem cell cultures from human embryo and adult humans.
[0083] We performed a passage of the retinal stem cells and showed
that 1) the stem cell is passageable and 2) the cell division
characteristics reveal that it is an asymmetrically dividing cell
as opposed to a symmetrically dividing cell like forebrain cells. A
single sphere always give rise to one sphere. We harvested these
cells from adults and embryos which shows that they are long lived.
The passage in culture of cells identified in both the embryonic
and adult retina establishes this cell as a stem cell--the ability
to self-renew throughout the life of the animal.
[0084] The retinal spheres were different from the neurospheres
generated by embryonic and adult telencephalic neural stem cells in
that the retinal spheres expressed high levels of Chx 10 (see
Example 3), a marker of neural precursor cells that is a gene
product demonstrated to be essential for normal development of the
mammalian eye (Burmeister et al., Nature Genetics 12:376-384,
1996).
EXAMPLE 3
ChX 10 Marker Occurs in Retinal Stem Cells
[0085] EGF, FGF2 and heparin together induced the proliferation of
retinal stem cells in serum-free medium, which produced a sphere of
undifferentiated precursor cells. EGF alone in serum-free medium
also produced undifferentiated precursor cells. These spheres were
not immunoreactive for glial fibrillary acidic protein (GFAP) (an
intermediate filament protein specific for astrocytes),
neuron-specific enolase (a neuron-specified enzyme), or myelin
basic protein (MBP) (a cell surface protein specific to
oligodendrocytes). The spheres were, however, immunoreactive for
nestin (characterized by Lehndahl et al., Cell 60:585 (1990)) which
is an intermediate filament protein found in undifferentiated CNS
cells. The precursor cells were also immunoreactive for the Chx 10
protein marker (characterized by Liu et al., Neuron, Vol. 13,
377-393 (1994); Burmeister et al. Nature Genetics 12:376-384,
1996). Chx 10 marker is a homeobox gene that is normally expressed
in vivo by all retinal precursors. Chx 10 can be found in forebrain
stem cell derived spheres although it is in lesser quantity than it
is in retinal stem cells. Chx 10 is also only expressed in very
limited regions of the forebrain. The mature cell types that
differentiate from these precursor cells were predominantly not
immunoreactive for nestin or Chx 10. When differentiated in vitro,
separate cells from the spheres derived from retinal stem cells
expressed glial and at least one pan neuronal marker.
EXAMPLE 4
Adult Retinal Stem Cell Originates from Adult Retinal Pigment
Epithelial Population
[0086] These experiments determined the origin of the adult retinal
stem cell. The retina consists of the inner neural retinal layers
and an outer cell monolayer of RPE cells. The RPE cells in
pigmented mice are visibly pigmented (melanin granules), whereas
the neural retinal cells are essentially nonpigmented. We separated
the retinal layers (previously described in Example 1) and found
that the retinal stem cells (retinal spheres) came from the RPE
layer in vitro. The RPE stem cells initially proliferated to form
small spheres of primarily pigmented cells (FIG. 1), but after
several more days of growth in serum-free media/growth factors, the
pigmented spheres started to produce nonpigmented progeny (FIG. 2).
The nonpigmented cells are neural retinal cells.
[0087] The result of this experiment suggests that the density of
pigment granules in individual cells within a sphere (isolated from
a single pigmented cell in the RPE layer) provide an independent
marker for the lineage and/or differentiation state of a retinal
cell: stem cells are heavily pigmented, different more restricted
progenitor cells have smaller and variable numbers of pigment
granules (perhaps distributed in different cellular compartments),
and differentiated neural retinal cells are nonpigmented. This is
an effective way to do lineage analysis and will be used in
conjunction with specific retinal cell type markers to determine
the correlation between the relative density of retinal stem
cell-derived progenitors and the onset of retinal cell type
specific differentiation. For example, utilizing
immunocytochemistry to determine the temporal pattern of retinal
cell differentiation from sphere-derived cells to determine: (1)
how pigmentation levels and distribution correlate with the stage
of differentiation as detected by cell-specific antibodies such as
neurofilament (ganglion cells), rhodopsin (rod cells), HPC
(horizontal cells), VC1.1 (amacrine cells), and GFAP (Mueller
glia); and (2) if the temporal order of emerging differentiated
retinal cell types is conserved in vitro, with ganglion cells
differentiating first, etc.
EXAMPLE 5
Stimulation of Proliferation of the Embryonic and Adult Eye Stem
Cell
[0088] We stimulate proliferation of the adult and embryonic
retinal stem cell in a chemically defined serum-free medium in the
presence of growth factors. Basic fibroblast growth factor (FGF2),
epidermal growth factor (EGF), nerve growth factor (NGF), ciliary
neurotrophic factor (CNTF), brain-derived neurotrophic factor
(BDNF), insulin-like growth factor (IGF-1) and stem cell factor
(SCF) are all tested separately to stimulate proliferation of the
adult and embryonic retinal stem cell to produce spheres.
Similarly, various combinations of these growth factors are used to
determine the extent of combinatorial effects between growth
factors to facilitated retinal stem cell proliferation and sphere
formation in vitro.
[0089] Stimulating production of Chx 10 may encourage the
generation of retinal precursors. For example, growth factors may
be used to stimulate Chx 10 production which encourages later
differentiation of more specific retinal stem cells (for example,
bipolar cells).
EXAMPLE 6
Proliferation of the Embryonic and Adult Eye Stem Cell in the
Absence of Growth Factors
[0090] The retinal stem cell can be isolated from the adult mouse
in serum free conditions independent of any growth factors
(including insulin). This is shown in FIG. 3. The neural stem cells
of the adult and embryonic forebrain do not proliferate in the
absence of growth factors. In fact, of the many growth factors that
have been utilized to try to stimulate the generation of forebrain
neurospheres, only three factors have thus far been successfully
used. EGF, FGF2 and IGF-1 have all been shown to stimulate the
generation of neurospheres from forebrain tissue. Thus, the retinal
cell is a cell type that can proliferate and be passed in the
absence of any growth factors something that no other primary
mammalian cell type is capable of doing.
EXAMPLE 7
Proliferation of the Embryonic and Adult Eye Stem Cell in the
Absence of Growth Factors
[0091] Another clear difference between forebrain neurospheres and
retinal stem cells is that the retinal stem cell is pigmented
whereas the forebrain neurosphere is not. The biochemistry involved
with pigmentation is important in the proliferative capacity of the
stem cell (Jeffrey, T.I.N.S., 20:4:165-169 (1997)) because we get
differences in the size and number of retinal spheres generated
from pigmented mice or rats compare to their non-pigmented
counterparts. Spheres generated from non pigmented cells are
smaller and fewer in number.
EXAMPLE 8
Characterization of Differentiated Retinal Cell Types
[0092] We determine which differentiated cell types are produced
with various growth factors (and combinations) in culture.
Identification of one growth factor (or combination of specific
growth factors) which biases the production of one specific
differentiated retinal cell type is extremely useful in trying to
repair eye injuries in humans that involve primarily one specific
cell type. Using immunocytochemistry or in situ hybridization, we
determine the presence of differentiated retinal cell types using
established markers such as neurofilament (ganglion cells), HPC
(horizontal cells), Chx 10 (bipolar cells), VC1.1 (amacrine cells),
rhodopsin (rod cells), and GFAP (Mueller glia) (Liu et al., Neuron,
Vol. 13, 377-393 (1994)).
EXAMPLE 9
New Retinal Markers
[0093] We identify other novel markers (besides Chx 10) that: (1)
identify retinal precursor cells (stem and progenitor cells) and
distinguish them from forebrain precursor cells; (2) allow for the
identification of genes involved in transition from retinal
precursor phenotypes to differentiated retinal phenotypes; and (3)
identify genes involved in cell cycle progression during retinal
differentiation, using established methods such as differential
display. This provides unique markers and targets for ocular
cancers. Specific antibodies directed to novel proteins or the
presence of novel mRNAs that mark the retinal stem cells or retinal
progenitor cells are identified.
EXAMPLE 10
Human Neural Stem Cells
[0094] We isolate human neural stem cells in culture from the adult
and embryonic retina using the aforementioned media and growth
factor conditions, and subsequently utilize aforementioned
techniques to determine the identity of stem cell, progenitor cell,
and differentiated retinal cell types.
EXAMPLE 11
In Vivo Administration of Exogenous Growth Factors
[0095] We administer exogenous growth factors in the in vivo adult
mammalian eye to stimulate the adult retinal neural stem cell to
proliferate in vivo and produce new retinal neurons. We perform
this experiment in control mouse eyes and previously injured mouse
eyes in vivo to model the recovery of the human eye from injury or
disease (procedure set out in Craig et al., The Journal of
Neuroscience, 16(8):2649-2658 (1996)). We perform intraoccular
infusions of the aforementioned growth factors and determine
retinal cell differentiation using immunohistochemical techniques
with the detection of established markers (see Example 8).
[0096] The cells of this invention also proliferate in the absence
of growth factors, so new retinal neurons may also be produced in
vivo in the absence of exogenous growth factors.
EXAMPLE 12
Interface with Biomaterials and Stem Cells to Provide Therapies for
Ocular Dysfunction or Disease
[0097] We deliver encapsulated modified retinal stem cells using
biomaterial and genetic engineering technologies (procedures set
out in Shiochet et al, 1995). This cell therapy delivers a
continuous source of a given factor (of a gene of choice) to the
eye. We provide a source of stimulatory or inhibitory factors
required for ganglion cell axonal growth into the brain. We also
provide more transient therapies using biodegradable materials that
in combination with the stem cells require the transient expansion
of cell numbers (see Example 11), the reduction of inhibitory
components and the increase in stimulatory components to axonal
cell growth.
[0098] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
[0099] The present invention has been described in terms of
particular embodiments found or proposed by the present inventors
to comprise preferred modes for the practice of the invention. It
will be appreciated by those of skill in the art that, in light of
the present disclosure, numerous modifications and changes can be
made in the particular embodiments exemplified without departing
from the intended scope of the invention. All such modifications
are intended to be included within the scope of the appended
claims.
* * * * *