U.S. patent application number 13/440912 was filed with the patent office on 2012-08-30 for production of extracellular matrix, conditioned media and uses thereof.
This patent application is currently assigned to UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY. Invention is credited to Vamsi Gullapalli, Ilene Sugino, Marco Zarbin.
Application Number | 20120219737 13/440912 |
Document ID | / |
Family ID | 46719161 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219737 |
Kind Code |
A1 |
Sugino; Ilene ; et
al. |
August 30, 2012 |
Production of extracellular matrix, conditioned media and uses
thereof
Abstract
Provided is a matrix for promoting survival and differentiation
of cells transplanted thereon, comprising a base matrix and a
cell-made matrix thereon. Methods and means for making and using
same are also provided. Also provided are conditioned media,
related compositions, related methods, and related packaging
products.
Inventors: |
Sugino; Ilene; (Madison,
NJ) ; Gullapalli; Vamsi; (Corpus Christi, TX)
; Zarbin; Marco; (Chatham, NJ) |
Assignee: |
UNIVERSITY OF MEDICINE AND
DENTISTRY OF NEW JERSEY
Somerset
NJ
|
Family ID: |
46719161 |
Appl. No.: |
13/440912 |
Filed: |
April 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12738839 |
Aug 2, 2010 |
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PCT/US2008/080408 |
Oct 19, 2008 |
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13440912 |
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60999601 |
Oct 19, 2007 |
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61471951 |
Apr 5, 2011 |
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61561224 |
Nov 17, 2011 |
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Current U.S.
Class: |
428/35.6 ;
424/520; 424/93.7; 435/325 |
Current CPC
Class: |
C12N 2533/90 20130101;
C12N 5/0621 20130101; A61K 35/30 20130101; Y10T 428/1348 20150115;
C12N 2501/115 20130101; A61K 35/00 20130101; A61P 27/02
20180101 |
Class at
Publication: |
428/35.6 ;
435/325; 424/93.7; 424/520 |
International
Class: |
B32B 1/06 20060101
B32B001/06; A61P 27/02 20060101 A61P027/02; C12N 5/0735 20100101
C12N005/0735; C12N 5/0775 20100101 C12N005/0775; C12N 5/071
20100101 C12N005/071; A61K 35/12 20060101 A61K035/12 |
Goverment Interests
GOVERNMENTAL SUPPORT
[0002] The Research leading to the present invention was supported
in part, by National Institutes of Health Grant No. NIH RO3
EY013690. Accordingly, the U.S. Government has certain rights in
this invention.
Claims
1. A method of increasing survival and/or differentiation of target
cells on a base matrix, the method comprising: creating a cell-made
extracellular matrix on said base matrix to produce a modified base
matrix and administering said target cells to said modified base
matrix.
2. The method of claim 1, wherein the step of creating the
cell-made extracellular matrix on said base matrix to produce the
modified base matrix is performed in vitro.
3. The method of claim 1, wherein the step of administering said
target cells to said modified base matrix is performed in vivo.
4. The method of claim 1, wherein the step of creating the
cell-made extracellular matrix on said base matrix to produce the
modified base matrix is performed in vivo.
5. A modified base matrix for survival and/or differentiation of
target cells thereon, the modified base matrix comprising a
cell-made extracellular matrix thereon.
6. The modified base matrix of claim 5, wherein the modified base
matrix is produced by culturing cells capable of forming said
cell-made extracellular matrix on the base matrix.
7. The modified base matrix of claim 5, wherein said modified base
matrix is produced by treating the base matrix with at least an
active fraction of a conditioned media from culturing cells capable
of forming said cell-made extracellular matrix on the base
matrix.
8. The modified matrix of claim 5, wherein said base matrix is
Bruch's membrane.
9. The modified base matrix of claim 8, wherein the Bruch's
membrane is undergoing macular degeneration.
10. The modified matrix of claim 5, wherein said base matrix is a
synthetic polymer-based matrix.
11. The modified base matrix of claim 10, wherein the synthetic
polymer is selected from polylactic acid (PLA), polyglycolic acid
(PGA), poly(lactide-co-glycolide) (PLGA), poly(methyl methacrylate)
(PMMA), polyorthoesters, and any combinations thereof.
12. The modified base matrix of claim 11, wherein the synthetic
polymer is polycaprolactone (PCL).
13. The modified base matrix of claim 5, wherein said target cells
are selected from RPE, umbilical cells, placental cells, adult stem
cells, embryonic stem cells, fetal RPE, adult iris pigment
epithelial (IPE) cells, bone marrow-derived stem cells, Schwann
cells, neural progenitor cells, and any combination thereof.
14. The modified base matrix of claim 5, wherein said target cells
are autologous.
15. The modified base matrix of claim 5, wherein said target cells
are fetal RPEs.
16. The modified base matrix of claim 5, wherein the target cells
are adult or embryonic stem cells or are differentiated from adult
or embryonic stem cells.
17. A conditioned media from culturing cells capable of forming a
cell-made extracellular matrix on the base matrix of claim 5.
18. The conditioned media of claim 17, which is serum-free.
19. The conditioned media of claim 17, wherein said cells capable
of forming said cell-made extracellular matrix on the base matrix
are selected from corneal endothelial cells, RPE cells, IPE cells,
embryonic stem cells, bone marrow-derived stem cells, placental
cells, and/or umbilical cells.
20. The conditioned media of claim 19, wherein said cells are
corneal endothelial cells.
21. The conditioned media of claim 19, wherein the conditioned
media or at least a fraction thereof is produced by culturing cells
capable of forming said cell-made extracellular matrix.
22. An active fraction of a conditioned media of claim 17,
characterized by a depletion of biologically active components
having MW of less than about 100 kD.
23. The active fraction of claim 22, wherein the active fraction of
the conditioned media from culturing cells capable of forming said
cell-made extracellular matrix on the base matrix is formed by a
depletion of biologically active components having MW of less than
about 100 kD.
24. A kit for promoting the survival and/or differentiation of
target cells on a base matrix, comprising: a) a set of instructions
and at least one of: b) an efficient amount of cells capable of
forming a cell-made extracellular matrix; and c) at least an active
fraction of a conditioned media or an extracellular matrix from the
cells capable of forming a cell-made extracellular matrix.
25. The kit of claim 24, wherein the cells capable of forming the
cell base and at least the active fraction of the conditioned media
or the extracellular matrix from the cells capable of forming the
cell-made extracellular matrix are according claim 5.
26. A kit for promoting survival and differentiation of target
cells comprising a set of instructions and a modified base matrix
according to claim 5.
27. The kit of claim 24 further comprising an effective amount of
target cells selected from RPE, umbilical cells, placental cells,
adult stem cells, cells differentiated from adult stem cells, ES
cells, cells differentiated from ES cells, bone marrow-derived stem
cells, fetal RPE, adult iris pigment epithelial (IPE) cells,
Schwann cells, and any combination thereof, derived from autologous
or allogenic source.
28. A method for making a conditioned medium, comprising obtaining
a plurality of cells capable of forming a cell-made extracellular
matrix; culturing the cells in a first medium for a period of time
to form a second medium; and collecting the second medium thereby
making the conditioned medium.
29. The method of claim 28, wherein the first medium is free of
serum.
30. The method of claim 28, wherein the cells are selected from the
group consisting of corneal endothelial cells, RPE cells, IPE
cells, embryonic stem cells, bone marrow-derived stem cells,
placental cells, and umbilical cells.
31. The method of claim 30 wherein the cells are corneal
endothelial cells.
32. The method of claim 28, wherein the period of time is 1-5
days.
33. The method of claim 32, wherein the period of time is 2-4
days.
34. The method of claim 33, wherein the period of time is 3
days.
35. A composition comprising a fraction of a conditioned medium of
claim 17.
36. The composition of claim 35, wherein the fraction comprises
molecules having MW of less than 3 kD.
37. The composition of claim 35, wherein the fraction comprises
molecules having MW of less than 50 kD.
38. The composition of claim 37, wherein the fraction comprises
molecules having MW of 10-50 kD.
39. The composition of claim 38, wherein the fraction comprises
molecules having MW of 30-50 kD.
40. The composition of claim 35, comprising a first fraction and a
second fraction of the medium, wherein (i) the first fraction
comprises molecules having MW of less than 3 kD, and (ii) the
second fraction comprises molecules having MW of 10-50 kD.
41. The composition of claim 35, wherein the fraction comprises one
or more proteins selected from those in Table 6.
42. The composition of claim 35, wherein the composition is a
pharmaceutical composition and comprises a pharmaceutically
acceptable carrier.
43. The composition of claim 35, wherein the composition is a cell
culture medium for use in storing, preserving, or inducing
differentiation of cells or tissues.
44. A method for treating age-related macular degeneration (AMD),
comprising identifying a subject in need thereof; and administering
to the subject an effective amount of the composition of claim
35.
45. A packaging product comprising a composition of claim 44; a
cell or a piece of tissue, and a container holding the
composition.
46. The packaging product of claim 45, wherein the cell is selected
from the group consisting of retinal pigment epithelial (RPE)
cells, stem cells, and corneal cells.
47. The packaging product of claim 45, wherein the tissue is
selected from the group consisting of RPE derived from precursor
cells, RPE derived from human embryonic stem cells, RPE derived
from iPSC stem cells, whole retinae, whole cornea, tissues, and
neural tissues and organs.
48. The packaging product of claim 45, wherein the cell is in
suspension or in a support matrix designed for cell delivery.
49. The packaging product of claim 45, where in the composition has
a temperature within the range of 0-40.degree. C.
50. The packaging product of claim 49, wherein the composition has
a temperature within the range of 4-30.degree. C.
51. The packaging product of claim 50, wherein the composition has
a temperature within the range of 15-25.degree. C.
52. The packaging product of claim 44, wherein the container is
sealed.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application No. 61/471,951 filed on Apr. 5, 2011 and U.S.
Provisional Application No. 61/561,224 filed on Nov. 17, 2011. This
application is a continuation-in-part of U.S. patent application
Ser. No. 12/738,839, which is a 35 U.S.C. .sctn.371 National Phase
application of International Application Serial No.
PCT/US2008/080408, filed Oct. 19, 2008, which claims benefit of
priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application No. 60/999,601 filed on Oct. 19, 2007. The disclosures
of the aforementioned applications are incorporated herein by
reference.
FIELD OF INVENTION
[0003] This invention relates to the production of an extracellular
matrix, conditioned media, and related uses.
BACKGROUND
[0004] Disease-related changes may mask extracellular matrix ligand
availability to transplanted cells, impairing post-attachment
events and leading, in turn, to cell death or inability of the
cells to differentiate. In addition, disease-related changes in the
extracellular matrix can promote cell death, leading to the
clinical situation in which cell transplantation is
contemplated.
[0005] One of the conditions in which cell transplantation may be
useful is age-related macular degeneration. (In addition, other
conditions affecting the macula, such as retinitis pigmentosa and
Stargardt disease, may benefit from cell-based therapy.) The macula
lutea is an area of the retina that is about 5000 .mu.m in
diameter. The center of the macula, the fovea, contains specialized
photoreceptors and provides high acuity vision necessary for
reading, driving, and recognizing faces. In order for light-sensing
photoreceptors to function properly, they must be in intimate
contact with a cell layer called the retinal pigment epithelium
(RPE). The photoreceptors and RPE exchange nutrients and other
materials. The choroid is a vascular layer of the eye wall
interposed between the sclera and RPE, and its capillaries, termed
the choriocapillaris, provide the blood supply to the RPE and
photoreceptors. The RPE is separated from the choriocapillaris by a
thin layer of collagenous tissue called Bruch's membrane.
[0006] Age-related macular degeneration (AMD) is the most important
cause of new cases of blindness in patients older than 55 years of
age in the industrialized world. RPE cells may be one of the
targets of the pathological processes that cause AMD. Approximately
10% of patients with AMD lose central vision. Among the .about.75%
of AMD patients with central visual loss, abnormal blood vessels,
termed choroidal new vessels (CNVs), grow from the choriocapillaris
and leak fluid and blood under the RPE and macula (exudative or
"wet" AMD), which causes visual loss. The stimulus for CNV growth
in AMD is complex, and the biochemical pathways are now being
identified. One critical element is vascular endothelial growth
factor (VEGF), which is involved in CNV growth and leakage. Among
.about.25% of AMD patients with severe central visual loss, the RPE
and foveal photoreceptors die in the absence of CNVs (atrophic or
"dry" AMD, also termed geographic atrophy (GA)). No visually
beneficial treatment exits for .about.60-75% of AMD patients.
[0007] Existing therapy has significant limitations. Antioxidants,
for example, do not seem to be effective in the prevention of early
AMD (i.e., drusen, retinal pigmentary changes). The Age-Related Eye
Disease Study (AREDS) did not show a statistically significant
benefit of the AREDS vitamin and mineral formulation for either the
development of new geographic atrophy or for involvement of the
fovea in eyes with pre-existing geographic atrophy.
[0008] Pharmacological therapies (e.g., AVASTIN.RTM. and
LUCENTIS.RTM., both of which block the action of VEGF) that are
pathway-based have provided the best treatment results for AMD
patients that have ever been reported. Nonetheless, a need for
improved therapy remains. Although LUCENTIS.RTM. treatment is
associated with moderate visual improvement in 25-40% of patients
according to the results of two randomized studies, the remaining
60-75% of patients are in urgent need of an alternative approach.
Also, these medications currently are administered via repeated
intravitreal injection, which entails some risk and inconvenience
for the patient. Further, pharmacological therapy generally
involves administration of a finite number of compounds and usually
involves fluctuations in drug levels above and below the desired
level.
[0009] Accordingly, novel methods and compositions are desired
which would address these drawbacks of currently accepted treatment
of AMD.
SUMMARY OF INVENTION
[0010] The instant invention addresses the drawbacks of the prior
art by providing, in one aspect, a modified base matrix for
promoting survival and/or differentiation of target cells thereon,
the modified base matrix comprising a cell-made extracellular
matrix (which is a mixture of proteins and other substances) on its
surface.
[0011] In different embodiments of the invention, the step of
creating the cell-made extracellular matrix may be achieved by
culturing, on the base matrix, the cells capable of producing such
extracellular matrix, and/or by treating the base matrix with
solubilized components of the extracellular matrix and/or at least
an active fraction of the conditioned media from the cells capable
of producing such extracellular matrix. Combination of these
approaches is also contemplated.
[0012] In another aspect, the invention provides a method of
increasing survival and/or differentiation of target cells on a
base matrix, the method comprising: creating a cell-made
extracellular matrix on the base matrix to produce a modified base
matrix and administering the target cells to the modified base
matrix. In different embodiments of the invention, the matrices
include, without limitations, those described above.
[0013] In another aspect, the invention provides a method of
increasing survival and/or differentiation of target cells on a
base matrix through providing a soluble formulation of the
extracellular matrix or conditioned media to the apical surface of
the cells to stimulate self-assembly and deposition of
extracellular matrix and/or stimulation of mechanisms for cell
survival and differentiation.
[0014] In further embodiments of the invention the base matrix may
be a biological matrix, such as Bruch's membrane or a synthetic
polymer based matrix.
[0015] The cells capable of producing the extracellular matrix are
in different embodiments selected from corneal endothelial cells,
RPE cells, human embryonic stem (ES) cells and any combinations
thereof. In a preferred set of embodiments, the cells are corneal
endothelial cells, including, without limitations, bovine corneal
endothelial cells (BCE).
[0016] In different embodiments, the target cells suitable for the
methods of the instant invention are selected from RPE cells,
umbilical cells, placental cells, adult stem cells, human ES cells
(or other embryonic stem cells), cells derived from human ES cells
(e.g. RPE derived from ES cells, retinal progenitor cells), fetal
RPE cells, adult iris pigment epithelial (IPE) cells, Schwann
cells, and combinations thereof. The target cells may be derived
from an autologous or an allogeneic source.
[0017] In another aspect, the invention provides a conditioned
media from culturing the cells capable of producing the
extracellular matrix. The cells capable of forming the
extracellular matrix may be the cells as described above. In a
preferred embodiment, the media is collected after the cells reach
confluency.
[0018] In another aspect the invention provides an active fraction
of the conditioned media, as described in the previous paragraph.
The active fraction is characterized by the depletion of bioactive
components having molecular weight less than 20 kD, preferably less
than 30 kD, more preferably, less than 50 kD, more preferably, less
than 70 kD, more preferably, less than 80 kD, more preferably, less
than 90 kD, and most preferably, less than 100 kD. The active
fraction may also be comprised of a combination of any of the above
molecular weight fractions.
[0019] In yet another aspect, the invention provides a method of
treating an eye disease associated with degradation of an in situ
extracellular matrix in the eye; such treatment includes creating a
modified base matrix and administering the target cells to the
modified base matrix.
[0020] In different embodiments of this aspect of the invention,
the modified base matrix is created according to any of the
embodiments of the previous aspect of the invention. Further, the
target cells are chosen as described in any of the embodiments of
the previous aspects of the invention.
[0021] In yet another aspect, the invention provides a kit for
improving survival and differentiation of target cells on a matrix.
Generally, the kit includes at least an active fraction of the
conditioned media or solubilized extracellular matrix according to
any embodiments described herein. The kit may also include a base
matrix. In another set of embodiments, the kit comprises a modified
base matrix. Further, in any embodiments of this aspect of the
invention, the target cells may be provided.
[0022] In a further aspect, the invention provides a method for
making a conditioned medium. The method includes (i) obtaining a
plurality of cells capable of forming a cell-made extracellular
matrix; (ii) culturing the cells in a first medium for a period of
time to form a second medium; and (iii) collecting the second
medium thereby making the conditioned medium. In one embodiment,
the first medium is free of serum. The cells can be selected from
the group consisting of corneal endothelial cells, RPE cells, IPE
cells, embryonic stem cells, bone marrow-derived stem cells,
placental cells, and umbilical cells. Preferably, the cells are
corneal endothelial cells, such as bovine corneal endothelial
cells. The period of time can be 1-5 days, such as 2-4 days or 3
days.
[0023] The invention also provides a composition comprising,
consisting essentially of, or consisting of a fraction of the
aforementioned conditioned medium. In one embodiment, the fraction
comprises, consists essentially of, or consists of molecules having
MW of less than 3 kD. In another, the fraction comprises, consists
essentially of, or consists of molecules having MW of less than 50
kD, e.g., 10-50 kD or 50 kD. In a preferred embodiment, the
composition comprises, consists essentially of, or consists of a
first fraction and a second fraction of the medium, where (i) the
first fraction composition comprises, consists essentially of, or
consists of molecules having MW of less than 3 kD, and (ii) the
second fraction com composition comprises, consists essentially of,
or consists of molecules having MW of 10-50 kD. For example, the
fraction can comprise, consist essentially of, or consist of one or
more proteins selected from those listed in Table 6 below. The
just-described composition can be a pharmaceutical composition and
contains a pharmaceutically acceptable carrier.
[0024] The above-described composition can be used for treating
age-related macular degeneration (AMD). To that end, one can
identify a subject in need of such treatment and administer to the
subject an effective amount of the composition.
[0025] The above-described composition can also be used as a cell
culture medium for use in storing, preserving, or inducing
differentiation of cells or tissues. Accordingly, this invention
also provides a packaging product containing the composition, a
cell or a piece of tissue, and a container holding the composition.
The cell can be selected from the group consisting of retinal
pigment epithelial (RPE) cells, stem cells, and corneal cells. The
tissue can be selected from the group consisting of RPE derived
from precursor cells, RPE derived from human embryonic stem cells,
RPE derived from iPSC stem cells, whole retinae, whole cornea,
tissues, and neural tissues and organs. The cell can be in
suspension or in a support matrix designed for cell delivery. The
composition can be used at a temperature within the range of
0-40.degree. C., such as 4-30.degree. C. or 15-25.degree. C. In one
embodiment, the composition and the cell or tissue can be sealed in
the container.
[0026] In any embodiment of this aspect of the invention, suitable
non-limiting examples of the base matrices, the modified base
matrices, and the target cells are those described in the other
aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 demonstrates that long-term survival of fetal RPE on
aged submacular human Bruch's membrane is impaired if the surface
(basement membrane or superficial surface of the inner collagenous
layer (ICL)) is not treated.
[0028] FIG. 2 demonstrates fetal RPE resurfacing on aged human
submacular Bruch's membrane is improved following resurfacing with
bovine corneal endothelial cell extracellular matrix (BCE-ECM).
[0029] FIG. 3 demonstrates that resurfacing aged human submacular
Bruch's membrane with a biologically deposited extracellular matrix
(ECM) improves long-term RPE survival compared to untreated Bruch's
membrane by over 200%.
[0030] FIG. 4 illustrates RPE survival on submacular human Bruch's
membrane of an AMD donor (age 79 years) cultured in
serum-containing bovine corneal endothelial cell-conditioned media
(BCE-CM) vs. routine RPE culture media. In the absence of Bruch's
membrane treatment, these RPE cells generally show poor survival on
human submacular Bruch's membrane of AMD eyes after 21 days in
organ culture.
[0031] FIG. 5 illustrates improved RPE survival with 21 day
exposure to serum-containing BCE-CM compared to 2 day exposure on
human peripheral Bruch's membrane from a non-AMD donor (age 80
years).
[0032] FIG. 6 demonstrates that overnight treatment with serum-free
BCE-conditioned media results in improvement of RPE survival and
differentiation on human submacular Bruch's membrane.
[0033] FIG. 7 demonstrates that RPE derived from human embryonic
stem cells successfully survive on aged human submacular Bruch's
membrane treated with serum-free BCE-conditioned media (BCE-CM) (A,
B), compared to untreated submacular Bruch's membrane (C, D).
[0034] FIG. 8 illustrates that soaking a polycaprolactone (PCL)
scaffold in serum-free BCE-conditioned media (BCE-CM) results in
improved initial RPE attachment (A) compared to no BCE-CM treatment
(B).
[0035] FIG. 9 illustrates that fetal RPE attachment and survival at
5 days is improved if the RPE are initially cultured in serum-free
BCE-conditioned media for 2 days (A) vs. no BCE-CM treatment
(B).
[0036] FIG. 10 illustrates that fetal RPE cultured in RPE complete
media can attach and resurface an untreated PCL scaffold, but by 7
days the cells do not exhibit density arrest.
[0037] FIG. 11 shows a two-dimensional gel with spot identification
of BCEC-CM (uncut, consisting of all fractions).
[0038] FIG. 12 provides graphical analysis of cell survival
(nuclear density) on aged and AMD Bruch's membrane in molecular cut
filtrates utilizing 3-300 kDa filters: the nuclear densities after
culture in the retentates (comprising molecules above the filter
size) are compared to BCEC-CM that has not be subject to
ultrafiltration (uncut).
[0039] FIG. 13 represents results of cell survival (nuclear
density) on aged and AMD Bruch's membrane in molecular cut
filtrates utilizing 30-100 kDa filters. The nuclear density after
culture in the filtrates is compared to BCEC-CM that has not been
subject to ultrafiltration (uncut).
[0040] FIG. 14 shows comparison of cell viability at day-3 in an
RPE medium at 4.degree. C. (left) and room temperature (RT, right),
where Live/dead tests performed at day-3 showed more cell death in
the RPE medium at 4.degree. C. than at room temperature (red:
ethidium homodimer staining of dead nuclei; green: calcein staining
of live cells). There did not appear to be any intact cells
remaining in the 4.degree. C. RPE medium well after staining.
[0041] FIG. 15 shows cell viability at day-7 in CM, CM molecular
cut filtrates, and RPE medium at 4.degree. C. (left column) and
room temperature (right column); at day-7, the majority of cells
remaining after live/dead staining were dead in CM and CM fractions
at 4.degree. C. The majority of cells in CM and CM fractions at
room temperature were alive and confluent with small defects while
only cell debris remain in the well stored in the RPE medium.
[0042] FIG. 16 shows cell viability after storage using CM batch
29AD.
[0043] FIG. 17 shows change in cell viability after storage at
4.degree. C. using CM batch 58.
[0044] FIG. 18 shows change in cell viability after storage at room
temperature using CM batch 58.
[0045] FIG. 19 shows change in live cells after storage at
4.degree. C.
[0046] FIG. 20 shows change in live cells after storage at room
temperature.
[0047] FIG. 21 shows comparison of cell behavior after seeding and
culture on tissue culture plastic in CM (left column) vs. RPE
(right column) medium with media change 3.times./week; the images
were taken at approximately the same location for all 3 time
points.
[0048] FIG. 22 shows comparison of cell behavior after seeding and
culture on tissue culture plastic in RPE medium (top row) vs. CM
(bottom row) with media change 3.times./week.
[0049] FIGS. 23A-G show paired submacular explants from a
74-year-old female with soft drusen, seeded with hES-RPE. CM
vehicle: (A) Postmortem clinical photograph shows soft drusen
(arrow) in the macula. Inset is a higher magnification image of the
area indicated by the arrow. The drusen are not easily visualized
in this photomicrograph due to post mortem changes. (B and C) No
intact cells are seen on the cultured explant. BCEC-CM: (D) Arrow
points to a patch of confluent soft drusen in the macula of the
fellow eye, shown in the high magnification inset. (E) Cells almost
fully resurface the explant with small defects in coverage. Cells
are variable in size and shape. Insert. Cells are generally flat
with most exhibiting short apical processes on their surfaces. (F
and G) Cells resurfacing the explant are in a monolayer of very
flat and elongate cells. Arrowhead (G) points to cell containing
vesicles. CM vehicle nuclear density (ND), 0; BCEC-CM ND,
19.90.+-.0.35. Scale bar: (E) 100 .mu.m; (E, inset) 20 .mu.m; (F)
50 .mu.m; (G) 20 .mu.m. Toluidine blue staining.
[0050] FIGS. 24A-G show paired explants from an 81-year-old male
with no submacular pathology, seeded with fetal RPE. (A, D) No
submacular pathology is seen in the post mortem clinical
photographs. CM vehicle: (B) Cellular debris but no intact cells
are seen on the surface of Bruch's membrane. Few remaining patches
of RPE basement membrane (arrows, insert) are present. (C) Rare
single cells are seen on the explant surface. Arrow points to a
single, very flat cell. BCEC-CM: (E) The explant is almost fully
resurfaced with small defects in cell coverage (arrows). Patches of
small, rounded cells are interspersed with localized areas where
cells are more variable in size and shape. Cells express abundant
short apical surface processes on their surfaces (inset). (F, G)
The explant is resurfaced by a mono- and bilayer of cells.
Arrowhead (G) points to a cell overlying a cell on Bruch's
membrane. CM vehicle ND, 0.51.+-.0.16; BCEC-CM ND, 26.8.+-.0.41.
Scale bar: (E) 100 .mu.m; (E, inset) 20 .mu.m; (F) 50 .mu.m; (G) 20
.mu.m. Toluidine blue staining.
[0051] FIGS. 25A-G show paired explants from a 75-year-old female
with large bilateral subfoveal choroidal new vessels (CNVs), seeded
with fetal RPE after mechanical CNV removal. (A, D) Arrows point to
CNVs in postmortem clinical photographs; insets show CNVs after
surgical dissection from Bruch's membrane. CM vehicle: (A) The CNV
was approximately 4.8.times.3 mm. (B, C) No intact cells are seen
on the explant surface. BCEC-CM: (D) The CNV was approximately
4.times.3.5 mm. (E) Fetal RPE fully resurface the explant with some
areas of thick multilayers (arrowhead). The cell surfaces are
covered with apical processes (inset). (F) Cells resurfacing the
explant are predominantly monolayered with localized areas where
thin or spindle-shaped cells overlay cells on Bruch's membrane. The
cells resurfacing the explant are more variable in size and shape
than those observed on explants from donors with geographic
atrophy. (G) Fetal RPE are able to resurface small drusen (arrow
points to druse on Bruch's membrane) and basal laminar deposits
(asterisk). Arrowhead points to a cell with a darkly staining
irregular-shaped nucleus. CM vehicle ND, 0; BCEC-CM ND,
25.10.+-.0.30. Scale bar: (E) 100 .mu.m; (E, inset) 20 .mu.m; (F)
50 .mu.m; (G) 20 .mu.m. Toluidine blue staining.
[0052] FIGS. 26A-G show paired explants from an 82-year-old female
with geographic atrophy, seeded with fetal RPE. The patient's
clinical history noted AMD for 20 years. (A, D) Postmortem clinical
photographs showing subfoveal geographic atrophy before RPE
seeding. CM vehicle: (B) Only a few dead cells (arrows) and
cellular debris are present on the explant surface. (C) No cells
are present on the Bruch's membrane surface. BCEC-CM: (E) RPE fully
resurface Bruch's membrane in the area of geographic atrophy with a
few very small defects (arrows). Localized areas of multilayering
are present. Cell surfaces show abundant apical processes (inset).
(F) In this field, cells resurfacing the BCEC-CM explant are
predominantly bilayered. Cells directly on Bruch's membrane are
small and tightly packed; flat cells appear to overlie the cells in
contact with Bruch's membrane. (G) Flattened cell processes
overlying cells on top of Bruch's membrane are indicated by
arrowheads. The cell processes contain vesicles. CM vehicle ND, 0;
BCEC-CM ND, 19.61.+-.0.43. Scale bar: (E) 100 .mu.m; (E, inset) 20
.mu.m; (F) 50 .mu.m; (G) 20 .mu.m. Toluidine blue staining.
[0053] FIGS. 27A-G show paired explants from an 80-year-old male
with intermediate-size drusen in the CM vehicle-cultured eye and
intermediate and large drusen in the BCEC-CM-cultured eye, seeded
with cultured adult RPE (isolated from a 70-year-old donor). CM
vehicle: (A) Two drusen (closely clustered) were present in the
macula (arrow and arrowhead, high magnification inset). The drusen
are not easily visualized in these photomicrographs due to post
mortem changes. (B) Very few large cells are observed on the
explant surface (6 cells in this image field). Arrow points to a
pair of very large, flat cells. (C) No cells are present on the
surface of Bruch's membrane. BCEC-CM: (D) A cluster of drusen
(arrow and high magnification inset) can be seen in the macula of
the fellow eye. (E) The explant is fully resurfaced by a monolayer
of cells that are highly variable in size. Some of the cells within
the monolayer do not have intact cell membranes (cells that appear
white in the low magnification image), and some cells have died
with remnants of cellular debris (arrows). The high magnification
inset shows that most of the cells are covered with short apical
processes, including cells that are very large (fetal RPE that are
of this size on submacular Bruch's membrane generally have smooth
surfaces with no apical processes). One cell in the field exhibits
surface blebs. (F, G) Cells resurfacing the explant are generally
large and often pigmented. Localized areas of bilayering are
present (F, arrowheads). CM vehicle ND, 0.14.+-.0.14; BCEC-CM ND,
12.0.+-.0.77. Scale bar: (E) 100 .mu.m; (E, inset) 20 .mu.m; (F) 50
.mu.m; (G) 20 .mu.m. Toluidine blue staining.
[0054] FIGS. 28A-B show nuclear densities of cells seeded on aged
submacular Bruch's membrane explants after 21-day culture in
conditioned medium vehicle (CM vehicle) or BCEC-conditioned medium
(BCEC-CM) (paired explants from the same donor). (A) Nuclear
density comparison of RPE derived from human embryonic stem cells
(hES-RPE, N=6), cultured human fetal RPE (fRPE, N=22), and cultured
human adult RPE (RPE donor ages 58, 71, 78 years; N=7). Within each
group, significant differences were observed between cells cultured
in CM vehicle and cells cultured in BCEC-CM. The nuclear density of
cells cultured in CM vehicle was not statistically different
between groups. The nuclear densities of hES-RPE and fRPE were not
significantly different from each other but were significantly
higher than the nuclear density of adult RPE after culture in
BCEC-CM. (B) Comparison of nuclear densities of fRPE on
age-matched, non-AMD vs. AMD Bruch's membrane at day-21. Explants
seeded with fRPE on aged Bruch's membrane (N=9) were compared to
explants seeded on AMD submacular Bruch's membrane (N=13). No
significant differences were observed in the nuclear densities of
fRPE on non-AMD vs. AMD explants for a given medium although the
nuclear density was significantly higher in the presence of BCEC-CM
vs. CM vehicle. Nuclear density values are counts of nuclei of
cells directly in contact with Bruch's membrane, expressed as mean
nuclear density/mm Bruch's membrane. Bars are mean nuclear
density.+-.standard error. *P<0.05; **P<0.001.
[0055] FIG. 29 shows nuclear densities of fetal RPE cultured in
BCEC-CM for 3- (N=7), 7- (N=8) or 14-days (N=6) followed by culture
in CM vehicle for a total culturing period of 21 days. Submacular
Bruch's membrane explants from fellow eyes were cultured in BCEC-CM
for the entire 21-day period. Nuclear densities were significantly
higher when cultured for the entire 21-day period in BCEC-CM
compared to shorter periods of time in BCEC-CM. Nuclear density
after three-day culture was significantly lower than nuclear
density after 14-day culture in BCEC-CM. Bars are mean nuclear
density.+-.standard error. *P<0.05.
[0056] FIG. 30 shows comparison of fetal RPE nuclear density after
21-day culture in different media and on different surfaces.
Nuclear densities of fetal RPE after culture in BCEC-CM on aged and
AMD Bruch's membrane (N=43), BCEC-ECM-resurfaced aged Bruch's
membrane cultured in RPE medium (N=11), and young Bruch's membrane
cultured in RPE medium (N=5) were not significantly different.
Culture on Bruch's membrane from aged and early AMD donors in RPE
medium (N=33) resulted in significantly lower nuclear densities
than that observed in BCEC-CM cultured, BCEC-ECM-resurfaced, and
young Bruch's membrane explants. RPE nuclear density after culture
in CM vehicle on aged and AMD Bruch's membrane (N=22) was
significantly lower than culture in RPE medium on aged and early
AMD explants. BCEC-CM explant nuclear densities are combined data
from 21-day fetal RPE nuclear density counts of the Effects of
BCEC-CM on Long-Term Cell Survival study (FIG. 28A) and 21-day
BCEC-CM controls from the Cell Survival Following Different BCEC-CM
Culture Times Study (FIG. 29). Data for BCEC-ECM resurfaced
explants and young donor explants are from Sugino et al. Invest
Ophthalmol Vis Sci 2011; 52:1345-1358. data for fetal RPE on aged
(including early AMD) Bruch's membrane explants were combined data
from Sugino et al. Invest Ophthalmol Vis Sci 2011; 52:4979-4997 and
Sugino et al. Invest Ophthalmol Vis Sci 2011; 52:1345-1358 (data
were not significantly different, P=0.745). Bars are mean nuclear
density.+-.standard error. *P<0.05.
[0057] FIGS. 31A-F show fetal RPE ECM deposition onto tissue
culture dishes after 7-, 14-, and 21-day culture in BCEC-CM or RPE
medium. ECM is deposited to a higher degree when cells are cultured
in BCEC-CM (A-C) over the 21-day period compared to that observed
after culture in RPE medium (D-F). Increase in the numbers of thick
fibers can be seen in BCEC-CM culture with time while thick fiber
deposition seems to be less extensive at all time points after
culture in RPE medium. ECM coating of the tissue culture plastic is
evident by the disappearance of the culture plastic striations
(barely discernable in BCEC-CM cultures at day-7) at day-14 and
-21. In RPE medium, culture plastic striations can be seen at day-7
and -14 but not at day-21, indicating that some material coats the
culture dish. Scale bar, 50 .mu.m; 0.1% Ponceau S stain, phase
contrast.
[0058] FIGS. 32A-L show immunocytochemical labeling
(epifluorescence) of collagen IV, laminin, and fibronectin
deposition onto tissue culture dishes after 7-day culture in
BCEC-CM or RPE medium. BCEC-CM: Collagen IV labeling (A) is
visualized as a network of fibers with some thickened fibers and
localized areas of continuous coating. Laminin labeling (B, E) is
similar of that of collagen IV although not as extensive. Laminin
appears to colocalized with some collagen IV fibers (C, collagen
IV, laminin overlay). Fibronectin labeling (D) is an open network
of fibers with some areas of where fibers appear to have heavier
deposition. Localized non-fibrous coating of the tissue culture
dish can be seen adjacent to fibers. Fibronectin-laminin overlay
(F) shows some co-localization of label. RPE medium: Collagen IV
(A) labeling is more extensive than laminin (H, K). Very little
fibronectin labeling (J) is present. Some co-localization of
collagen and laminin is seen in the overlay (I). Labeling of all
three ECM proteins is not as extensive as that seen after BCEC-CM
culture (images for each protein photographed at same exposures).
Scale bar, 200 .mu.m.
[0059] FIGS. 33A-L show immunocytochemical labeling
(epifluorescence) of collagen IV, laminin, and fibronectin
deposition onto tissue culture dishes after 14-day culture in
BCEC-CM or RPE medium. BCEC-CM: Collagen IV (A), laminin (B, E) and
fibronectin (D) deposition is more extensive than that seen at
day-7 (FIG. 32). All three proteins show extensive resurfacing of
the tissue culture dish with small defects in coverage. Collagen IV
and laminin are highly co-localized (C) while fibronectin and
laminin are co-localized in part (F). RPE medium: Collagen IV (G)
and laminin (H, K) labeling are more extensive than at day-7 (FIG.
32) but are not as extensive as labeling seen after culture in
BCEC-CM for the same time period. Very little fibronectin (J) is
present. Images for each protein in the two conditions were
photographed at the same exposure. Collagen IV and laminin are
extensively co-localized (I) while fibronectin and laminin (L) are
co-localized in part. (Intensity of fibronectin labeling has been
increased for the overlay.) Scale bar, 200 .mu.m.
[0060] FIGS. 34A-L show immunocytochemical labeling
(epifluorescence) of collagen IV, laminin, and fibronectin
deposition onto tissue culture dishes after 21-day culture in
BCEC-CM or RPE medium. BCEC-CM: Similar to 14-day culture, collagen
IV (A) and laminin (B, E) extensively resurface the culture dish
and are highly co-localized (C). Fibronectin (D) does not appear to
be as extensively deposited as collagen IV and laminin and is
colocalized in part with laminin (F). RPE medium: Collagen IV (G)
and laminin (H, K) appear to deposited at levels similar to those
seen at day-14 and are not as extensive as that deposited after
culture in BCEC-CM. Both proteins appear to be co-localized (I).
Very little fibronectin was detected (J). Images for each protein
in the two conditions were photographed at the same exposure. Scale
bar, 200 .mu.m.
[0061] FIGS. 35A-M show ECM deposition under fetal RPE on Bruch's
membrane from a 70-year-old donor (no submacular pathology) after
21-day culture in BCEC-CM or RPE medium. BCEC-CM: (A) Calcein
imaging of cells on Bruch's membrane prior to removal with ammonium
hydroxide. The explant is resurfaced almost completely with small,
highly fluorescent cells. Small defects are present in the RPE
layer. (B) SEM of the surface of Bruch's membrane revealed after
cell removal in an area where the ECM has been damaged (possibly at
the time of cell removal or during confocal imaging manipulation),
demonstrating the difference in surface morphology of the inner
collagenous layer (ICL) vs. the newly deposited ECM. Arrows point
to the folded edge of the ECM. (C) A network of open and fused
fibers covered the surface of the ICL. The ECM forms a fairly
continuous sheet in some areas. High magnification inset shows the
ECM surface details. (D-F) Both collagen IV (D) and laminin (E)
covered the explant with an extensive mesh-like deposition. There
was some colocalization of label (F, overlay). (G) Control (no
primary antibody, overlay) imaged at similar pinhole settings as
(D) and (E) with higher detector gain in both FITC and rhodamine
channels. Very little fluorescence is seen in either channel with
some choroidal autofluorescence (FITC) seen in the upper left
(arrow) of the image (tissue was not flat). (H-J) Fibronectin
labeling (H) of ECM fibers was evident on the explant while laminin
labeling (I), similar to that seen in (E), was seen in fibers,
between fibers, and as punctate labeling associated with fibers.
(Punctate laminin labeling shows up best in the overlay (J)).
Fibronectin and laminin did not appear to be co-localized to any
significant degree (J, overlay). (K) Control (no primary or
secondary antibodies, overlay) imaged at the same settings as H-J.
Only faint autofluorescence could be detected. RPE medium: (L)
Calcein imaging of the explant shows cells resurfaced Bruch's
membrane with several small defects in the RPE layer (arrows point
to small RPE defects in the submacular area and a large defect on
one edge (asterisk). Photographed at the same intensity settings as
(A), the overall intensity of calcein imaging appears to be less
than on (A) except at the edge of the large defect. (M) SEM
examination of the surface of this explant revealed no ECM
deposition confirming negative labeling (not shown) of all three
markers by confocal examination. Collagen fibers are partially
obscured by deposits. (A, L) epifluorescence; (B, C, M) SEM; (D-K)
confocal compressed z-stacks. SEM scale bar, 10 .mu.m; inset (C), 5
.mu.m. Confocal scale bar, 50 .mu.m.
DETAILED DESCRIPTION
[0062] In order to alleviate the drawbacks of the prior art,
cell-based therapy may offer advantages over pharmacological
therapy. Cell-based therapy to replace lost or diseased RPE has the
potential to preserve and restore vision in: 1) age-related macular
degeneration (AMD) patients with evolving atrophy and/or choroidal
neovascularization, 2) patients suffering from traumatic
RPE-Bruch's membrane injury, and 3) patients with other diseases
associated with RPE dysfunction (e.g., Stargardt disease and some
forms of retinitis pigmentosa). In addition to replacing lost or
diseased RPE with cells capable of performing RPE functions,
transplanted RPE may be able to rescue nearby dying photoreceptors
through their known capacity to secrete substances such as
neurotrophic factors and cytokines.
[0063] As noted above, pharmacological therapy involves
administration of a finite number of compounds and usually involves
fluctuations in drug levels above and below the desired level. In
contrast, cells placed in situ express a plethora of molecules
(e.g., neurotrophic factors, cytokines) that can inhibit
pathological processes and rescue neurons that are damaged by
disease. Moreover, they can express these molecules in amounts,
combinations, and frequencies that are tailored precisely to
molecular changes that occur from moment to moment. Thus, cells
have the capacity to function as "factories" that produce many more
substances at appropriate doses and times than can be managed with
conventional pharmacological therapy. This pharmacological salutary
capacity of cell-based therapy is termed "rescue".
[0064] Another capacity of cell-based therapy is "replacement,"
which refers to the ability of transplanted cells to replace native
cells that have died. In diseases such as AMD, RPE and
photoreceptor cell death constitutes a component of "irreversible"
visual loss in many patients. Among AMD patients with evolving
atrophy, RPE transplantation could be curative.
[0065] The first efforts to develop cell-based therapy for AMD
involved RPE transplantation after CNV excision. Before current
pharmacological therapy was available, CNV excision was proposed as
a treatment for CNVs. In most AMD patients, CNV excision is
associated with iatrogenic RPE defects due to the intimate
association of RPE cells and the CNV. Combined RPE transplantation
and CNV excision has been attempted in AMD eyes, but it has not yet
led to significant visual improvement in most patients. In
contrast, RPE transplantation in animal models of retinal
degeneration has been proved to rescue photoreceptors and preserve
visual acuity. Although animal studies validate cell
transplantation as a means of achieving photoreceptor rescue, an
important distinction between humans with AMD and laboratory
animals in which RPE transplantation has been successful is the
age-related modification of Bruch's membrane in human eyes, which
may have a significant effect on RPE graft survival.
[0066] With normal aging, human Bruch's membrane, especially in the
submacular region, undergoes numerous changes (e.g., increased
thickness, deposition of extracellular matrix (ECM) and lipids,
cross-linking of protein, non-enzymatic formation of advanced
glycation end products). These changes and additional changes due
to AMD could decrease the bioavailability of ECM ligands (e.g.,
laminin, fibronectin, and collagen IV) and cause the poor survival
of RPE cells in eyes with AMD. Thus, although human RPE cells
express the integrins needed to attach to these ECM molecules,
long-term transplanted RPE cell survival on aged submacular human
Bruch's membrane is impaired.
[0067] Because the changes in Bruch's membrane from aging and AMD
are complex and may not be fully reversible, one approach is to
establish a new ECM over Bruch's membrane. Adding exogenous ECM
ligands (e.g., combinations of laminin, fibronectin, vitronectin,
and collagen IV) can improve RPE attachment to aged Bruch's
membrane to a limited degree. (Del Piore et al., Curr Eye Res.
2002; 25:79-89). These results are consistent with the hypotheses
that ECM ligand availability may decrease with Bruch's membrane
aging and that it is possible to increase ligand density on this
surface.
[0068] It is doubtful that attention to individual ECM ligands
without attention to their 3-dimensional organization will be
highly effective (as indicated by the results of previous studies).
The instant disclosure demonstrates that bovine corneal endothelial
cells (BCE) can attach to Bruch's membrane and, more importantly,
lay down ECM. Thus, Bruch's membrane can be resurfaced with a
complex ECM that is known to support excellent RPE growth and
differentiation and that is well-defined biologically (Tseng et
al., J Biol. Chem. 1981; 256:3361-5; Gospodarowitz et al, J Cell
Physiol. 1983; 114:191-202; Robinson et al., J Cell Physiol. 1983;
117:368-76; Nevo et al., Connect Tissue Res. 1984; 13:45-57; Sawada
et al., Exp Cell Res. 1987; 171:94-109; Kay et al., Invest
Ophthalmol Vis Sci. 1988; 29:200-7).
[0069] The inventors have surprisingly found that RPE focal
adhesion formation on aged submacular Bruch's membrane is abnormal
compared to that seen on BCE-ECM-coated culture dishes. Without
wishing to be bound by any particular theory, the inventors
hypothesized that this early event, probably resulting from poor
ECM ligand availability, underlies later degenerative changes in
RPE cells on aged Bruch's membrane after they attach. RPE focal
adhesion formation is markedly improved on BCE-ECM-coated aged
submacular Bruch's membrane six hours after seeding. RPE cells
seeded onto the BCE-ECM-coated Bruch's membrane uniformly resurface
the submacular explants with small, compact cells of variable
shape. As discussed in the examples, the inventors' data
demonstrate that resurfacing by BCE-ECM enhances RPE cell long-term
survival on aged submacular human Bruch's membrane by .about.230%
(see FIG. 2A-C and FIG. 3), which is in marked contrast to previous
studies in which only modest improvement was seen following
treatment with soluble ECM ligands. RPE long-term survival and
differentiation are enhanced via this approach.
[0070] The research described in the instant application has
demonstrated that survival of transplanted cells depends critically
on the surface on which the transplanted cells grow. In two animal
models, allogeneic RPE transplants can survive for at least short
periods of time in the subretinal space and that freshly harvested
RPE sheets or microaggregates are similarly successful. (Wang et
al., Invest Ophthalmol Vis Sci. 2001; 42:2990-9; Wang et al., Exp
Eye Res. 2004; 78:53-65). In pigs, there is more inflammation
associated with freshly harvested sheets than with cultured
dispersed cell transplants, possibly due to the greater trauma
associated with sheet transplantation.
[0071] AMD-related changes as well as iatrogenic changes associated
with choroidal new vessel (CNV) excision create a damaged Bruch's
membrane surface in eyes undergoing CNV excision. (Nasir et al.,
Brit. J. Ophthalmol. 1997; 81:481-9; Zarbin, Arch Ophthalmol. 2004;
122:598-614). In most cases, surgical damage to Bruch's membrane
includes removal of the RPE basement membrane and removal of
portions of the inner collagenous layer (ICL). Aged adult RPE can
resurface RPE defects on aged submacular human Bruch's membrane in
organ culture only to a limited extent. (Wang et al., Invest
Ophthalmol Vis Sci. 2003; 44:2199-2210) In addition to the surface
affecting the ability of aged adult RPE to resurface RPE defects,
aged RPE per se are impaired in their ability to attach and grow in
culture and on Bruch's membrane. (Tsukahara et al., Exp Eye Res
2002; 74(2):255-266; Zarbin. Trans Am Ophthalmol Soc 2003;
101:499-519; Wang, et al., J Rehabil Res Dev 2006; 43: 713-22;
Ishida, et al., Curr Eye Res. 1998; 17: 392-402).
[0072] Resurfacing is even more limited if RPE migration/ingrowth
must occur on the ICL. The in vitro wound healing data accurately
predict the outcome in AMD patients following CNV removal, who show
incomplete ingrowth of RPE with associated photoreceptor
degeneration. (Hsu et al., Retina. 1995; 15:43-52). Freshly
harvested, aged adult RPE cells, such as would be used in
autologous transplants, do not survive on aged submacular Bruchs
membrane. (Tsukahara et al., Exp Eye Res. 2002; 74:255-66).
Culturing adult RPE cells upregulates integrins necessary for cell
attachment. (Zarbin, Trans Am Ophthalmol Soc. 2003; 101:499-520).
However, aged adult RPE never grow as robustly on aged Bruch's
membrane as young RPE. Histopathology of an AMD eye that underwent
uncultured adult RPE transplantation confirms these predictions.
(Del Priore et al., Am J. Ophthalmol. 2001; 131:472-80).
[0073] Long-term studies of cultured fetal human RPE on aged
submacular human Bruch's membrane show that many cells do not
survive, and if they are present, they do not appear to be
adequately differentiated. (Gullapalli et al., Exp Eye Res. 2005;
80:235-48). Of the various cell types studied to date (including
adult stem cells, embryonic stem cells (differentiated into
RPE-like cells), adult and fetal RPE, and adult iris pigment
epithelial (IPE) cells), none appear to survive and differentiate
adequately on aged submacular human Bruch's membrane. (Zarbin et
al., 2003; Gullapalli et al., 2005; Gullapalli et al., Trans Am
Ophthalmol Soc. 2004; 102:123-37; discussion 137-8; Itaya et al.,
Invest Ophthalmol Vis Sci. 2004; 45:4520-8).
[0074] Thus, in one aspect, the invention is drawn to a modified
base matrix for survival and/or differentiation of RPE cells
thereon, the modified base matrix comprising a cell-made
extracellular matrix thereon.
[0075] In different embodiments, the base matrices suitable for the
instant invention may be protein-based matrices, including, without
limitations, collagen (including gelatin), solubilized human
basement membrane, and fibrinogen-based formulations. These
synthetic matrices can include mixtures optimized according to
concentration of base formulations and additional cell-supporting
molecules added to said formulations.
[0076] In other embodiments, the base matrices may comprise
non-proteinaceous polymers, such as, for example, polycaprolactone
(PCL), polylactic acid (PLA), polyglycolic acid (PGA),
poly(lactide-co-glycolide) (PLGA), poly(methyl methacrylate)
(PMMA), polyorthoester matrices, and any combinations thereof.
[0077] In yet another set of embodiments, the base matrices may be
biological membranes, such as, for example a Bruch's membrane. In
one embodiment, the Bruch's membrane used as a base matrix of the
instant invention is an aged Bruch's membrane. The term "aged"
essentially depends on a species source of the membrane used (e.g.,
assuming that the source of the membrane is human, the membrane
over 40 years old, or 50 years old, or 60 years old, or 70 years
old, or 80 years old, or 90 years old, or 100 years old). The
species source of the Bruch's membrane include, without limitations
primates, e.g., gorilla, chimpanzee, orangutan, and human. If the
source of the membrane is not human, the age of the membrane should
be adjusted accordingly, based on the life span of the source
species.
[0078] The matrices described and/or exemplified in any of the
embodiments of the invention may be located in vivo or in
vitro.
[0079] The methods of production of the base templates depend on
the nature of the template. For example, if the template is
polymer-based (e.g., PCL based), it may be chemically synthesized.
If the template is a biological membrane, as described above, it
can be surgically harvested and cultured according to the methods
known in the art, including, without limitations, those described
in the Examples below.
[0080] Once the base matrix is chosen and obtained, it is modified
with an extracellular cell-made matrix to produce a modified base
matrix. The suitable cells capable of forming matrices are well
known in the art and include, without limitations corneal
endothelial cells (including, but not limited to, bovine cells),
RPE cells (including, but not limited to, human), IPE cells
(including, but not limited to, human), and stem cells (including,
but not limited to, human embryonic stem cells, placental stem
cells, umbilical stem cells, bone marrow-derived stem cells, neural
progenitor cells).
[0081] The choice of the cells capable of forming the extracellular
cell-made matrices ultimately depends on the nature of target cells
that are to be grown on the modified base matrix. In a set of
embodiments, wherein the target cells that are grown on the
modified base matrix are RPE, corneal endothelial cells, e.g.,
bovine corneal endothelial cells (BCE) present a suitable
option.
[0082] Another aspect of the invention is the application of
conditioned media. It may be applied in one of three ways: 1) as a
modification of the base matrix, 2) as a solution or in a
biocompatible and degradable matrix applied to the apical surface
of transplanted cells, or 3) as part of the vehicle in which the
cells are transplanted.
[0083] The methods of culturing BCE cells are well known in the art
although specifics of the methods may vary slightly (see, e.g.,
Bonanno et al., Am J Physiol Cell Physiol 277: C545-C553, 1999;
Tseng et al., J. Cell Biol 1983; 97:803-809; Katz, et al, Invest
Ophthal Vis Sci 1994; 35:495-502; Gospodarowicz, et al., Exp Eye
Res 1977; 1:75-89; MacCallum et al., Exp Cell Res 1982; 139:1-13;
Vlodaysky Curr Protocols Cell Biol 1999; 10.4.1-10.4.14. Briefly,
according to the protocol published by Bonanno, the primary
cultures from fresh cow eyes are established in T-25 flasks with 3
ml of DMEM, 10% bovine calf serum, and an antibiotic-antimycotic
(100 U/ml penicillin, 100 .mu.g/ml streptomycin, and 0.25 .mu.g/ml
Fungizone); gassed with 5% CO.sub.2-95% air at 37.degree. C., and
media changed every 2-3 days. These are subcultured to three T-25
flasks and grown to confluence in 5-7 days. The resulting
second-passage cultures are then subcultured onto coverslips or
filters, reaching confluence within 5-7 days.
[0084] Another method of culturing BCE is to establish freshly
isolated cells on tissue culture dishes (diameter 35, 60, or 100
mm) in Dulbecco's modified Eagle's medium (DMEM) supplemented with
RPE complete media (DMEM with 2 mM glutamine, 15% fetal bovine
serum, 2.5 .mu.g/ml fungizone, 0.05 mg/ml gentamicin, 1 ng/ml basic
fibroblast growth factor (bFGF)). Cells are grown in a humidified
incubator at 10% CO.sub.2-95% air at 37.degree. C. until confluent
with media change every 2-3 days. Upon confluency, cells are
passaged at a split ratio of .about.1:3.7. First passage cells are
grown in RPE complete media until confluent; second passage cells
are generated by passaging first passage cells at a split ratio of
.about.1:7.3.
[0085] ECM can be generated by culturing (including but not limited
to) first, second, or fourth passage cells in ECM media (DMEM with
2 mM glutamine, 10% fetal bovine serum, 5% donor calf serum, 2.5
.mu.g/ml fungizone, 0.05 mg/ml gentamicin, 1 ng/ml bFGF, 4%
dextran). 1 ng bFGF is added every 2-3 days until cells are
confluent. ECM can be harvested from cells at confluence or up to 3
months post-confluency. Time of ECM harvesting is specific to the
cell depositing ECM. (BCE require less time to deposit ECM than
RPE, including RPE derived from human ES.) Cells can be removed for
ECM harvesting by exposure to 0.02M NH.sub.4OH and/or PBS and/or
detergents (e.g., 0.5% triton X-100) and/or urea (2M).
[0086] Conditioned media is generated by growing cells following
passage in maintenance media (ECM media without dextran). bFGF may
or may not be added every 2-3 days. 48-72 hours prior to
collection, cells are washed a minimum of 3.times. in DMEM with no
supplements to remove serum. Media is collected after 48-72 hour
culturing in MDBK-MM or other base medium.
[0087] In different embodiments of the invention, the modified base
matrix may generally be created by at least three techniques:
first, the matrix-forming cells are cultured on the base matrix;
second, the matrix is deposited by cells onto culture dishes and
harvested; and third, the matrix-forming cells are cultured
separately from the base matrix, and the tissue culture media from
the matrix-forming cells is collected. Harvested deposited ECM
and/or media from culture may be administered to the base matrix,
may be applied to the apical surface of cells, or may be used as a
vehicle for cell transplantation. Apical application of the ECM
and/or conditioned media can be by one of the following methods
(including but not limited to): injection of the ECM and/or
conditioned media solely or in a biocompatible, biodegradable
matrix and/or injection following transplant cell attachment or
placement onto Bruch's membrane; incorporated into the overlying
material (e.g., gelatin) used for transplanting cell sheets or
embedded single cells or cell aggregates. The combination of these
techniques is also contemplated.
[0088] If the first or second option is employed, the matrix-formed
cells may be stripped from the base matrix by chemical methods,
such as, for example, NH.sub.4OH or Urea or detergent wash or PBS
soaking. Enzymatic methods (e.g., trypsin digestion) are less
desirable due to possible protein damage.
[0089] If the third option is employed, it is important to keep in
mind that serum, which may be present in the conditioned media,
usually contains ligands of the cell-made (extracellular) matrix in
the media. Accordingly, the suitable media should preferably be
serum free, or at the very least, serum depleted to reduce the
likelihood of inducing an inflammatory/immune response in the
transplant recipient.
[0090] After sufficient time, e.g., at least 7 days or at least
until cultures reach 100% confluency, or at least 1 week after
confluency, or at least 2 weeks after confluency, or at least 3
months after confluency) the modified base matrix is formed to a
degree sufficient to improve survival and differentiation of the
cells which are to be grown on the modified base matrix (i.e.,
target cells). In other words, the sufficient time may be less than
3 months, or less than 2 weeks post-confluency, or less than 1 week
post-confluency, or less than 7 days. As discussed above, the
target cells may include, without limitations, RPE, umbilical
cells, placental cells, adult stem cells, ES cells, bone
marrow-derived stem cells, fetal RPEs, adult iris pigment
epithelial (IPE) cells, neural progenitor cells, Schwann cells, and
any combination thereof, and may be derived from an autologous or
an allogeneic source.
[0091] In another aspect, the invention provides a method of
increasing survival and/or differentiation of target cells on the
base matrix, the method comprising: creating cell-made
extracellular matrix on said base matrix to produce a modified base
matrix and administering to said modified base matrix said target
cells.
[0092] According to this aspect, the base matrix and the modified
base matrix include, without limitations, the base matrices and the
modified base matrices as described according to the previous
aspect of the invention or as disclosed in the examples below.
[0093] The target cells include, without limitations, the target
cells described above. In one embodiment, the cells are RPE. The
RPE cells may be chosen or differentiated from multiple sources.
For example, RPE may be differentiated from stem cells, such as
embryonic or adult stem cells, or RPE may be fetal RPE. The methods
of in vitro differentiation of RPE are known in the art.
[0094] For example, if one desires to differentiate the RPE from ES
cells, US Publication 20070196919 discloses a suitable exemplary
method for doing so. Briefly, the H-1 (WA-01) human embryonic stem
cell line may be obtained from a commercial or a non-commercial
source, such as Wicell Research Institute. The cells are cultured
and passaged on a feeder layer made of irradiated mouse embryonic
fibroblasts. Embryoid bodies are formed by treating
undifferentiated hES colonies with 1 mg/ml of type IV collagenase
(Invitrogen) and resuspending them in a 6-well ultra-low attachment
plate (VWR) in the presence of media containing DMEM:F12 (Gibco),
10% knockout serum (Invitrogen), B-27 supplement (Invitrogen), 1
ng/ml mouse noggin (R&D Systems), 1 ng/ml human recombinant
Dkk-1 (R&D Systems), and 5 ng/ml human recombinant insulin-like
growth factor-1 (IGF-1) (R&D Systems). The cells are cultured
as embryoid bodies for 3 days. On the fourth day, the embryoid
bodies are plated onto poly-D-lysine-Matrigel (Collabora-tive
Research, Inc)-coated plates and cultured in the presence of DMEM:
F12, B-27 supplement, N-2 Supplement (Invitrogen), 10 ng/ml mouse
noggin, 10 ng/ml human recombinant Dkk-1, 10 ng/ml human
recombinant IGF-1, and 5 ng/ml human recombinant basic fibroblast
growth factor (bFGF) (R&D Systems). The media is changed every
2-3 days.
[0095] Adult cells may also be used for creating RPE cells. For
instance, retinal and corneal stem cells themselves may be utilized
for cell replacement therapy in the eye. In addition, neural stem
cells from the hippocampus have been reported to integrate with the
host retina, adopting certain neural and glial characteristics (see
review of Lund, R. L. et al., 2003, J. Leukocyte Biol. 74:
151-160). Neural stem cells prepared from fetal rat cortex were
shown to differentiate along an RPE cell pathway following
transplantation into the adult rat subretinal space (Enzmann, V. et
al., 2003, Investig. Ophthalmol. Visual Sci. 44: 5417-5422). Bone
marrow stem cells have been reported to differentiate into retinal
neural cells and photoreceptors following transplantation into host
retinas (Tomita, M. et al., 2002, Stem Cells 20: 279-283; Kicic, A.
et al., 2003, J. Neurosci. 23: 7742-7749). An ocular surface
reconstruction in a rabbit model system, utilizing cultured mucosal
epithelial stem cells, has also been reported.
[0096] In other embodiments, other cell types may be used for the
methods of the instant invention. For example, US Publication
20050037491 (the '491 publication) reports that placental or
umbilical cells injected into an eye of a dystrophic RCS rat
differentiate into cells exhibiting at least some RPE
characteristics, as assessed by ERG recording, rod and cone
responses, a- and b-wave recording, histological examination, and
Nissl staining.
[0097] In the experiments of the '491 publication, cultures of
human adult umbilical and placental cells (passage-10) were
expanded for 1 passage. All cells were initially seeded at 5,000
cells/cm.sup.2 on gelatin-coated T75 flasks in Growth Medium. For
subsequent passages, all cells were treated as follows. After
trypsinization, viable cells were counted after trypan blue
staining. Briefly, 50 microliters of cell suspension was combined
with 50 microliters of 0.04% w/v trypan blue (Sigma, St. Louis
Mo.), and the viable cell number, was estimated using a
hemocytometer. Cells were trypsinized and washed three times in
supplement free-DMEM:Low glucose medium (Invitrogen, Carlsbad,
Calif.). Cultures of human umbilical placental and fibroblast cells
at passage-11 were trypsinized and washed twice in Leibovitz's L-15
medium (Invitrogen, Carlsbad, Calif.). For the transplantation
procedure, dystrophic RCS rats were anesthetized with
xylazine-ketamine (1 mg/kg i.p. of the following mixture: 2.5 ml
xylazine at 20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml
distilled water), and their heads secured by a nose bar. Cells
devoid of serum were resuspended (2.times.10.sup.5 cells per
injection) in 2 microliters of Leibovitz, L-15 medium (Invitrogen,
Carlsbad, Calif.) and transplanted using a fine glass pipette
(internal diameter 75-150 micrometers) trans-sclerally. Cells were
delivered into the dorso-temporal subretinal space of anesthetized
3-week old dystrophic-pigmented RCS rats (total N=10/cell
type).
[0098] As discussed throughout this disclosure, treatment of the
base matrix with a conditioned media from BCE cells is sufficient
for improved survival and/or differentiation of the target cells.
Accordingly, in another aspect, the invention provides a
conditioned media from cultured cells capable of producing the
cell-made matrix, according to any embodiment, as described above.
In addition, the inventors have surprisingly discovered that
experiments with media harvested from passage-2 cultures show that
media harvested from cells that have been in culture for 2 weeks
after reaching confluency is not as supportive as media harvested
at earlier time points (50% confluent, confluent, 1 week after
confluency). Thus, in a preferred embodiment, the conditioned
culture medium is harvested from the cells that have not been
confluent for more than 2 weeks.
[0099] The inventors have also discovered that the whole
conditioned media is not necessary for the improved survival and/or
differentiation of the RPE on the modified base matrix. Thus, in
another aspect, the invention is drawn to the active fraction of
the conditioned culture media, according to any of the embodiments
described above. Specifically, the inventors have found that high
molecular weight components are sufficient for the initial
beneficial effect of the conditioned culture media. Specifically,
such an active fraction may be characterized by having its low
molecular weight components depleted. However, low molecular weight
components may be important in long-term survival and
differentiation.
[0100] In different embodiments, the active fraction is
characterized by the depletion of bioactive components having
molecular weight less than 20 kD, preferably less than 30 kD, more
preferably, less than 50 kD, more preferably, less than 70 kD, more
preferably, less than 80 kD, more preferably, less than 90 kD, and
most preferably, less than 100 kD. The active fraction may be
characterized by any combination of components separated according
to size or other methods (e.g., high pressure liquid chromatography
(HPLC)).
[0101] The depletion of low molecular weight or other non-essential
components may be achieved by many methods, including, without
limitation, filtration, size fractionation by gel filtration or
gradient centrifugation, HPLC (separation according to charge,
size, or hydrophobicity), immunoprecipitation, affinity column
separation, and the like. However, it is important that the methods
of depletion of low-molecular weight compounds should not result in
protein cleavage nor should it disrupt secondary and tertiary
protein structures of any needed components in the medium.
[0102] While it is possible to surgically remove CNVs, CNV excision
is associated with iatrogenic RPE defects due to the intimate
association of RPE cells and the CNV. (Thomas et al., Am J
Ophthalmol 1991; 111:1-7; Nasir et al., Br J Ophthalmol 1997;
81:481-489; Castellarin et al., Retina 1998; 18:143-149; Hsu et
al., Retina 1995; 15:43-52; Rosa et al., Arch Ophthalmol 1996;
114:480-487). Combined RPE transplantation and CNV excision has
been attempted in AMD eyes, but it has not led to significant
visual improvement in most patients. (Algvere et al., Graefes Arch
Clin Exp Ophthalmol 1994; 232:707-716; Del Priore et al., Am J
Ophthalmol 2001; 131:472-480; Binder et al., Am J Ophthalmol 2002;
133:215-225; Tezel et al., Am J Ophthalmol 2007; 143:584-595;
Joussen et al., Am J Ophthalmol 2006; 142:17-30). Potential causes
of RPE transplant failure in human patients include immune
rejection, inability of transplanted RPE cells to survive and
differentiate on aged submacular Bruch's membrane, and
choriocapillaris atrophy, all causing death of the RPE graft. In
contrast, RPE transplants rescue photoreceptors and preserve visual
acuity in animal models of retinal degeneration. (Li et al., Exp
Eye Res 1988; 47:911-917; Coffey et al., Exp Neurol 1997; 146:1-9;
Lund et al., Proc Natl Acad Sci USA 2001; 98:9942-9947; Wang et
al., Invest Ophthalmol Vis Sci 2008; 49:416-421; Gias et al., The
European journal of neuroscience 2007; 25:1940-1948).
[0103] An important distinction between humans with AMD and
laboratory animals is the age-related modification of Bruch's
membrane that occurs in human eyes. With normal aging, human
Bruch's membrane, especially in the submacular region, undergoes
numerous changes (e.g., increased thickness, deposition of
extracellular matrix (ECM) and lipids, cross-linking of protein,
nonenzymatic formation of advanced glycation end products). (Guymer
et al., Prog Retin Eye Res 1999; Marshall et al., The Retinal
Pigment Epithelium. New York: Oxford University Press;
1998:669-692; 18:59-90; Abdelsalam et al., Surv Ophthalmol 1999;
44:1-29). Pauleikhoff and coworkers reported an age-related decline
in the presence of laminin, fibronectin, and collagen IV in the RPE
basement membrane. (Pauleikhoff et al., Ophthalmologe 2000;
97:243-250). It is possible that changes in submacular Bruch's
membrane permeability and choriocapillary density may contribute to
age-related RPE death. However, it was found that RPE survival is
also impaired on aged submacular Bruch's membrane explants in organ
culture, where diffusion of nutrients is not a factor in cell
survival. This finding suggests that there are additional factors
within aged Bruch's membrane itself that adversely affect RPE
survival and that modification of Bruch's membrane may have a
significant effect on RPE graft survival in patients with AMD.
(Gullapalli et al., Exp Eye Res 2002; 74:255-266).
[0104] As discussed above and shown in the examples below, in the
instant invention, the use of modified base matrix according to any
embodiment of the invention promotes survival and/or
differentiation of cells transplanted onto this matrix. In
embodiments where the base matrix is an aged Bruch's membrane or a
Bruch's membrane from an eye undergoing macular degeneration,
survival and/or differentiation of transplanted RPE was improved
when the Bruch's membrane was modified with extracellular matrix
from BCE cells. Importantly, the experiments were performed in
human eyes, thus validating the methods and compounds of the
instant invention for human treatment.
[0105] Accordingly, in one aspect, the methods according to the
instant inventions may be performed for treatment of humans
suffering from AMD (whether the wet AMD or the dry AMD). As
mentioned above, retinal degenerative diseases constitute the
leading causes of blindness in the industrialized world. AMD, the
most prevalent of these, can be treated pharmacologically, although
at this time the majority of patients do not recover lost vision.
RPE cells may be a primary target of the pathological processes
that cause AMD.
[0106] The goal of cell-based therapy as a treatment for AMD
patients is to replace diseased or dying RPE cells, which provide
metabolic support for the photoreceptors. RPE transplantation could
prevent further vision loss and might even, in some cases, lead to
vision improvement in selected AMD patients. Cell-based therapy in
AMD patients has not reached its full potential due to the failure
of cells to survive and become functional in the diseased AMD
eye.
[0107] As disclose herein, the present invention provides a
biologically synthesized mixture (bovine corneal endothelial
cell-conditioned media, BCEC-CM) that improves transplanted RPE
survival by more than 10-fold when tested in an organ culture
bioassay utilizing aged and AMD human donor eyes. It was found that
BCEC-CM significantly improved cell survival on aged and AMD
Bruch's membrane (the surface on which cells must survival in
patients) using fetal RPE, aged adult RPE, and RPE generated from
human embryonic stem cells. Identification of the bioactive
molecules in BCEC-CM allows for the development of an adjunct to
cell transplantation therapy in patients with AMD to ensure
successful cell transplant integration and functionality.
[0108] In one embodiment, the present invention provides BCEC-CM
and conditioned media comprising bioactive molecules derived
therefrom for therapeutic use in AMD. Specifically, it provides
fractions of BCEC-CM identified via molecular cut filtration
possessing bioactivity, one in the <3 kDa filtrate and one found
in a 10-50 kDa fraction. These fractions, in combination, ensure
cell survival on Bruch's membrane. The bioactive molecule or
molecules in each of these fractions in BCEC-CM can serve as an
adjunct to cell transplantation therapy in patients with AMD.
[0109] In other embodiments, the methods of treatment comprise
modifying Bruch's membrane with the cell made extracellular matrix,
according to any embodiments described herein, and wherein the
Bruch's membrane is located in vivo. Essentially, in different
embodiments, Bruch's membrane is modified when the at least the
active fraction of the conditioned media (or the whole conditioned
media) of any of the embodiments described above or exemplified
below can be applied basally as a substrate to coat the surface of
Bruch's membrane, in a mixture with cells, or apically in a
biocompatible matrix.
[0110] It is also worth noting that this invention has been shown
to support adult and embryonic stem cells and retinal pigment
epithelial cells (adult and fetal) on human Bruch's membrane,
including Bruch's membrane from AMD eyes. Accordingly, in different
embodiments, different types of cells may be applied within the
methods of this aspect of the invention.
[0111] The compositions containing the extracellular matrix (e.g.,
at least the active fraction of the conditioned media according to
any embodiment of the instant invention) can be applied to Bruch's
membrane in living patients through a variety of strategies, e.g.,
direct application to the subretinal space.
[0112] In another embodiment, the scaffold (i.e., the base matrix),
such as, for example, a polymeric scaffold such as PCL, can be
delivered into the subretinal space. In different embodiments, the
scaffold is modified with the extracellular matrix (resulting in
the modified base matrix), as described above and exemplified
below. Further, such modified base matrix may be delivered in
combination with a scaffold that contains cells to be transplanted
to the patient's eye. The suitable cells have been described
above.
[0113] As used herein, the terms "treat" or "treatment" or
"treating" etc., refer to executing a protocol in an effort to
alleviate signs or symptoms of a disease in a subject. Alleviation
may occur either before or after appearance of these signs or
symptoms. In addition, these terms do not require a complete
alleviation of the signs or symptoms, do not require a cure, and
include protocols resulting in only marginal effects on a
patient.
[0114] A "subject" refers to a human and a non-human animal.
Examples of a non-human animal include all vertebrates, e.g.,
mammals, such as non-human primates (particularly higher primates),
dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals,
such as birds, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model.
[0115] In another aspect, the invention provides a kit for
treatment of AMD (both wet AMD and dry AMD). Generally, the kit
would include a set of instructions and at least the active
fraction of the conditioned media, as described in any of the
embodiments of the instant invention, and may comprise the
unfractionated conditioned media, also, according to any of the
embodiments of the instant invention. Alternatively, the kit may
comprise the cells capable of producing the cell-made extracellular
matrix, according to any of the embodiments of the instant
invention. Specifically and without limitations, the cells capable
of producing the cell-made extracellular matrix include BCE cells.
Alternatively, the kit may include ECM generated and harvested from
cell-deposited matrices in solubilized or non-solubilized form.
Optionally, the kit may provide the base matrix, according to the
embodiments described above. The base matrix may be a natural
polymer (e.g., a protein-based base matrix), a synthetic polymer
(e.g., PCL), a biological membrane (e.g., Bruch's membrane), or a
combination thereof.
[0116] In another set of embodiments, the kit may comprise a
modified base matrix, according to any of the embodiments described
herein. In any of the embodiments of the kit, suitable target cells
may also be provided, according to any of the embodiments described
above.
[0117] The set of instructions may be provided in any media,
including, without limitations, written, graphic, audio recording,
video recording, and electronic media.
[0118] In yet another aspect, the invention provides a novel medium
for and method of storing and differentiating cells for
transplantation. Cell-based therapy represents a promising
approach, which may be sight-preserving and/or restoring for
patients with these diseases. However, cell-based therapies are
complicated by the necessary step of growing, differentiating and
storing cells for transplantation. Given the severe consequences of
AMD and the often irreversible vision loss which may occur, there
exists a need for specifically-designed and improved methods of
preparing cells for transplantation.
[0119] The present invention provides a novel medium for and method
of differentiating, storing and preserving cells for
transplantation. The medium was developed as a measure to improve
the viability and functionality of cells or tissues, prior to
transplantation. It can be used with various types of cells and
tissues, including, but not limited to, ocular cells, ocular tissue
and neural tissue. One application of the medium is the storage and
shipment from cell manufacturer to an end user (e.g., surgeon). The
medium can further serve as a cell culture medium, to induce rapid
differentiation of RPE cells, as well as other cells and tissues.
Accordingly, this invention provides a novel medium for storage and
differentiation which provides the significant advantage of
improving the health and functionality of cells, tissues or organs,
at the time of transplantation.
[0120] In one embodiment, the present invention provides a storage
and preservation medium for use prior to transplantation of various
biological transplants, including, but not limited to, cells,
retinal pigment epithelial (RPE) cells, RPE derived from various
cells, RPE derived from human embryonic stem cells, RPE derived
from iPSC stem cells for neurodegenerative diseases, corneal cells,
whole retinae, whole cornea, tissues, neural tissues and organs.
This medium will support RPE viability during long-term storage,
without media replacement. Furthermore, the cells can be in
suspension or in a support matrix designed for cell delivery.
[0121] In another embodiment, the present invention provides a cell
culture medium to induce and maintain rapid differentiation of
retinal pigment epithelial cells. In a further embodiment, the
present invention provides a cell culture medium, for RPE, as well
as other cells, which allows and assures rapid attachment onto
untreated surfaces, including, but not limited to, tissue culture
plastic without coating for attachment (e.g., laminin, fibronectin,
and matrigel).
[0122] As disclosed herein, the medium is based on secreted
molecules generated as conditioned media (CM) containing bovine
corneal endothelial cell (BCEC) secreted molecules, either the CM
itself, components of the CM or a combination of components based
on the identification of active molecules in BCEC-conditioned
media. The medium of present invention can be used as a
storage/preservation medium without changing or replenishing. This
medium can also be used as a cell culture medium for inducing rapid
differentiation in RPE when changed three times per week.
[0123] The present invention offers distinct advantages over
technology currently in existence. Storage and preservation media
currently exist for corneas but there is no medium specifically
developed for use with RPE or for other similar types of cells. The
composition and concentration of the components of the present
invention vary significantly from that of existing cornea storage
media. Furthermore, when used as a differentiation medium, the
present invention features a rapid induction of differentiation,
which exceeds that of standard RPE culture media. Morphological
indicators of differentiation in fetal RPE demonstrate that the
onset of differentiation occurs very rapidly (e.g., within one week
when seeded at 3164 cells/mm.sup.2), and the cells achieve a level
of differentiation that is only observed (if at all) in long-term
fetal RPE culture in standard RPE culture media.
[0124] Finally, when used as a cell culture medium for RPE, a
BCEC-conditioned medium induces unexpected rapid attachment of
cells onto untreated tissue culture plastic (attachment as soon as
one hour following seeding) and effects accelerated growth and
differentiation. On the other hand, a standard RPE culture medium
attachment takes approximately 24 hours on untreated tissue culture
plastic, with a marked difference in the rate of growth. This
aspect of the present invention, a reduction of the time required
to achieve differentiated RPE (or other cells, tissues or organs),
provides a substantial advantage in the field of manufacturing. For
example, in the case of induced pluripotent stem cells (iPSC),
BCEC-conditioned media can reduce the time a patient has to wait
for autologous cell transplantation.
[0125] The role of the medium of the present invention as a storage
medium presents a marked improvement over existing technology. In
order to solidify its usefulness, quantitative analysis of cell
viability at different time points can be performed, in order to
compare cell death rates in different media. Additionally,
maintenance of RPE markers can be compared in different media with
time in culture. Since another method of cell introduction at the
time of transplantation is single-cell suspensions, it can also be
determined, as part of the present invention, whether RPE can
maintain viability in suspension in BCEC-CM. Generally, RPE are
anchorage-dependent cells that undergo apoptosis if not attached to
a suitable substrate. Since BCEC-CM contains many soluble ECM
ligands, this medium can likely support cells in suspension.
Injection of fresh, as opposed to frozen, cells could be
advantageous for cell transplantation of since frozen cells must
recover after thaw and tend to attach and grow sluggishly compared
to fresh cells. Additionally, frozen cells must be washed to remove
DMSO (in freezing solution) while fresh cells could be injected
directly from the storage vial.
[0126] The present invention aims to identify the cell-supporting
components in BCEC-CM and to manufacture a solution comprised of
small molecules and human recombinant proteins for commercial
development. Although an RPE medium appears to support cells to a
similar degree as BCEC-CM (except at the condition noted
previously), the presence of fetal bovine serum in the medium is
not ideal (xenogeneic proteins). The storage solution for
commercial development can be a newly-developed product with a
unique formation or can be molecules added to Optisol to increase
effectiveness.
[0127] Since the presence of mRNA does not necessarily predict
protein presence, the present invention can include a determination
of the expression of RPE differentiation markers (proteins) with
time in culture. Long-term cultures of fetal RPE on BCEC-ECM and on
tissue culture plastic in standard RPE media and BCEC-CM can be
compared. Preliminary data indicate that protein expression of late
differentiation markers (RPE65 and bestrophin) cannot be detected
in the conditions tested to date (3 weeks). If fetal RPE
differentiates more rapidly in BCEC-CM than in standard RPE media,
similar studies on hES-RPE (Advanced Cell Technology) can be
performed.
[0128] The invention will now be described in the following
non-limiting examples.
EXAMPLES
Example 1
Long-Term Survival of Fetal RPE on Aged Submacular Human Bruch's
Membrane is Impaired
[0129] Fetal RPE (3164 cells/mm.sup.2) were seeded on aged human
submacular Bruch's membrane debrided to expose the superficial
surface of the inner collagenous layer. To create surfaces exposing
the RPE basement membrane, RPE were gently wiped off the
RPE/choroid/sclera explant using a wet surgical sponge. To create
surfaces exposing the surface of the inner collagenous layer
beneath the RPE basement membrane (i.e., superficial ICL),
following RPE removal as indicated previously, a moistened surgical
sponge was use to abrade the RPE basement membrane. In general, the
area of RPE basement membrane debridement was created by
approximately 5 wipes of the moistened sponge in each of 4
directions (rotating the explant 90 degrees after each series of 5
wipes). (V. K. Gullapalli, et al., Exp Eye Res 2005;
80(2):235-248). Cells were seeded onto the sclera/choroid explant
and cultured for 21 days and evaluated for resurfacing with
scanning electron microscopy (SEM) and light microscopy (LM).
Nuclear density counts (mean.+-.SD) of fetal RPE on aged submacular
human Bruch's membrane at day-1 (basement membrane, N=7;
superficial ICL, N=7), day-7 (basement membrane, N=6; superficial
ICL N=6), day-14 (basement membrane N=7), day-21 (superficial ICL,
N=6) were performed on 5 non-adjacent slides in the central 3 mm of
the section (includes the submacular region of Bruch's membrane).
Cells on tissue culture dishes coated with BCE-ECM (N=1) are
included for comparison. Cells were seeded at a density of 3164
cells/mm.sup.2 for all time points and surfaces. Fetal RPE survival
on submacular Bruch's membrane decreased with time, regardless of
the surface on which the cells are seeded (e.g., RPE basement
membrane or the surface of the inner collagenous layer (superficial
ICL)) (See FIG. 1, modified from V. K. Gullapalli, et al., Exp Eye
Res 2005; 80(2):235-248.) (Transplanted RPE will encounter
superficial ICL in situ if native RPE are removed by CNV excision.)
In contrast, density increased to 45 nuclei/mm.sup.2 if RPE are
grown on bovine corneal endothelial cell extracellular matrix
(BCE-ECM)-coated culture dishes.
Example 2
Fetal RPE Resurfacing on Aged Bruch's Membrane Resurfaced with
Bovine Corneal Endothelial Matrix (BCE-ECM)
[0130] BCE (3164 cells/mm.sup.2) were cultured on the inner
collagenous layer of aged human submacular Bruch's membrane (65 yr.
old donor) for 14 days to allow ECM deposition. Cells were culture
in the same way as cells cultured for ECM deposition on culture
dishes (see paragraph 0056). Following BCE removal with NH.sub.4OH
to expose the newly deposited ECM and extensive washing with PBS,
explants were seeded with fetal RPE (3164 cells/mm.sup.2) and
cultured for 21 days. The results of these experiments are
illustrated in FIG. 2.
[0131] FIG. 2A is a scanning electron micrograph (SEM), showing
that fetal RPE fully resurfaced the treated explant with large,
flat polymorphic cells. Cells showed varying amounts of short
apical processes on their surfaces (insert). Mag. bar 50 .mu.m;
insert mag bar 10 .mu.m.
[0132] FIGS. 2B and 2C are light micrographs (LMs). As shown in
FIG. 2B, cells fully resurfaced the treated explant and are in a
monolayer. Mag. bar 100 .mu.m. FIG. 2C is a higher magnification of
the explant shown in FIG. 2B, allowing one to discern the variable
morphology of the cells. Cells are tightly adherent to the explant
surface. Arrow in FIG. 2C points to the nucleus of a cell in the
monolayer; arrowhead to a choriocapillaris vessel. Mag. bar 20
.mu.m.
[0133] As a negative control, submacular Bruch's membrane of the
fellow eye was incubated in serum-free media with no BCE for 14
days followed by exposure to NH.sub.4OH, rinsing with PBS and fetal
RPE seeding and culturing for 21 days.
[0134] FIG. 2D is a SEM of the RPE on the untreated Bruch's
membrane surface. Notably, fetal RPE incompletely resurfaced the
untreated inner collagenous layer. Islands of large, flattened
cells are present (arrows). Dead, dying, or poorly attached cells
are also present on the surface or attached to the flattened cells
(arrowhead). Asterisk, exposed inner collagenous layer surface.
Mag. bar 50 .mu.m.
[0135] FIGS. 2 E and 2F show a representative view of the RPE on
untreated Bruch's membrane. In this section, there is only a single
clump of cells (arrow). Mag. bar 100 .mu.m. FIG. 2F is a high
magnification of the clump of cells shown in FIG. 2E. Arrow points
to a cell in the clump that is not intact. Arrowhead points to a
choriocapillaris vessel. Mag. bar 20 .mu.m.
Example 3
Resurfacing Bruch's Membrane with a Biologically Deposited
Extracellular Matrix (ECM) Improves Cell Survival
[0136] Bovine corneal endothelial cells (BCE, passage-2) were
seeded onto human submacular superficial ICL of Caucasian donors
over 55 years old at a density of 3164 cells/mm.sup.2 and cultured
for 14 days to allow ECM deposition or treated for 14 days with
serum-free media only. Following BCE removal with NH.sub.4OH and
extensive rinsing, fetal RPE (passage-2-5) were seeded at the same
density onto the treated Bruch's membrane surface and cultured for
21 days. The fellow eye was treated similarly except no BCE were
seeded.
[0137] RPE seeding density was 3164 cells/mm.sup.2 for 21-day
incubations to determine long-term survival and morphology. FIG. 3
shows the cumulative data from 9 explant pairs. Counts are mean
fetal RPE nuclei/mm Bruch's membrane (.+-.SEM).
[0138] A statistically significant 230% (p=0.006) increase in cell
density is seen at day-21 on treated explants compared to explants
treated with serum-free DMEM only (FIG. 3) or explants in which
cells were seeded directly on Bruch's membrane with no prior
treatment (FIG. 1, day-21 superficial ICL (striped bar)).
Example 4
Bovine Corneal Endothelial Cells (BCE) Secrete ECM Components into
the Overlying Media
[0139] During ECM formation, in addition to basal secretion, BCE
secrete ECM components into the media (BCE-conditioned media,
BCE-CM), and the composition and relative amounts of the components
vary with culture time and passage number. Secretion of ECM
components into the overlying media is most abundant in early
passage cells (up to passage-2) and exceeds basal ECM deposition in
quantity. (Tseng et al. J Biol Chem 1981; 256:3361-3365).
[0140] Serum-free BCE-conditioned media (BCE-CM) was prepared from
passage-2 cells that were cultured in serum-free Dulbecco's
modified Eagle's medium (DMEM) for 48 hrs. An initial sample
concentrated using a 30 kD cut-off filter identified 20 proteins by
MS/MS-MALDI. The proteins in an additional sample of conditioned
media, unfiltered, were subjected to 2D LC-MS/MS, and samples were
analyzed with MALDI-TOF and QTOF.
[0141] These analyses identified 84 proteins (at least one peptide
having C.I. values of >95%). Conditioned media from the same
preparation was also analyzed by 2D gel separation, and selected
spots (142) were analyzed by MALDI-TOF. This analysis identified 45
different proteins. A combined total of 109 proteins were
identified using these methods (Table 1).
TABLE-US-00001 TABLE 1 Protein components of two different samples
of bovine corneal endothelial cell conditioned media (BCE-CM) as
determined by MS/MS-MALDI, LC-MS/MS (MALDI-TOF and Q-TOF), and
MS/MS-MALDI of selected 2D gel spots. Name Protein Description
IGF-1 prepro-insulin-like growth factor I IGFBP-2 Insulin-like
growth factor-binding protein-2 IGFBP-4 insulin-like growth
factor-binding protein-4 IGFBP-7 PREDICTED: similar to insulin-like
growth factor binding protein 7 (predicted), partial FN Fibronectin
(FN) hypothetical hypothetical protein LOC504471 protein H factor 1
H factor 1 (complement) C3 Chain B, Structure Of Mammalian C3 With
An Intact Thioester At 3a Resolution C3 Complement component 3 C3d
Complement component C3d PREDICTED: similar to Complement C3
precursor, partial Complement C3 precursor [Contains: Complement C3
beta chain; Complement C3 alpha chain; C3a anaphyl C4 PREDICTED:
similar to Complement C4 precursor CCP modules CCP modules 3-12,
with parts of CCP 2 and 13 collagen, type I collagen, type I, alpha
2 collagen, type III collagen, alpha-1 (III) chain collagen, type V
type V preprocollagen alpha 2 chain collagenase type collagenase
type IV precursor IV TIMP2 tissue inhibitor of metalloproteinase 2
MMP2 matrix metalloproteinase 2 EGF-containing PREDICTED: similar
to EGF-containing fibulin-like extracellular matrix protein 1
fibulin-like isoform a precursor extracellular matrix protein 1
PREDICTED: similar to EGF-containing fibulin-like extracellular
matrix protein 1 isoform b fibulin-3, FIBL-3 EGF-containing
fibulin-like extracellular matrix protein 1 precursor fibulin-1
fibulin-1 C SPARC protein SPARC protein Osteonectin secreted
protein, acidic, cysteine-rich (osteonectin) ESM-1 PREDICTED:
similar to Endothelial cell-specific molecule 1 precursor (ESM-1
secretory protein) apolipoprotein A apolipoprotein A-I precursor
apolipoprotein E apolipoprotein E apolipoprotein E precursor EC-SOD
PREDICTED: similar to Extracellular superoxide dismutase [Cu--Zn]
precursor (EC-SOD) isoform 2 PCSK9 PREDICTED: similar to proprotein
convertase subtilisin/kexin type 1 inhibitor precursor alpha-actin
alpha-actin Alpha-cardiac PREDICTED: similar to Actin, alpha
cardiac (Alpha-cardiac actin) isoform 1 actin Beta-actin Actin,
cytoplasmic 1 (Beta-actin) actinin PREDICTED: similar to actinin
alpha 4 isoform 3 actinin, alpha 1 POTE-2 PREDICTED: similar to
Prostate, ovary, testis expressed protein on chromosome 2 Dkk-3
PREDICTED: similar to Dickkopf related protein-3 precursor (Dkk-3)
(Dickkopf-3) (hDkk-3) dickkopf homolog 3 cathepsin L cathepsin L
Fibulin-1 PREDICTED: similar to Fibulin-1 precursor isoform 1
Thrombospondin- Chain A, Crystal Structure Of The Thrombospondin-1
N-Terminal Domain 1 Vimentin Vimentin PGDS prostaglandin D2
synthase precursor ITI PREDICTED: similar to inter-alpha trypsin
inhibitor heavy chain precursor 5 isoform 1 nidogen nidogen
Entactin PREDICTED: similar to Nidogen precursor (Entactin) isoform
3 Osteonidogen PREDICTED: similar to Nidogen-2 precursor (NID-2)
(Osteonidogen) MGP matrix Gla protein HSPG PREDICTED: heparan
sulfate proteoglycan 2 heparan sulfate proteoglycan perlecan
PREDICTED: similar to Basement membrane-specific heparan sulfate
proteoglycan core protein precursor nephronectin PREDICTED: similar
to nephronectin isoform b FSTL1 Follistatin-related protein 1
precursor (Follistatin-like 1) (TGF-beta-inducible protein TSC-36)
FSTL3 PREDICTED: similar to Follistatin-related protein 3 precursor
(Follistatin-like 3) (Follistatin-rel LTBP-2 Latent-transforming
growth factor beta-binding protein 2 precursor (LTBP-2) albumin
albumin transthyretin transthyretin NPC Alveolar macrophage
chemotactic factor (Neutrophil chemotactic protein) (NPC) CTGF
connective tissue growth factor Dimeric Bovine Chain B, Dimeric
Bovine Tissue-Extracted Decorin, Crystal Form 2 Tissue-Extracted
Decorin GOLPH2 PREDICTED: similar to golgi phosphoprotein 2
KIAA1133 PREDICTED: similar to KIAA1133 protein GST glutathione
S-transferase, GST {N-terminal} {EC 2.5.1.18} [cattle,
erythrocytes, Peptide Partial, 2 serine protease serine protease
SMC1 mitosis-specific chromosome segregation protein SMC1 homolog
superfast myosin PREDICTED: similar to superfast myosin heavy chain
AEBP1 AE binding protein 1 angiomodulin angiomodulin anti-PPS
anti-pneumococcal capsular polysaccharide immunoglobulin heavy
chain variable region N-cadherin Cadherin-2 precursor
(Neural-cadherin) (N-cadherin) (CD325 antigen) CD44 CD44 antigen
precursor (Phagocytic glycoprotein I) (PGP-1) (HUTCH-I)
(Extracellular matrix receptor APT Chain A, Crystal Structure Of
The First Active Autolysate Form Of The Porcine Alpha Trypsin ACTH
preproadrenocorticotropic hormone (ACTH) corticotropin
corticotropin-like interm lobe peptide cystatin C cystatin C
(amyloid angiopathy and cerebral hemorrhage) [Bos taurus]
fibromodulin fibromodulin galanin galanin polypeptide precursor
polypeptide (AA -31 to 1139) PTGDS prostaglandin H2 D-isomerase
microglobulin similar to beta 2-microglobulin lumican lumican
gelsolin gelsolin cadherin 11 cadherin 11, type 2 preproprotein
Transferrin Serotransferrin precursor (Transferrin) (Siderophilin)
(Beta-1- metal-binding globulin) PDI Protein disulfide-isomerase A3
precursor (Disulfide isomerase ER-60) (ERP60) Nucleobindin 1
Nucleobindin 1 Calnuc Chain A, Nmr Solution Structure Of The
Calcium-Binding Domain of Nucleobindin (Calnuc) Osteonectin
Secreted protein, acidic, cysteine-rich Beta-globin Hemoglobin
subunit beta (Hemoglobin beta chain) (Beta-globin) Alpha enolase
Alpha enolase FHOS2S splicing FHOS2S splicing variant variant [
Aldehyde aldehyde dehydrogenase family 1, subfamily A1
dehydrogenase LDHA Lactate dehydrogenase-A ATIC-ALK Tropomyosin
4-anaplastic lymphoma kinase fusion protein Tyrosine 3-/tryptophan
5-monooxygenase activation protein, epsilon polypeptide Human
Annexin Chain A, Structure Of Human Annexin A2 In The Presence Of
Calcium Ions A2 Peroxiredoxin 1 Peroxiredoxin 1 Peroxiredoxin 6
Peroxiredoxin 6 Anti-oxidant Anti-oxidant protein 2 (non-selenium
glutathione peroxidase, acidic calcium- protein 2 independent
phospholipa) NME4 Expressed in non-metastatic cells 1 protein
biglycan biglycan clusterin clusterin osteoglycin plasminogen
activator inhibitor type 1, member 2 transketolase Proteins were
identified based on at least one peptide with C.I. value 95% or
more.
Example 5
Bovine Corneal Endothelial Cell Conditioned Media (BCE-CM) Can
Improve RPE Cell Survival on Aged Human Submacular Bruch's
Membrane
[0142] BCE-CM containing serum was prepared by exposing newly
confluent cultures of BCE to RPE complete media (DMEM with 2 mM
glutamine, 15% fetal bovine serum, 2.5 .mu.g/ml fungizone, 0.05
mg/ml gentamicin, 1 nag/ml bFGF) for 3 days. Media was centrifuged
and supernatant stored frozen. Submacular aged human Bruch's
membrane explants were debrided to expose the superficial inner
collagenous layer; 3164 cells/mm.sup.2 were seeded on each explant
and cultured for 21 days. Explants with cells were cultured in
serum-containing BCE-CM or RPE complete media.
[0143] Preliminary data using serum-containing BCE-CM as media for
RPE following seeding onto peripheral (N=2) and submacular Bruch's
membrane (N=1, with submacular drusen) shows BCE-CM used as media
supports better RPE attachment and long-term survival than RPE
complete media (FIG. 4). In the experiments illustrated in FIG. 4,
submacular human Bruch's membrane of an AMD donor (age 79 years)
was treated with serum-containing bovine corneal endothelial
cell-conditioned media (BCE-CM) vs. routine RPE culture media;
BCE-CM was prepared by exposing passage-2 BCE for 3 days in RPE
complete media containing serum. The explant to be treated had a
greater number of large submacular drusen than the control explant,
which means it was the more severely diseased of the two eyes. A.
RPE cultured in BCE-CM, day 21. Cells fully resurface the treated
explant with a few small defects (arrow). High magnification insert
shows short apical processes on the surface of some cells and along
cell borders. B. RPE cultured in RPE complete media, day-21. The
explant is sparsely resurfaced with patches or clumps of RPE.
Original magnifications 200.times.; insert 1000.times..
[0144] The inventors have also shown serum-containing BCE-CM used
as media during the duration of the incubation (21 days) showed
better cell morphology and resurfacing on peripheral inner
collagenous layer of Bruch's membrane than explants where BCE-CM
was changed to standard RPE media (which also contains serum) after
2 days (FIG. 5). In these experiments, RPE survival on peripheral
Bruch's membrane from a non-AMD donor (age 80 years) was
investigated. A. Aged human peripheral Bruch's membrane explant
cultured for 21 days in BCE-CM. Explant is fully resurfaced with a
fairly uniform monolayer of cells. High magnification insert shows
short apical processes covering the surface of the cells. B. Aged
human peripheral Bruch's membrane (isolated from fellow eye of that
shown in A) explant cultured for 2 days in BCE-CM then placed in
RPE complete media for 19 days. Explant is incompletely resurfaced
(arrows point to defects) with cells of varying size and
morphology. High magnification insert shows the cells have a few
very short apical processes, and the cells are fairly large and
smooth. Original magnifications: 200.times.; inserts:
1000.times..
Example 6
Bovine Corneal Endothelial Cell Conditioned Media (BCE-CM) Supports
Rapid RPE Attachment and Spreading on Non-Tissue Culture Treated
Plastic to a Similar Degree as Cells on BCE-ECM-Coated Tissue
Culture Plastic
[0145] Soluble ECM can affect cell shape and metabolism in addition
to stimulating production of ECM molecules. The inventors performed
studies to determine: 1) whether soluble components in BCE-CM can
be used instead of BCE-ECM to coat culture dishes and support fetal
RPE growth and differentiation; and 2) whether BCE-CM used as media
for cell suspension and seeding can support cells on non-tissue
culture treated dishes (NTC). Since serum contains ECM ligands
(e.g., vitronectin and fibronectin), these studies were performed
in serum-free media as the most stringent test of cell support.
Because of RPE dependence on serum in the media for long-term
survival, experiments were performed for 3 days only.
[0146] Serum-free conditioned media (sfBCE-CM) was prepared from
passage-2 cultures as described above. sfBCE-CM was applied in
media or by coating non-tissue culture treated dishes (NTC)
unconcentrated or in concentrated form (8-fold, using a 30 kD
cut-off filter). Negative control was cells seeded and cultured in
DMEM only. Fetal RPE (passage-3) were seeded at a density of 526
cells/mm.sup.2 for all attachment studies. To determine whether
non-protein components of BCE-CM contribute to early attachment and
spreading of fetal RPE, sfBCE-CM was heated to 80.degree. for 15
minutes, centrifuged, and the supernatant was used as media for
attachment and seeding of fetal RPE. The importance of intact
protein components in BCE-CM evidenced by cell behavior in
heat-treated sfBCE-CM was confirmed by treatment with proteinase K
agarose beads (removed prior to cell suspension and seeding) before
and after heat treatment.
[0147] sfBCE-CM used either as media (Table 2, A) or as a substrate
to coat tissue culture dishes (Table 2, B) supported rapid RPE
adhesion and cell division in serum-free conditions.
sfBCE-CM-treated dishes supported rapid attachment and spreading by
1 hour (Table 2, B), similar to BCE-ECM-treated dishes (Table 2,
D). Fetal RPE seeded in heat inactivated and/or proteinase
K-treated BCE-CM behaved similar to those on NTC (Table 2A, C).
Cells seeded onto 8.times. sfBCE-CM did attach and spread but to a
slightly lesser degree than on unconcentrated CM. The best
morphology (uniform spreading, less filopodia formation) was
observed in cells on BCE-ECM and in sfBCE-CM used as media or as a
coating substrate. Experiments are in progress to determine whether
differences in cell behavior are observed in media harvested from
BCE of different passages and times in culture.
TABLE-US-00002 TABLE 2 Fetal RPE behavior in serum-free BCE
conditioned media (sfBCE-CM) under different culture conditions.
Experimental Condition 1 Hour Day-1 Day-3 A. RPE CELL ATTACHMENT
AND GROWTH IN sfBCE-CM sfBCE-CM ~40% ~70-80% spread, almost
Confluent, uniform cell size, spread confluent few vacuoles
sfBCE-CM, heat Rounded Few cells, filopodia More cells than at
day-1 but inactivated very few, poor morphology. sfBCE-CM,
proteinase Rounded Rounded Rounded K treatment with and without
prior heat inactivation B. sfBCE-CM SURFACE COATING OF NTC DISHES
FOLLOWED BY RPE SEEDING WITH DMEM OR sfBCE-CM MEDIA sfBCE-CM, DMEM
40-50% High density, confluent in Confluent, small cells, good,
spread center, some multi- morphology, few vacuoles nucleate cells
sfBCE-CM, sfBCE-CM ~70% Moderate density, Elongate cells with
filopodia spread filopodia and lamellipodia and lamellipodia,
subconfluent 8X sfBCE-CM, ~60% High density of cells, Almost
confluent, more MW > 30K, DMEM spread confluent in center,
similar variable morphology than 1X to 1X BCE-CM, DMEM BCE-CM, DMEM
8X sfBCE-CM, 50-60% Moderate density of cells, Elongate cells with
filopodia MW > 30K, sfBCE-CM spread filopodia and lamellipodia
and lamellipodia, subconfluent C. NEGATIVE CONTROL NTC plastic,
DMEM Rounded Rounded Few elongated cells D. POSITIVE CONTROL
BCE-ECM surface ~50-90% Majority cells are spread Cells spread and
proliferating, spread but not confluent, some almost confluent
filopodia Cells were seeded at the same density for all
experiments. A. Effect of sfBCE-CM as media for attachment and
growth. sfBCE-CM, heat inactivated sfBCE-CM, and sfBCE-CM treated
with proteinase K are compared. B. Effect of sfBCE-CM as a surface
treatment for attachment and growth on non-tissue culture treated
(NTC) dishes, either unconcentrated or concentrated 8X using a 30
kD cut-off filter. Cells were suspended and cultured in either DMEM
or sfBCE-CM. C. Control (cells on untreated NTC dishes with DMEM as
media). D. Control (cells on BCE-ECM with DMEM as media).
[0148] Preliminary experiments with media harvested from passage-2
cultures show that media harvested from cells that have been in
culture for 2 weeks after reaching confluency is not as supportive
as media harvested at earlier time points (50% confluent,
confluent, 1 week after confluency) (data not shown). Protein
composition analysis is currently underway to determine changes in
the media harvested at these different time points to determine
what proteins may account for the decreased cell support.
Example 7
Soaking in Serum Free BCE-CM can Improve Cell Survival on Aged AMD
Bruch's Membrane
[0149] FIG. 6 demonstrates that even a relatively short treatment
(i.e., overnight) leads to improvement in RPE survival on and
resurfacing of Bruch's membrane. In this experiment, submacular
Bruch's membrane from an 80 year-old Caucasian male donor was
debrided to expose the superficial inner collagenous layer. Large
submacular drusen were present on Bruch's membrane of both eyes,
and the eye treated with BCE-CM showed more deposits (i.e., was the
more severely diseased of the two eyes). Bruch's membrane was
treated by overnight soaking of the explant in serum-free BCE-CM;
the fellow eye explant was soaked for the same period of time in
regular serum-free media (DMEM). Fetal RPE were seeded onto both
Bruch's membrane explants at a seeding density of 3164
cells/mm.sup.2. Both explants were cultured in RPE complete media
for 21 days.
[0150] The explant treated with the conditioned media as described
in the previous paragraph shows almost 100% resurfacing with a few
small defects (FIG. 6A, B, original magnification 200.times.). The
high magnification images (1000.times.) show small cells with
varying amounts of apical processes (a differentiation feature)
(FIG. 6C, D). In contrast, the untreated explant shows incomplete
resurfacing by very large flat smooth cells (FIG. 6 E, F, original
magnification 200.times.). Areas of cellular debris are evident
where the cells have died (arrows). Asterisks indicate areas not
resurfaced.
Example 8
Treatment of Aged Bruch's Membranes with BCE Conditioned Media
Improves Survival of RPE Derived from Human ES Cells
[0151] In order to investigate whether treatment of surfaces with
BCE-conditioned media improves survival and/or differentiation of
RPE other than fetal RPE, the following experiments were performed.
Fresh (not frozen) RPE derived from human ES cells (hES-RPE
obtained from Advanced Cell Technology, Inc.) of intermediate
pigmentation were seeded onto the inner collagenous layer of
submacular Bruch's membrane from a 63 year-old Caucasian female at
a seeding density of 3164 cells/mm.sup.2. There was no evident
pathology in the macula of either eye. The treated explant was
cultured in serum-containing BCE-CM while the untreated explant was
cultured in RPE complete media. Explants were harvested after 21
days in culture.
[0152] The results of these experiments are illustrated in FIG. 7.
The treated explant (FIG. 7 A, B) demonstrated some degree of
resurfacing by the hES-RPE with defects in coverage. The cells were
very flat and did not show prominent differentiated features. The
untreated explant (FIG. 7C, D) showed only sparse coverage, with
only a few cells in the submacular region of the explant. Thus,
BCE-CM treatment improved hES-RPE survival on aged human Bruch's
membrane.
Example 9
Active Components in BCE-CM Supporting Early Attachment and
Spreading in Cell Culture
[0153] Different preparations of sfBCE-CM of different molecular
weight cut-off were prepared to determine the MW fraction of active
components in sfBCE-CM. Media were reconstituted to 1.times.
following filtration. Retentate solutions of MW>3 kD, >10 kD,
>30 kD, and >50 kD and filtrate solutions of MW<3 kD,
<10 kD, <30 kD, and <50 kD were prepared. RPE (passage-4)
were suspended in each solution and seeded onto non-tissue cultured
treated plastic (NTC) as detailed above. In a separate study,
sfBCE-CM was filtered using a 100 kD molecular cut off filter,
yielding filtrates of <100 kD and retentates of >100 kD.
Fetal RPE (passage-3) behavior was observed in the 2 solutions up
to day-2.
[0154] The active cell-supporting components in BCE-CM appear to at
least include molecular weight (MW) 30 kD and higher. Based on
day-3 observations of vacuole formation (early apoptotic changes)
in RPE cultured in retentate fractions containing proteins of
molecular weight less than 30 kD, it appears that proteins present
in the low molecular weight fractions may have a negative effect on
the cells. Molecular weight fractions of 100 kD and higher
supported rapid initial RPE attachment in serum-free media. In this
assay, it was not found that molecular weight fractions below 100
kD supported rapid attachment and spreading in serum-free media to
any degree. Yet, as will be discussed in Example 12 below, two
additional bioactive fractions were identified that contributed to
cell survival on human submacular Bruch's membrane.
[0155] Thus, it appears that high molecular weight fractions
(>100 kD) are important in initial RPE attachment and spreading
in serum-free conditions.
TABLE-US-00003 TABLE 3 Fetal RPE behavior in serum-free BCE
conditioned media of different molecular weights. Molecular weight
fraction of sfBCE-CM 1 Hour Day-1 Day-3 Low MW (<3, 10, 30,
Rounded Rounded Rounded or 50 kD) High MW (>3, 10, 30, 30-40%
spread >3 kD confluent, >10, 30, All confluent, >3 kD
smallest or 50 kD) or 50 kD almost confluent cells with most
vacuoles; >10 to confluent with kD vacuoles, uniform cell
intercellular gaps size; >30 kD less vacuoles, uniform cell
size; >50 kD mixed sizes, no vacuoles sfBCE-CM (unfiltered
30-40% spread Confluent with Confluent, mixed sizes, few control
for above intercellular gaps vacuoles studies) >100 kD ~70%
attached ~90% spread, some No observations and spread filopodia
(more than seen in cells on BCE-ECM) <100 kD 80-90% attached,
20-30% spread, others No observations round are round, abundant
filopodia sfBCE-CM (unfiltered ~70% attached ~90% spread, some No
observations control for 100 kD cut- and spread filopodia off
studies) RPE were seeded at the same density for all experiments.
The effects of sfBCE-CM as media for attachment and growth,
prepared by centrifugal filtration of different MW cut-offs
(retentates above MW 3, 10, 30, 50, 100 kD and filtrates below MW
3, 10, 30, 50, 100 kD) are shown.
Example 10
BCE Conditioned Media is Effective at Dilutions up to 20.times.
[0156] Fetal RPE (passage-3, 526 cells/mm.sup.2) were seeded onto
non-tissue culture treated plastic in dilutions of serum-free BCE
conditioned media (sfBCE-CM, 1:1 to 1:80 dilutions) to determine
the maximum effective dilution of BCE-CM for support of initial RPE
attachment and spreading. Negative control was cells seeded in
serum-free DMEM. Results (Table 4). Support of attachment and
spreading was seen in BCE-CM diluted up to 1:10 in serum-free DMEM.
Cells in 1:20 and higher dilutions show increasingly poor
attachment and morphology at day-1 after seeding.
TABLE-US-00004 TABLE 4 Fetal RPE behavior in diluted serum-free
BCE-conditioned media. Dilution of sfBCE-CM 1 Hour Day-1 1:1
~60-70% attached ~90-95% attached and well spread and spread 1:5
~60-70% attached ~90-95% attached and well spread and spread 1:10
~50-55% attached ~90% attached and well spread and spread 1:20 ~50
attached ~60% attached, not as well spread and spread as higher
concentrations. Cells aggregated. 1:40 ~30-40% attached ~30-40%
attached, variably spread. and spread Cells aggregated; variable
morphology with lamellipodia, filopodia. Some cells elongated, some
not spread. 1:80 <5% attached and <10% attached, some
elongated spread minimal spreading. Cells aggregated; all of poor
morphology with elongation, filopodia and lamellipodia. DMEM
Rounded, few All rounded. (negative spread control) Fetal RPE were
suspended in different dilutions of serum-free BCE-CM and seeded
onto non-tissue culture treated dishes.
Example 11
Rpe can Attach and Grow on PCL Scaffolds
[0157] 1052 fetal RPE/mm.sup.2 were seeded onto 5 mm diameter PCL
scaffolds and cultured for 1 day. To assess attachment onto the
scaffolds, cell behavior was compared on scaffolds with no
treatment (FIG. 8, B) vs. scaffold soaked in serum-free BCE
conditioned media (sfBCE-CM, soaked for .about.1 hr. at 37.degree.
C.) to allow protein adsorption (FIG. 8, A). Cells were cultured in
DMEM or in sfBCE-CM. RPE were visualized on the scaffolds with
calcein imaging. RPE appeared to attach only to scaffolds treated
with or cultured in sfBCE-CM (Table 5, 1 day and FIG. 8). Greatest
attachment and spreading were observed in cells seeded onto
sfBCE-CM-soaked scaffolds.
[0158] To determine whether cells could eventually adhere and
spread on the scaffolds, scaffolds were exposed to sfBCE-CM by
either soaking (FIG. 9, A), followed by cell seeding and culturing
in DMEM for 2 days or using sfBCE-CM as media for 2 days. Controls
were cells on untreated scaffolds in DMEM for 2 days (FIG. 9, B).
Cultures were changed to RPE complete media (DMEM with 2 mM
glutamine, 15% fetal bovine serum, 2.5 .mu.g/ml fungizone, 0.05
mg/ml gentamicin, 1 ng/ml bFGF) after day 2 and cultured for 3
days. RPE were able to resurface the scaffolds only if the scaffold
was pre-soaked in sfBCE-CM or sfBCE-CM was used as media for two
days (see FIG. 9 and Table 5, 5 days).
[0159] To determine whether untreated scaffolds could support
eventual resurfacing by RPE, assays were carried out to examine
cell behavior on untreated scaffolds that were cultured in RPE
complete media for 7 days. Cells were seeded at the same density as
that onto Bruch's membrane (3164 cells/mm.sup.2).
[0160] RPE fully resurfaced the untreated scaffold although the
cells did not appear to density arrest by this time point (FIG. 10,
arrows point to areas of multilayer formation). It was observe
similar multilayer formation in RPE seeded onto tissue culture
plastic and onto glass coverslips.
[0161] PCL scaffolds can support initial fetal RPE attachment and
resurfacing if exposed to sfBCE-CM as a substrate coating the
scaffold or as media overlying seeded cells. Although untreated
scaffolds may support long-term survival of RPE in serum-containing
media, modification of the scaffold or addition of ECM ligands may
be necessary to support differentiated cell monolayers.
TABLE-US-00005 TABLE 5 Fetal RPE behavior on PCL scaffolds that
were either untreated or soaked in serum-free BCE conditioned media
(sfBCE-CM). Time in Scaffold Culture Treatment Media Cell Behavior
1 day None DMEM Few rounded cells None sfBCE-CM Many cells, many
are spread sfBCE-CM DMEM Many cells, many are spread BCE-ECM on
DMEM Many cells, majority plastic are spread 5 days None 2 d DMEM,
3 d RPE Few rounded cells complete media None 2 d sfBCE-CM, 3 d
Fully resurfaced RPE complete media sfBCE-CM 2 d DMEM, 3 d RPE
Fully resurfaced complete media 7 days None RPE complete Fully
resurfaced, some media multi-layering BCE-ECM on RPE complete Fully
resurfaced, plastic media monolayer For 1-day studies, cells on
untreated scaffolds were cultured in sfBCE-CM or DMEM; cells on
sfBCE-CM-soaked scaffolds were cultured in DMEM. For 5 day studies,
cells were cultured on untreated or sfBCE-CM-soaked scaffolds for 2
days in DMEM or sfBCE-CM followed by media change to RPE complete
media. For 7-day studies, untreated scaffolds were cultured in RPE
complete media. Cell behavior on BCE-ECM-coated culture dishes (no
scaffold controls) is included for day-1 and day-7 data for
comparison.
Example 12
Identification of Bioactive Fractions that Support Cells on Human
Aged and AMD Bruch's Membrane and Molecules of BCEC-CM
[0162] In this example, BCEC-CM was fractionated to identify
fractions having therapeutic activity. Briefly, BCEC-CM was
collected from passage-2 BCEC after 72 hour exposure to Madin-Darby
Bovine Kidney Maintenance Medium. The collected BCEC-CM was subject
to ultrafiltration utilizing centrifugal filters of sizes ranging
from 3 to 300 kDa. After separation, the fractions were tested for
bioactivity by analyzing RPE survival on human submacular Bruch's
membrane explants established from aged and AMD donor eyes. The
protein component of the bioactive fraction was analyzed by mass
spectrometry.
[0163] The bioactive fraction was identified as those molecules
found in the filtrate generated after ultrafiltration using a 50
kDa filter. Mass spectrometry of the 50 kDa filtrate of two
different BCEC-CM preparations identified 72 common secreted
proteins, including 5 growth factors. Subfractionation of the 50
kDa filtrate showed decreased bioactivity in the filtrate after
ultrafiltration utilizing a 30 kDa filter, indicating some
bioactivity was contained in the 30-50 kDa fraction, and complete
loss of bioactivity after removal of the 10-50 kDa fraction.
Ultrafiltration of the 50 kDa fraction utilizing a 3 kDa filter
also showed complete loss of activity in the retentate (3 kDa-50
kDa), indicating that bioactivity was present in the <3 kDa
filtrate.
[0164] Bioactive molecules were found in a fraction generated by
molecular weight cut off filtration. This bioactive fraction is
comprised of molecules found in the fraction generated after
filtration using a 50 kDa filter. Bioactive molecules supporting
long-term survival of cells on aged and AMD submacular Bruch's
membrane are found in several subfractions of the 50 kDa fraction:
a low molecular weight subfraction (below 3 kDa) and a 10-50 kDa
subfraction. This finding indicates there are at least two
bioactive molecules in BCEC-CM. Subsequent mass spectrometry
analysis of the protein component of the 50 kDa fraction identified
four candidate growth factors (proteins that stimulate cells in a
variety of ways including growth stimulation, cell death
prevention, and cell functionality and maturity acquisition).
Molecules found in the low molecular weight fraction support rapid
cell attachment, spreading, and growth in cell culture.
[0165] Preliminary two-dimensional gel and mass spectrometry spot
ID analysis (see FIG. 11, Table 6 below) showed abundant large
molecular weight extracellular matrix ligands (collagens and
fibronectin). These molecules (specifically, fibronectin) have been
shown to support RPE attachment in cell culture and to support
initial attachment on Bruch's membrane. To determine if high
molecular weight components contribute to cell survival on Bruch's
membrane, RPE survival was analyzed in large molecular weight
retentates. Molecular cut removal of low molecular weight
components showed little or no bioactivity in the retentate
fractions (see FIG. 12). The results of testing the high molecular
weight retentates (FIG. 12) indicate that a low molecular weight
fraction in the 3 kDa filter filtrate must be present in order for
BCEC-CM to show complete bioactivity.
TABLE-US-00006 TABLE 6 Proteins Obtained by Molecular Cut
Filtration and Identified by Mass Spectrometry IPI ID Protein MW
Gene Function IPI00691126 C-X-C motif chemokine 6 12 CXCL5 cytokine
IPI00699064 DKK3 protein 38 DKK3 cytokine IPI00714868 Protein FAM3C
25 FAM3C cytokine IPI00839037 Uncharacterized protein 13 PF4
cytokine IPI00696930 Uncharacterized protein 55 EFEMP1 enzyme
IPI00702154 Lysyl oxidase-like 1 65 LOXL1 enzyme IPI00710136
Angiogenin-1 17 ANG enzyme IPI00760446 Ribonuclease, RNase A
family, 4 17 RNASE4 enzyme IPI00686503 Platelet-derived growth
factor subunit 24 PDGFA growth factor IPI00698668 Connective tissue
growth factor 38 CTGF growth factor IPI00706240 growth
arrest-specific 6 74 GAS6 growth factor IPI00731393 PDGFD protein
42 PDGFD growth factor IPI01018572 Insulin-like growth factor I
variant 2 21 IGF-1 growth factor IPI00714018 Insulin-like growth
factor I 17 growth factor IPI00685095 Cystatin-C 16 CST3 other
IPI00685504 Alpha 1 type VIII collagen (Fragment) 73 COL8A1 other
IPI00688802 Uncharacterized protein 136 NID1 other IPI00688875
Follistatin-related protein 3 28 FSTL3 other IPI00690094 Galectin-1
15 LGALS1 other IPI00692839 Uncharacterized protein 44 other
IPI00698975 SPARC 35 SPARC other IPI00702294 Plasminogen activator
inhibitor 1 45 other IPI00704150 Vitamin K-dependent protein S 75
PROS1 other IPI00705697 Insulin-like growth factor-binding 34
IGFBP2 other IPI00706624 Procollagen C-endopeptidase 48 PCOLCE
other IPI00707101 Alpha-2-HS-glycoprotein 38 AHSG other IPI00707467
Follistatin-related protein 1 35 FSTL1 other IPI00707932 collagen
alpha-2(VIII) chain 67 COL8A2 other IPI00708244 Collagen alpha-2(I)
chain 129 COL1A2 other IPI00708990 Uncharacterized protein 148
LTBP1 other IPI00709059 Angiopoietin-related protein 7 39 ANGPTL7
other IPI00709084 Metalloproteinase inhibitor 1 23 TIMP1 other
IPI00710025 Factor XIIa inhibitor 52 other IPI00710385 Prolargin 44
PRELP other IPI00710453 Matrix Gla protein 12 MGP other IPI00711862
Epididymal secretory protein E1 17 NPC2 other IPI00712084
Thrombospondin-1 130 THBS1 other IPI00712366 Fibromodulin 43 FMOD
other IPI00712524 collagen, type IV, alpha 2, partial 165 COL4A2
other IPI00713428 ADM 21 ADM other IPI00713573 Uncharacterized
protein 109 COL6A1 other IPI00716121 Pigment epithelium-derived
factor 46 other IPI00718311 Isoform 1 of Proactivator polypeptide
58 PSAP other IPI00718620 Insulin-like growth factor-binding 28
IGFBP4 other IPI00730859 Uncharacterized protein 139 LTBP3 other
IPI00731756 Uncharacterized protein 24 SCG5 other IPI00824031 LTBP1
protein 147 LTBP1 other IPI00838716 Uncharacterized protein
(Fragment) 102 CHRD other IPI00840999 PCSK1N protein 27 PCSK1N
other IPI00883474 gelsolin a 86 GSN other IPI00905045 collagen
alpha-1(XI) chain 182 COL11A1 other IPI00906401 Uncharacterized
protein (Fragment) 25 IGFBP6 other IPI00733988 collagen type 5
alpha 1-like 30 other ECM IPI00716123 Mimecan 34 OGN other ECM
IPI00685447 72 type IV collagenase 74 MMP2 peptidase IPI00705266
Uncharacterized protein 102 ADAMTS5 peptidase IPI00712538 HtrA
serine peptidase 1 67 HTRA1 peptidase IPI00713459 A disintegrin and
metalloproteinase 136 ADAMTS3 peptidase IPI00713505 Complement C3
(Fragment) 187 C3 peptidase IPI00714873 Serine protease 23 42
PRSS23 peptidase IPI01004181 bone morphogenetic protein 1-like 107
BMP1 peptidase IPI00717574 Carboxypeptidase E 53 peptidase
IPI00689362 Transthyretin 16 TTR transporter IPI00690534
Serotransferrin 78 TF transporter IPI00708398 Uncharacterized
protein 70 ALB transporter IPI00712693 Apolipoprotein E 36 APOE
transporter IPI00713780 Uncharacterized protein 24 APOD transporter
IPI00715548 Apolipoprotein A-I 30 APOA1 transporter IPI00866855
IGFBP7 protein 29 IGFBP7 transporter IPI00697184 retinol-binding
protein 4 23 Secreted transporter IPI00867435 NID2 protein 143
Secreted? other
[0166] Testing of the filtrates revealed complete bioactivity can
be retained in filtrates utilizing the 50 kDa filter, indicating
that the bioactivity is comprised of molecules of molecular weight
at or near 50 kDa and lower. Although there appears to be a trend
towards decreased nuclear density in the <30 kDa filtrate, the
difference is not significant (Kruskal-Wallis One Way Analysis of
Variance on Ranks, P=0.092).
[0167] The results of these examples indicate that there are a
minimum of two bioactive molecules in BCEC-CM, one found in the
<3 kDa filtrate and one found in the 10-50 kDa fraction and that
bioactive molecules in both fractions must be present to ensure
cell survival on Bruch's membrane.
Example 13
Differentiation Medium for RPE
[0168] In this example, studies were performed to determine whether
fetal RPE mature more rapidly in BCEC-CM compared to standard RPE
medium, where onset of maturity was based on mRNA expression of
late RPE differentiation markers, bestrophin and RPE65. More
specifically, fetal RPE were seeded at a high seeding density (3164
cells/mm.sup.2) onto BCEC-ECM coated tissue culture dishes and
maintained in culture for 21 days in the aforementioned RPE medium
or BCEC-CM. The media were changed 3.times./week. At day-21, the
cells were harvested off the tissue culture dishes for real time
PCR mRNA analysis of bestrophin and RPE65.
[0169] It was found that the cells cultured in BCEC-CM appeared to
have patches of cells that looked more mature (morphologically)
than cells in the RPE medium. In contrast, the cells in the RPE
medium appeared to be more uniform in appearance. The cells
cultured in BCEC-CM expressed approximately 10.times. more RPE65
and 50.times. more bestrophin mRNA than the cells cultured in the
RPE medium.
Example 14
Bovine Corneal Endothelial Cell Conditioned Medium as Storage
Medium
[0170] In this example, assays were carried out to examine the
properties of CM in preserving confluent fetal RPE monolayers under
conditions likely to occur during shipping and storage of cells
attached to a substrate prior to use in patients.
[0171] a. Cell Behavior after Storage in RPE Medium, Optisol, and
CM (Batch 34AB)
[0172] In this assay, the substrate was tissue culture plastic
(TCP) or TCP coated with bovine corneal endothelial cell
extracellular matrix (ECM). Optisol is a medium developed for
corneal storage. Passage 2 fetal RPE were seeded on ECM or directly
on TCP and cultured in standard RPE medium for 11 days. At the
start of the storage period, cells were placed in one of the
following three media:
[0173] (i) RPE medium: DMEM base medium (HEPES, inorganic salts,
amino acids, vitamins, glucose, sodium pyruvate), bFGF, fetal
bovine serum, glutamine, gentamicin, and fungizone;
[0174] (ii) Optisol (Bausch and Lomb, Inc., proprietary cornea
preservation medium) "Optisol base powder", chondroitin sulfate,
dextran, sodium bicarbonate, antibiotics and fungizone, sodium
pyruvate, glutamine, mercaptoethanol, and amino acids; and
[0175] (iii) CM: MDBK-MM base medium (Sigma Aldrich proprietary
formulation including HEPES, human recombinant peptides (insulin
and possibly others), amino acids, and sodium bicarbonate), CM
harvested after 3 day exposure of MDBK-MM to confluent bovine
corneal endothelial cell cultures.
[0176] Experiments were carried out at 4.degree. C. (refrigerator)
and at room temperature in sealed culture plates or dishes with no
media change. Although it is unlikely a surgical site will have a
37.degree. C. CO.sub.2 incubator suitable for storing cells for
patient use, the viability of storing fetal RPE was compared in
such an incubator. In these preliminary studies, the onset of cell
death was estimated by initial appearance of defects in the RPE
monolayer.
TABLE-US-00007 TABLE 7 Onset of cell death after storage in
Optisol, CM, or RPE medium Substrate/ temperature Optisol CM RPE
TCP/4.degree. C. <7 days ~21 days ~21 days TCP/RT <3 days
>21 days <7 days ECM/4.degree. C. <10 days <10 days
<10 days ECM/RT <3 days <3 days ECM/37.degree. C. <7
days <7 days (unsealed) TCP/37.degree. C. 14-<21 days 14-21
days (unsealed)
[0177] The results were shown in Table 7. It was found that Optisol
was relatively poor at preserving RPE viability. All cells stored
in Optisol were of abnormal morphology at early time points. Both
CM and RPE media generally maintained fetal RPE viability to a
similar degree except CM was better at preserving RPE at room
temperature if the cells were on tissue culture plastic. Fetal RPE
cultured on an extracellular matrix that supports rapid cell
attachment and growth in cell culture, showed poor viability
regardless of the storage medium.
[0178] b. Storage of Confluent RPE Cultures in CM (Batch 58)
Molecular Cut Fractions
[0179] In this assay, confluent fetal RPE cultures (passage 3 or 4,
7-13 days in culture) grown on tissue culture plastic were stored
at room temperature or 4.degree. C. in sealed 48 well plates with
no medium change for up to 7 days. The storage media were RPE
medium and CM, including 3 kD and 50 kD CM molecular cut filtrates.
Live/death assessment was performed at day-3 or day-7. The results
are shown in Table 8 below, where the values reflect the days in
storage when loss of cells in the RPE monolayer was observed.
TABLE-US-00008 TABLE 8 Onset of cell death after storage in RPE
medium and CM molecular cut fractions Storage medium 4.degree. C.
RT RPE 3-4 D 3-4 D 3K CM 4 D-<7 D >7 D 50K CM 4 D-<7 D 7
D->7 D Uncut CM 4 D-<7 D >7 D
[0180] It was found that onset of cell death was sooner than in the
previous study (Table 7 vs. Table 8), possibly due to variability
between CM batches and/or the fetal RPE passage number and time in
culture. An additional consideration for comparisons between
experiments for storage at room temperature is the variability in
ambient temperature. RPE medium effective storage times were
consistently short for both temperatures. Storage times in CM,
including CM fractions were longer than RPE for storage at room
temperature. The 3 kD and 50 kD filtrates storage times were
similar to CM that had not be subject to molecular cut filtration
("uncut CM").
Example 15
Storage of Cell Suspensions
[0181] In this example, assays were performed to examine the
ability of CM for shipping and storing fresh cell suspensions on
wet ice or ambient temperature. Such ability offers an alternative
to using frozen cells for patient transplants. Frozen cells require
storage in liquid nitrogen, shipment on dry ice or under liquid
nitrogen, and thaw, rinse, and resuspension in delivery vehicle for
patient use. Cells recovered from thaw attach and grow slower than
fresh cells with some cell death occurring from freezing and
subsequent manipulations. Ideally, fresh cells would be stored in a
solution similar to that of the delivery vehicle so no or few
manipulations are required.
[0182] a. Cell Viability after Storage in CM vs. RPE Medium
[0183] Previous studies indicated that RPE undergo apoptosis if not
attached to a suitable substrate by 24 hours (Tezel et al. Graefes
Arch Clin Exp Ophthalmol 1997; 235:41-47). In this assay, studies
were performed to determine if cell suspensions can retain any
degree of viability after storage.
[0184] Briefly, fetal RPE cell suspensions (100,000 in 100 ul) in
the CM or RPE medium (contains fetal bovine serum, glutamine, and
basic fibroblast growth factor) were placed in sealed microfuge
tubes. The tubes were stored at room temperature or 4.degree. C.
for 1, 2, 3, or 7 days. At the end of the storage period, the
numbers of live and dead cells were determined by trypan blue
staining and the remaining cells plated on tissue culture plates
and cultured in fresh storage medium to assess viability.
[0185] FIG. 16 illustrates cell viability, expressed as the percent
of live cells at the end of each storage period, for fetal RPE
suspensions in CM or RPE medium at room temperature (RT) or
4.degree. C. The results showed that fetal RPE suspensions can
retain a high degree of viability when stored in sealed microfuge
tubes at 4.degree. C. and room temperature for up to 3 days in
storage. At 7 days in storage, cells stored in the RPE medium at
4.degree. C. showed a marked drop in percent of live cells. At room
temperature, cells were clumped in both media, making viability
assessment difficult. Harvested cells from each storage time showed
rapid attachment, spreading, and growth when cultured on tissue
culture plastic. All cultures were confluent by day-7 in culture
except for the 7-day storage, room temperature cells that were
stored in CM. These data show that it is possible to store cell
suspensions for a short period of time with little loss in cell
viability.
[0186] b. Storage of Cell Suspensions in Molecular Cut Fractions of
CM (CM Batch 58)
[0187] The studies of part a in Example 14 above were repeated with
additional storage in 3 kD and 50 kD CM molecular cut filtrates. To
determine the change in cell numbers with time in culture, time 0
viability counts were performed for each tube of cells to be
stored. To better assess cell numbers in tubes stored at room
temperature, cell clumps were treated with trypsin for the 3 and 7
day storage time points. Cell viabilities in all media were below
the levels measured at time 0 at days 3 and 7.
[0188] It was found that, consistent with the previous experiment
(FIG. 16), poorest viability was observed in cells stored at
4.degree. C. in the RPE medium (FIG. 17). At room temperature
storage, all cells maintained viability levels above that measured
at time zero (FIG. 18). Cells stored in CM and CM fractions showed
better preservation of live cells than those stored in RPE medium
at 4.degree. C. (FIG. 19). The change in cell numbers in the RPE
medium at day-7 is consistent with the drop in cell viability at
this time point. The day-1 data are not shown since the counting
method was not the same as days 3 and 7. The increase in live cell
numbers at day-3 compared to time 0 indicated that the cells were
dividing. Cells in 50 kD CM appear to maintain cell division at
day-7.
[0189] As shown in FIG. 20, storage in 3 kD CM and the RPE medium
at room temperature showed increased number of live cells at day-1
while live cell numbers dropped to similar levels in 50 kD CM and
uncut CM. Similar to storage at 4.degree. C., cells in the RPE
medium show a drop in live cells with time in culture. The cells in
50 kD CM appeared to be dividing during 3 and 7 day storage periods
with number of live cells at day-7 well above the time 0 levels.
The cells in uncut CM showed a marked rise in the number of live
cells at day-7 compared to time 0 levels.
Example 16
CM as Culture Medium
[0190] In this example, assays were performed to examine whether CM
could offer an advantage over a standard RPE culture medium as a
defined, serum-free medium.
[0191] a. Comparison of Cell Behavior in CM vs. RPE Medium
[0192] Studies of fetal RPE behavior (when cultured in CM vs. RPE
medium) show that CM can support fetal RPE to some degree when
cultured directly on tissue culture plastic. It was found that
cells in CM rapidly attached, spread, and grew to confluence. The
ability of CM to support cells in long-term cultures is highly
variable and may depend on the batch of CM and/or the fetal RPE
starting culture (passage number, length of time in culture prior
to harvest). In some CM cultures, some cell death occurred between
day-14 and day-21. In other cases, CM cultures were similar in size
at 14 and 21 days although the cells may be more pigmented at
day-21.
[0193] Lastly, it was found that some CM supported cells to a
higher degree with mature cells at day-21 compared to day-14. In
RPE medium, some cell death and/or cessation of cell division
appeared to occur between day-7 and day-21 in some cultures as the
cultures were similar in appearance or the cells are larger at
day-21. Additionally, the presence of pigmented clumps of dead
cells could be seen in many cultures at these time points. In
comparing parallel cultures of CM and RPE medium, CM appeared to
preserve cell viability longer than RPE medium. Studies are in
progress to determine cell behavior in CM vs. RPE medium on single
human ECM proteins (e.g. laminin, collagen I, collagen IV).
[0194] The set of figures in FIGS. 21A-F are parallel cultures at
different culture times. It was found that fetal RPE attached and
spread rapidly in early cultures. As shown in the figures, by 7
days, both cultures appeared similar. At day-14, the appearance of
mature RPE could be seen in both cultures; some of the cells were
very small and appeared to have rounded (columnar) vs. flat
surfaces. There appeared to be more of these highly differentiated
cells in the CM culture vs. RPE medium culture. The white arrow
points to a cluster of highly pigmented RPE. These could be
dead/dying RPE that are shed from the culture. By 21 days, the
cells were larger in both media, indicating that some cell death
occurred.
[0195] b. Culture with Limited Viability of RPE in RPE Medium
[0196] In this example, additional assays were performed to examine
effects of CM and the RPE medium on cell behavior.
[0197] In a set of parallel cultures (see FIG. 22), RPE in the RPE
medium on tissue culture plastic showed some degree of cell death
as early as 8 days in culture with the appearance of pigmented
clusters on top of the RPE monolayer. The cells in the RPE medium
were of similar size at all three time points. The RPE appeared to
be smaller when cultured in CM indicating cell division
occurred.
[0198] c. Expression of RPE Differentiation Markers in Cells
Cultured in CM vs. RPE Medium
[0199] Assays were carried out to examine mRNA expression levels 21
days after seeding on tissue culture plastic utilizing a different
CM batch than that used in Example 13. It was found that the fetal
RPE cultured in CM expressed 9.6.times. more Bestrophin and
1.28.times. more RPE65 than the cells cultured in the RPE medium.
Western blot also showed Bestrophin protein present as strong bands
in 14- and 21-day CM cultures with faint (if present at all) bands
in parallel RPE medium cultures at the same time points.
[0200] d. Culture of RPE in 3 kD Filtrate
[0201] While CM low molecular weight components alone do not
support cells on Bruch's membrane, the 3 kDa filtrate alone
supported attachment and spreading of human fetal RPE on human
collagen I, a major component of the inner collagenous layer of
Bruch's membrane, as well as on uncoated tissue culture plastic
although not to the same degree.
[0202] On collagen I, cell spreading was observed initially by
day-1 and by day-3 in culture, .gtoreq.50% of RPE are spread. Cells
reached confluence by 3 days or later, depending on the CM batch.
On tissue culture plastic, RPE attachment, spreading, and growth
occurred but, depending on CM batch, to a lesser degree than was
observed on collagen I, with the onset of spreading later on tissue
culture plastic. The 10-50 kDa fraction alone supported only
limited attachment and spreading on both tissue culture plastic and
collagen I at early times in culture with only a few elongate cells
observed at day-3. Addition of the 3 kDa filtrate to this fraction
restored the activity to the level observed in <50 kDa filtrate.
These studies indicate that the 3 kDa filtrate contains bioactive
molecules that are necessary for cell attachment and growth in
long-term cell culture.
Example 17
Enhancing Cell Survival on Bruch's Membrane in Eyes Affected by Age
and AMD
[0203] In this example, assays were carried out to determine
whether BCEC-CM can support transplanted cells on aged and AMD
Bruch's membrane (BM).
[0204] Currently, no proved treatment options exist for patients
with geographic atrophy, an advanced form of AMD. For selected
patients with extensive drusen or geographic atrophy threatening
the fovea, cell transplants might prevent central vision loss
through replacement of dysfunctional or dead RPE cells.
Anti-vascular endothelial growth factor therapy is currently the
best treatment available for AMD-associated CNVs, but randomized
studies indicate that only 25-40% of treated patients experience at
least moderate visual improvement. Thus, even today, a significant
number of patients become blind despite the availability of
pathway-based therapy for AMD-associated CNVs. If cell transplants
could prevent CNV development or rescue photoreceptors following
CNV excision, then these transplants also might have an impact on
CNV-related blindness.
[0205] A major obstacle to the success of RPE transplants in AMD
patients is the failure of transplanted RPE cells to survive and
become functional in the diseased AMD eye. RPE transplantation in
patients with AMD (atrophic and neovascular) typically has produced
limited visual recovery regardless of the type of cell transplanted
(e.g., autologous or allogeneic, adult or fetal RPE) or whether the
cells are transplanted with or without choroid. In contrast, RPE
transplantation in animal models of retinal degeneration has been
proved to rescue photoreceptors and preserve visual acuity.
Although animal studies validate cell transplantation as a means of
achieving photoreceptor rescue, an important distinction between
humans with AMD and laboratory animals in which RPE transplantation
has been successful is the age- and AMD-related modifications of
the surface on which human RPE reside in situ (i.e., Bruch's
membrane), which may have a significant effect on RPE graft
survival. Evidence from human donor eye organ culture experiments
indicates that healthy RPE cannot survive for an extended period of
time on aged submacular Bruch's membrane, and the poorest survival
is observed on AMD Bruch's membrane. These in vitro studies were
performed on human submacular Bruch's membrane with no treatment to
improve cell survival. Previous studies to improve cell survival on
aged Bruch's membrane included adding ECM ligands singly or in
combination to "coat" Bruch's membrane, detergent treatment to
eliminate debris accumulated within Bruch's membrane followed by
ECM ligand coating, and resurfacing Bruch's membrane with a
cell-deposited matrix. The first two methods showed limited
improvement in attachment and early survival. Long-term survival
was not demonstrated. The last method improved long-term cell
survival more than 200%. However, from a therapeutic standpoint,
resurfacing submacular Bruch's membrane with the cell-deposited ECM
was problematic due to the inability to solubilize ECM components
in a manner compatible with clinical application. These studies
demonstrate the need for development of a method to improve
long-term cell transplant survival in AMD patients.
[0206] BCECs secrete an ECM that supports rapid attachment, growth,
and differentiation of RPE. During BCEC-ECM formation, in addition
to basal secretion, BCECs secrete ECM components into the overlying
medium, including collagens, proteoglycans, and entactin/nidogen.
Secretion of ECM components into the overlying medium is most
abundant in early passage cells and exceeds basal ECM deposition in
quantity. Since soluble ECM can affect cell shape and metabolism in
addition to stimulating production of ECM molecules, the presence
of these proteins suggests that conditioned medium harvested from
BCEC cultures could be a source of cell-supporting soluble proteins
and, if effective, could lead to development of an adjunct to
cell-based therapy for AMD. In this example, assays were performed
to characterize the behavior of RPE cells transplanted onto Bruch's
membrane of aged and AMD donor eyes cultured in BCEC-CM or CM
vehicle utilizing a previously characterized human submacular
Bruch's membrane bioassay.
Material and Methods
[0207] Conditioned Medium Preparation
[0208] Cow eyes (ages 6 months -3 years) were obtained from local
slaughterhouses. Each globe was rinsed briefly in 70% ethanol, and
the cornea was separated from the rest of the globe by making a
circumferential cut anterior to the limbus. The cornea was rinsed
quickly in PBS and positioned with the epithelial surface down on a
sterile support placed on a Petri dish. The cup formed by the
cornea was filled with 0.05% trypsin-0.02% EDTA (Invitrogen-Gibco,
Life Technologies, Carlsbad, Calif.) and placed in a 37.degree. C.,
10% CO2 incubator for 30-60 minutes. BCECs were scraped off gently
using a blunt metal spatula and collected into a 15 ml tube
containing Dulbecco's modified Eagle's medium (DMEM, Cellgro,
Manassas, Va.) supplemented with 2 mM glutamine, 15% fetal bovine
serum (FBS), 2.5 .mu.g/ml amphotericin B, 50 .mu.g/ml gentamicin,
and 1 ng/ml bFGF (all from Invitrogen-Gibco) (termed "RPE medium").
Cells were spun down, resuspended in RPE medium, seeded onto 60 mm
dishes, and cultured at 37.degree. C. in 10% CO2. Cultures were
passaged at confluence. For BCEC-CM harvest, passage-2 or -4 cells
were cultured in RPE medium with 10% FBS and 5% donor bovine serum
(Invitrogen-Gibco), instead of 15% FBS, until confluent. BCEC-CM
was obtained by incubating confluent BCEC cultures for 72 hours in
Madin-Darby Bovine Kidney Maintenance Medium (MDBK-MM,
Sigma-Aldrich, St. Louis, Mo.) supplemented with 2.5 .mu.g/ml
amphotericin B and 50 .mu.g/ml gentamicin. The vehicle, MDBK-MM
(hereafter referred to as "CM vehicle"), is a serum- and
protein-free, defined medium designed for maintaining high-density
cultures of MDBK cells. Following collection, BCEC-CM was
centrifuged briefly to remove cellular debris, and the supernatant
was stored at -80.degree. C. Twelve batches of BCEC-CM were used in
this study.
[0209] Cell Culture
[0210] RPE were isolated from fetal eyes (Advanced Bioscience
Resources, Inc., Alameda, Calif.; gestational age 18-22 weeks) or
adult eyes (donor age 58, 71, 78 yrs.) after incubation of
RPE/choroid pieces in 0.8 mg/ml (fetal eyes) or 0.4 mg/ml
collagenase type IV (Sigma-Aldrich) (adult eyes) as described
previously. RPE were cultured in RPE medium on bovine corneal
endothelial cell-extracellular matrix (BCEC-ECM)-coated tissue
culture dishes prepared in this laboratory according to a
previously described protocol. After achieving confluence, primary
fetal RPE cultures were passaged at a 1:6 split ratio onto
BCEC-ECM-coated dishes using 0.25% trypsin-EDTA to harvest the
cells. Subsequent cultures were passaged at a 1:4 split ratio.
Adult RPE seeded onto Bruch's membrane were from day-11-15 primary
cultures; fetal RPE were harvested from cultures of passage 1-3,
3-7 days in culture after seeding. Human embryonic stem
cell-derived RPE (hES-RPE, Advanced Cell Technology, Worcester,
Mass.), were established from a stem cell culture designated as
MA09. Cells were maintained in MDBK-MM medium (Sigma-Aldrich) until
removal from flasks and seeding onto Bruch's membrane explants.
Cells of passage-32 as stem cells and passage-2 or -3 as hES-RPE
were utilized and were removed from culture dishes using
trypsin/EDTA after 50-85 days in culture.
[0211] Bruch's Membrane Organ Culture
[0212] Adult donor eyes were received from the Lions Eye Institute
for Transplant and Research (Tampa, Fla.) and eyebanks placing
donor eyes through their website (Ocular Research Biologics System
(ORBS), orbsproject.org), Midwest Eyebanks (includes eyebanks in
Illinois, Michigan, and New Jersey), the San Diego Eyebank (San
Diego, Calif.), and eyebanks placing tissue through the National
Disease Research Interchange (NDR1, Philadelphia, Pa.). Acceptance
criteria for donor eyes included: 1) death to enucleation time no
more than 7 hours; 2) death to receipt time no more than 48 hours;
3) no ventilator support prior to death; 4) no chemotherapy within
the last 6 months prior to death; 5) no radiation to the head
within the last 6 months prior to death; 6) no recent head trauma;
7) no ocular history affecting the posterior segment except for
AMD. These acceptance criteria have been found in previous studies
to yield well-preserved explants. Posterior segments were examined
through a dissecting microscope for submacular pathology and
documented by photography. A previously published method was used
to create inner collagenous layer (ICL) surfaces by mechanical
debridement. Six-millimeter diameter corneal trephines (Bausch and
Lomb, Rochester, N.Y.) were used to create macula-centered, Bruch's
membrane explants. Explants were placed in wells of 96-well plates
for cell seeding and organ culture. Cells were seeded at a seeding
density of 3164 cells/mm.sup.2, a seeding density that has been
shown to yield a monolayer of cells on a 6 mm diameter Bruch's
membrane explant in organ culture one day after seeding. Explants
were harvested at day-21, fixed in phosphate-buffered 2%
paraformaldehyde and 2.5% glutaraldehyde, bisected, and processed
for light or scanning electron microscopy.
[0213] Scanning Electron Microscopy (SEM)
[0214] Explant halves for SEM were post-fixed in phosphate buffered
osmium tetroxide, dehydrated using a graded series of ethanol,
critical point dried (Tousimis, Rockville, Md.), and sputter-coated
(Denton, Moorestown, N.J.) according to standard SEM protocols. SEM
image acquisition (JEOL JSM 6510, Tokyo, Japan) was performed with
routine photography at 30.times., 50.times., 200.times., and
1000.times.. SEM evaluation of Bruch's membrane involved assessment
of cell surface morphology and, in areas not resurfaced by cells,
the level of Bruch's membrane exposed by debridement.
[0215] Light Microscopy (LM)
[0216] Bruch's membrane explant halves processed for histology were
embedded in LR White (Electron Microscopy Sciences, Hatfield, Pa.);
4-6 sections of 2 .mu.m thickness were mounted on slides, dried
overnight, and stained with 0.03% toluidine blue (Electron
Microscopy Supply). LM evaluation focused on RPE morphology (cell
shape, density, pigmentation, polarization) and evaluation of
Bruch's membrane and choroid. Nuclear density counts were performed
to assess treatment success quantitatively, comparing paired
explants from fellow eyes. Nuclear density counts were performed by
counting the number of RPE nuclei in intact cells in contact with
Bruch's membrane in the central 3 mm of 4-5 non-consecutive slides
(approximately every 5th slide). Linear measurements of Bruch's
membrane in the analyzed area were obtained by digital image
acquisition and measurement with the freehand line tool using NIH
Image J (http://rsb.info.nih.gov/ij/index.html). Nuclear density
was expressed as the number of nuclei per mm of Bruch's
membrane.
[0217] Statistical Analysis
[0218] Statistical differences between pairs were determined by
Wilcoxon Signed Rank tests. For comparisons between time points and
comparison between groups, existence of significant differences was
determined by Kruskal-Wallis One Way Analysis of Variance on Ranks.
If significance was observed, All Pairwise Multiple Comparison
Procedures testing (Dunn's method) determined the significance
between pairs of groups. Comparisons between two groups in unpaired
studies were by Mann-Whitney Rank Sum tests. Comparisons between
ages of two groups were by unpaired t-tests or between multiple
groups by One Way ANOVA. Ages are indicated as mean age with
standard deviation. A P value <0.05 was considered statistically
significant.
[0219] Extracellular Matrix Deposition
[0220] Fetal RPE (3164 cells/mm.sup.2) were seeded onto tissue
culture-treated plastic (48-well plates) or Bruch's membrane and
cultured in BCEC-CM or RPE medium. ECM on tissue culture plates was
analyzed at day-7, -14, and -21 (N=3). ECM on Bruch's membrane (6
donor pairs; three pairs with extensive drusen; 3 pairs normal;
mean donor age, 79.2.+-.3.17 yrs.) was analyzed at day-21 only.
Primary antibodies were: mouse monoclonal collagen IV (1:500
dilution, Sigma-Aldrich), rabbit polyclonal laminin (1:25 dilution,
Sigma-Aldrich), and mouse monoclonal fibronectin (1:50 dilution,
Abcam, Cambridge, Mass.). Secondary antibodies were fluorescein
(FITC)-conjugated goat anti-mouse IgG (H+L) and rhodamine
(TRITC)-conjugated goat anti-rabbit IgG (H+L) applied at 1:50
dilution (both from Jackson ImmunoResearch Laboratories, West
Grove, Pa.). All antibodies were diluted in 2% normal goat serum,
0.3% Triton X-100 (both from Sigma-Aldrich) in phosphate buffered
saline (PBS).
[0221] Cell culture: Cells were removed from culture wells by
incubating in 0.02M NH4OH for 5 minutes followed by rinsing in PBS.
The exposed ECM was fixed for 15 minutes in cold 4%
paraformaldehyde followed by 3 PBS washes. Wells were then
incubated for 45 minutes at room temperature in blocking solution
(2% normal goat serum, 0.5% BSA in PBS). Primary antibodies were
applied to culture wells and incubated for 2 hours at room
temperature. After washing with PBS plus 0.3% triton, secondary
antibodies were applied, and wells were incubated for 1 hour at
room temperature. After washing with PBS plus triton, mounting
medium (Vectashield, Vector Laboratories, Burlingame, Calif.) was
added to the wells. Epifluorescence images for each protein at the
same time point were photographed at the same exposure to determine
relative differences in the amount of deposited protein using an
inverted microscope equipped with the appropriate fluorescein and
rhodamine filters (10.times. neoflaur objectives, Axiovert, Carl
Zeiss, Thornwood, N.Y.). Following immunostaining photography, ECM
was stained with 0.1% Ponceau S (Sigma-Aldrich) for 10 minutes at
room temperature and photographed using a 32.times. phase
objective.
[0222] Bruch's membrane: At day-21, live RPE were imaged by calcein
staining (Live/Dead viability/toxicity assay, Molecular Probes,
Eugene, Oreg.) to determine surface coverage by cells. Following a
brief rinse in PBS, explants were incubated in 2 .mu.M calcein for
1 hour at room temperature. Explants were rinsed briefly, then
photographed at 2.5.times. magnifications using a fluorescence
microscope equipped with a fluorescein filter set (Axiophot, Carl
Zeiss). Montages of calcein-imaged explants were created in
Photoshop CS4 (Adobe Systems, Mountain View, Calif.). After calcein
imaging and RPE removal by 5 minute incubation in 0.02M NH4OH,
explants were fixed in 4% paraformaldehyde for 1 hour at 4.degree.
C. Following washing in PBS, explants were blocked at room
temperature for 45 minutes. The explants were then bisected prior
to immunostaining, and the surface of Bruch's membrane was
immunostained for laminin and collagen IV (one half) and
fibronectin and laminin (other half) or cut into thirds with the
third piece used for controls. Primary antibodies were applied to
explants, which were then incubated overnight at 4.degree. C. The
following day, explants were rinsed with PBS, and secondary
antibodies were applied. Explants were incubated for 2 hours at
room temperature following by washing with PBS. Explants were
stored and examined in mounting medium. Single images or z-stacks
were acquired from the surface of Bruch's membrane using a
40.times. water immersion lens on a confocal microscope (LSM510,
Carl Zeiss, Thornwood, N.Y.). Lasers lines and corresponding
emission filters were: 488 nm excitation, 505-530 nm band pass
filter for FITC; 543 nm excitation, 560-615 nm band pass filter for
rhodamine. Following confocal microscopy evaluation, explants were
processed for SEM.
Results
Effect of BCEC-CM on Long-Term Cell Survival on Aged and AMD
Bruch's Membrane
[0223] RPE derived from human embryonic stem cells (hES-RPE), fetal
RPE, and aged adult RPE were seeded onto the inner collagenous
layer of submacular Bruch's membrane of donor eyes age 62 and older
(see Table 9 below for donor information) and cultured in BCEC-CM
or CM vehicle (representative images, FIGS. 23-27). On explants
cultured in CM vehicle, limited resurfacing was seen at day-21 with
no or few surviving cells on the majority of explants (hES-RPE, 3
of 6 total explants resurfaced with few cells, and no cells on the
remaining 3 explants; fetal RPE, 20 explants with no or few cells
of 22 total explants; adult RPE, 5 explants with no or few single
cells of 7 total explants) (FIGS. 23-27, A-C). When present, intact
cells were large and flat, regardless of cell type (FIGS. 24C,
27B). Cytoplasmic vacuoles were common. All explants cultured in
BCEC-CM showed cells remaining at day-21 with many explants almost
fully (i.e., more than 75% of the surface covered by cells) or
fully resurfaced (hES-RPE, 3 explants completely or almost fully
resurfaced of 6 total explants; fetal RPE, 16 explants completely
or almost fully resurfaced of 22 total explants; adult RPE, 6
explants almost fully resurfaced of 7 total explants). hES-RPE
(FIGS. 23 E-G) cells were predominantly monolayered with highly
variable morphology and were larger and flatter than fetal RPE
(FIGS. 24-26, E-G). Fetal RPE showed focal areas of bi- and
multilayers with the most extensive multilayering found in explants
that underwent CNV removal prior to cell seeding (FIG. 25E).
Resurfacing was extensive and many cells were compact with abundant
expression of well-developed surface apical processes regardless of
submacular pathology (FIGS. 22-26E, insets). Adult RPE, generally
larger than fetal RPE, were predominantly monolayered with
localized multilayered clumps of cells (FIGS. 27E-G). Adult RPE
exhibited abundant short apical processes (FIG. 27E). Since adult
RPE were from primary cultures, the cells were generally more
pigmented than hES-RPE or fetal RPE (FIG. 27G).
[0224] The nuclear density of hES-RPE cells grown in BCEC-CM (mean,
20.1 nuclei per mm Bruch's membrane) was significantly higher than
that of cells grown in CM vehicle (mean, 5.1 nuclei per mm Bruch's
membrane) (P=0.031). Fetal RPE nuclear density after culture in
BCEC-CM (mean, 20.6 nuclei per mm Bruch's membrane) was
significantly higher than cells cultured in CM vehicle (mean, 2.2
nuclei per mm Bruch's membrane) (P<0.001). Adult RPE cultured in
BCEC-CM nuclear density (mean, 10.0 nuclei per mm Bruch's membrane)
was also significantly higher than the nuclear density of cells
cultured in CM vehicle (mean, 1.2 nuclei per mm Bruch's membrane)
(P=0.016). (See FIG. 28A.) ANOVA on ranks showed significant
differences in the median values among the cell types cultured in
BCEC-CM (P=0.004). Nuclear densities of fetal RPE and hES-RPE
cultured in BCEC-CM were significantly higher than the nuclear
density of adult RPE cultured in BCEC-CM (P<0.05). Fetal RPE and
hES-RPE nuclear densities were not significantly different when
cultured in BCEC-CM (P>0.05). Nuclear densities of hES-RPE,
fetal RPE, and cultured adult RPE were not significantly different
when cultured in CM vehicle only (P=0.060). There were no
statistically significant differences in ages of donor eye explants
between groups (P=0.345: hES-RPE mean donor age, 80.2.+-.8.4 yrs.;
fetal RPE mean donor age, 80.2.+-.7.8 yrs.; aged adult RPE mean
donor age, 75.7.+-.3.64 yrs.). It was assessed whether RPE survival
on age-matched AMD vs. non-AMD Bruch's membrane was similar in the
presence of BCEC-CM (FIG. 28B). Explants seeded with fetal RPE on
aged Bruch's membrane from eyes without significant AMD changes
(including donor eyes with submacular focal RPE hyerplasia and few
small (<100 .mu.m) drusen were compared to explants seeded on
AMD Bruch's membrane. AMD donors included donor eyes with CNVs
(removed prior to cell seeding with no subsequent debridement),
geographic atrophy, and/or extensive large (.gtoreq.125 .mu.m)
drusen. After culture in BCEC-CM, non-AMD donor eye mean nuclear
density was 19.6 nuclei/mm Bruch's membrane, and AMD donor eye mean
nuclear density was 21.3. After culture in CM vehicle, non-AMD
donor eye mean nuclear density was 2.4 nuclei/mm Bruch's membrane,
and AMD donor eye mean nuclear density was 2.0. The differences in
fetal RPE nuclear density on Bruch's membrane in the presence of
BCEC-CM vs. CM vehicle were statistically significant for both
non-AMD and AMD donors (non-AMD, P=0.004; AMD, P<0.001). For a
given culture medium, the nuclear densities of fetal RPE on non-AMD
vs. AMD explants were not significantly different (culture in
BCEC-CM, P=0.548; culture in CM vehicle, P=0.231). Ages of the two
groups were not statistically different (P=0.226: aged, non-AMD
mean donor age, 77.8.+-.5.0 yrs.; AMD mean donor age, 82.2.+-.9.3
yrs.).
RPE Cell Survival Following Different BCEC-CM Culture Times
[0225] To determine whether RPE behavior on submacular Bruch's
membrane explants depends on the time of exposure to BCEC-CM,
explants seeded with fetal RPE and cultured for differing periods
of time in BCEC-CM were compared to fellow eye explants cultured
for the entire incubation period (21 days) in BCEC-CM. One explant
of the pair was cultured in BCEC-CM for 3-, 7-, or 14-days followed
by culturing in CM vehicle to bring the total number of days in
culture to 21 (FIG. 29). Fellow eye explants were cultured in
BCEC-CM for the entire 21-day period. Fetal RPE nuclear densities
on explants cultured for 3 days in BCEC-CM (mean nuclear density,
3.5 nuclei/mm Bruch's membrane) vs. 21 days in BCEC-CM (mean
nuclear density, 21.8) were significantly different (P=0.016).
Nuclear densities on explants after 7-day BCEC-CM culture (mean
nuclear density, 10.9 nuclei/mm Bruch's membrane) vs. 21 days (mean
nuclear density, 28.0) were significantly different (P=0.008).
Nuclear densities after 14-day BCEC-CM culture (mean nuclear
density, 17.0 nuclei/mm Bruch's membrane) vs. 21-day BCEC-CM
culture (mean nuclear density, 27.9) were significantly different
(P=0.031). Nuclear densities of explants cultured for 14 days in
BCEC-CM were significantly higher than those of explants cultured
for 3 days in BCEC-CM (P<0.05) while 3- vs. 7-day and 7- vs.
14-day nuclear densities were not significantly different
(P>0.05). There were no statistically significant differences
between the control groups cultured for the entire 21-day period in
BCEC-CM (P=0.074). Ages between groups were not significantly
different (P=0.881: 3-day cohort, mean donor age, 78.3.+-.7.6 yrs.;
7-day cohort, mean donor age, 76.5.+-.6.0 yrs.; 14-day cohort, mean
donor age, 77.2.+-.7.1 yrs.)
Comparison of RPE Nuclear Density after 21-Day Culture in Different
Media and on Different Surfaces
[0226] Nuclear densities of fetal RPE after 21-day culture in
BCEC-CM or CM vehicle on AMD (including late AMD) and aged, non-AMD
explants (current study: CM vehicle, mean donor age, 80.2.+-.7.8
yrs. (data represented in FIG. 28A); BCEC-CM mean donor age,
78.8.+-.7.3 yrs. (combined data represented in FIGS. 28A and 21-day
controls of FIG. 29)), were compared with: 1) 21-day fetal RPE
nuclear densities on young explants (mean donor age, 44.8.+-.2.3
yrs.) cultured in RPE medium, 2) aged and early AMD Bruch's
membrane explants (mean donor age, 73.6.+-.6.4 yrs.) cultured in
RPE medium, and 3) BCEC-ECM-resurfaced aged Bruch's membrane (mean
donor age, 73.9.+-.7.4 yrs.) cultured in RPE medium (FIG. 30). Of
the aged explants studied, mean donor age of explants cultured in
BCEC-CM and CM vehicle (includes eyes with early as well as late
AMD) were significantly higher than the mean donor age of aged,
including early AMD, explants cultured in RPE media (P<0.05) but
not significantly different from the mean donor age of explants
that were resurfaced with BCEC-ECM (aged, non-AMD) (P>0.05). The
nuclear densities of fetal RPE on BCEC-ECM-resurfaced aged Bruch's
membrane (mean nuclear density, 23.2 nuclei/mm Bruch's membrane)
and unresurfaced young Bruch's membrane (mean nuclear density,
27.2) were significantly higher than the nuclear density on aged,
untreated Bruch's membrane after culture in RPE medium (mean
nuclear density, 11.221) (P<0.05) and were not significantly
different from the nuclear density on explants cultured in BCEC-CM
(mean nuclear density, 23.2) (P>0.05). Nuclear densities of
fetal RPE cells in these three conditions (i.e., BCEC-CM-cultured,
BCEC-ECM-resurfaced, and young Bruch's membrane) were significantly
higher than the nuclear density on aged and early AMD Bruch's
membrane cultured in RPE medium (mean nuclear density, 2.2)
(P<0.05). Nuclear densities of cells cultured in RPE medium on
aged and early AMD Bruch's membrane were significantly higher than
nuclear densities of cells cultured in CM vehicle on aged and AMD
Bruch's membrane (P<0.05).
ECM Deposition Following Culture in BCEC-CM
[0227] Providing a newly-deposited ECM on the surface of Bruch's
membrane can significantly improve cell survival in long-term organ
culture, and the resulting nuclear densities are similar to those
observed after culture in BCEC-CM (FIG. 30). To determine whether
incubation in BCEC-CM stimulates ECM deposition, which might
account for the cell-preserving effect of BCEC-CM, it was
investigated whether RPE ECM deposition on Bruch's membrane was
increased after culture in BCEC-CM. Since little RPE cell survival
on Bruch's membrane was seen in CM vehicle, assays were performed
to compare ECM deposition after culture in BCEC-CM with ECM
deposition after culture in standard RPE culture medium where some
degree of RPE resurfacing was more likely (see FIG. 30). To assess
ECM deposition on a non-toxic substrate, ECM deposition onto tissue
culture dishes was examined at days-7, -14, and -21 (N=3).
[0228] Stained fibers were present on the surface of culture dishes
in both media at all three time points. As the time in culture
increased, the amount of ECM deposition increased. ECM visualized
by Ponceau S staining showed deposition after BCEC-CM culture to be
more extensive than deposition after RPE medium culture at all time
points (FIG. 31). In BCEC-CM cultures, collagen IV and laminin were
present as a thick coating with defects that became smaller with
time in culture (FIGS. 32-34). Collagen IV deposition appeared to
be more extensive than laminin and fibronectin deposition at day-7.
At day-14 and -21, collagen IV and laminin showed more uniform
coating of the culture dish surface compared to day-7. Collagen IV
and laminin appeared to be extensively co-localized at all three
time points (FIGS. 32-34, C). Fibronectin labeling was on a network
of thin fibers at week-1 with diffuse labeling of the culture dish
between fibers seen at day-14 and -21. Some colocalization of
fibronectin with laminin was seen (FIGS. 32-34, F), but it was not
as extensively co-localized as laminin was with collagen IV. In RPE
medium cultures (FIGS. 32-34, G-L), collagen IV and laminin
labeling at day-7 was not as extensive as that seen in BCEC-CM
cultures with labeling seen as an open mesh with localized areas
coating the culture dish between the fibers of the mesh. Localized
areas of surfaces coated with collagen IV and laminin were more
extensive at day-14 and -21 compared to day-7, but there was
little, if any, increase in labeling between day-14 and -21. Faint
fibronectin labeling was detected in RPE medium cultures at all
time points with sparse to moderate labeling seen at day-21.
Similar to that observed in BCECCM cultures, collagen IV and
laminin showed extensive co-localization. Fibronectin
co-localization was difficult to determine due to sparse labeling
in some cultures, but it did seem to partially co-localize with
laminin (see FIG. 32L). Controls showed diffuse, faint fluorescence
with rhodamine filters with secondary antibody only in the 3-week
RPE medium cultures. No detectable fluorescence was found with FITC
or rhodamine filters in preparations at other time points or in CM
vehicle cultures at all time points (not shown). Controls in which
the antibody was omitted showed no fluorescence with either filter
set (not shown). Calcein imaging confirmed the presence of RPE on
Bruch's membrane explants cultured in both media for 21 days with
more extensive resurfacing and smaller, more compact cells on
explants cultured in BCEC-CM (FIGS. 35A and L). On explants
cultured in BCEC-CM, SEM and confocal evaluation showed extensive
ECM on the surface of the inner collagenous layer for all three
markers (N=6; mean donor age, 79.2.+-.3.17 yrs) (FIGS. 35A-F and
H-J). The extent and complexity of the ECM varied within and
between BCEC-CM-treated explants, ranging from a complex mesh of
thick and thin fibers to a fairly continuous ECM sheet with a rough
surface (FIGS. 35A-F and H-J). Collagen IV and laminin labeling
were found as thick and thin fibers with some localized thickening
giving the labeling a punctate appearance. Both collagen IV and
laminin formed a continuous sheet in localized areas, visualized
between fibers. Collagen IV labeling was more variable than that of
fibronectin and laminin, sometimes not as extensive. There was some
co-localization of collagen IV with laminin. Fibronectin was found
mainly in fibers and did not appear to co-localize with laminin. On
explants cultured in RPE medium, no or sparse localized labeling
was observed despite RPE presence on Bruch's membrane as visualized
by Calcein imaging (FIGS. 35 L and M). When present, laminin
labeling could be seen on short strands. If present, fibronectin
was present in short (thin) strands; sometimes labeling was
punctate. Collagen IV labeling generally was sparse or not present.
ECM tended to be more prevalent outside the submacular area on the
periphery of the explant. SEM evaluation of the explants showed
predominantly bare ICL or a few strands on the surface of the bare
ICL in explants cultured in RPE medium (FIG. 35M). Control explant
pieces (omission of primary antibody or both primary and secondary
antibodies) showed slight autofluorescence of the tissue (FIGS. 35G
and K) at the microscope settings used to obtain immunolabeling
images.
[0229] In previous studies examining fetal RPE attachment to aged
and AMD submacular Bruch's membrane, it was showed that RPE can
attach to a high degree to the RPE basement membrane and to the
inner collagenous layer, indicating that attachment to these layers
may not be the limiting factor in cell transplant success.
Immunochemistry studies showed that at days-3 and -7 after seeding,
fetal RPE are present on Bruch's membrane and appear to be fairly
healthy based on the appearance of stained nuclei. Between days-7
and -14, a high degree of cell death has occurred, and additional
cell death was observed at days-14 and -21. At these later time
points, abundant condensed and fragmented nuclei are present. These
studies provide evidence that a method to ensure cell survival (vs.
attachment alone) must be developed for RPE cell replacement
therapy to be successful in AMD patients.
[0230] Previously, it was demonstrated that cell survival on aged
submacular Bruch's membrane can be enhanced greatly by resurfacing
Bruch's membrane with a cell-deposited ECM. This resurfacing
strategy was chosen because ECM deposited by BCECs supports rapid
fetal RPE attachment and growth in cell culture. It was showed that
resurfacing aged Bruch's membrane with BCEC-ECM improved long-term
cell survival significantly (>200%). A limitation to the
feasibility of utilizing this ECM in clinical applications was the
insolubility of the ECM and the resulting low yield of proteins
with ECM harvest, both for transfer to Bruch's membrane and for
quantitative analysis. In the present study, BCEC-CM was harvested
from BCECs cultured under conditions similar to those that yield
BCEC-ECM-coated culture dishes and BCEC-ECM deposition on Bruch's
membrane. The rationale for this choice was based on the reported
abundance of potentially cell-supporting proteins secreted into the
medium, such as ECM and ECM-associated molecules. In the present
study, it was demonstrated that cell survival is greatly enhanced
when RPE cells are exposed to this BCEC-CM in long-term culture
and, importantly, that cell survival is enhanced on submacular
Bruch's membrane of AMD eyes. It was showed previously that RPE
survival on AMD submacular Bruch's membrane explants was severely
impaired after culture in RPE medium. However, the nuclear density
of fetal RPE on submacular human Bruch's membrane cultured in RPE
medium is significantly higher compared to the nuclear density of
fetal RPE cultured in CM vehicle (FIG. 30). This difference could
be related to the significantly lower mean donor age of the
explants cultured in RPE media and, therefore, possibly fewer aged
and AMD changes to Bruch's membrane. However, culture on non-AMD
and AMD explants was similarly poor in CM vehicle. It seems likely
that the serum in the RPE medium aided in supporting cell survival
to a slight degree but not to the degree seen in BCEC-CM.
[0231] BCEC-CM may supply soluble matrix proteins for ECM
deposition, stimulate ECM deposition, and/or stimulate the RPE in
some other fashion, thus allowing better survival that could lead
to increased ECM deposition on aged and AMD submacular Bruch's
membrane. The presence of increased ECM deposition under the cells
cultured in BCEC-CM compared to cells cultured in RPE medium may
reveal a mechanism by which cell survival is enhanced, perhaps in
the same manner that BCEC-ECM resurfaced explants support long-term
cell survival on submacular human Bruch's membrane. It is not known
if the ECM deposition per se enabled cell survival in the same way
as resurfacing Bruch's membrane with BCEC-ECM or if the ECM
deposition was a reflection of long-term survival of the cells by
another mechanism. When RPE cell survival is observed on explants
cultured in RPE medium, the cells are not as differentiated as
those cultured in BCECCM and have not deposited ECM to any degree.
The degree of ECM deposition by RPE on culture dishes in RPE medium
or in BCEC-CM is much greater than that observed on Bruch's
membrane explants. This difference may arise because RPE appear to
mature more slowly on Bruch's membrane, and a certain degree of
maturity must be obtained before ECM deposition can occur.
Differences in the amount and composition of ECM deposition may
also be related to differences in cellular response to the
underlying substrate. Since the antibodies used in this study were
not human-specific, it was not known if deposited proteins
originated from the BCEC-CM (bovine proteins) or from protein
synthesis by the RPE or both. As mentioned previously, when
cultured in standard RPE medium, fetal RPE can survive to a high
degree up to 7 days in organ culture on submacular human Bruch's
membrane. After day-7, survival is impaired with decreasing
presence of cells as time in culture increases further. The
necessity of long-term presence of BCEC-CM to assure cell survival
is consistent with the notion that sustained cell stimulation is a
factor in assuring cell survival. The RPE survival after culture in
BCEC-CM as measured by nuclear density was statistically similar to
that of cells cultured on BCEC-ECM and also similar to that
observed on young Bruch's membrane, the latter both cultured in RPE
medium (FIG. 30). The nuclear densities observed in these studies
are much lower than the nuclear densities of submacular in situ RPE
even when comparing age matched nuclear densities and also much
lower than the nuclear densities of fetal RPE in culture. The lower
nuclear density on submacular Bruch's membrane after culture in
BCEC-CM is partly due to the existence of defects in surface
coverage on some explants. Many explants are almost fully
resurfaced or are fully resurfaced after culture in BCEC-CM, and
many cells appear to show some morphological features of
differentiation (e.g., apical processes, tight junctions). However,
there is variability in cell size with some cells fairly large,
especially compared to the size of cultured fetal RPE. One source
of the variability in cell behavior between explants might be
biological variability in the composition of BCEC-CM between
batches, as some batches appeared to be less effective than others,
showing more and larger defects in surface coverage and larger and
flatter cells. Another source of variability in cell behavior
arises from differences in cell survival on localized areas of
Bruch's membrane within explants, as some explants showing
excellent overall resurfacing also exhibited small localized
defects in RPE coverage (e.g., FIGS. 24 and 26). Lastly,
particularly in reference to the AMD cohort, variability in cell
behavior on explants with CNVs is likely related, at least in part,
to the differences in the Bruch's membrane surface following CNV
removal since no additional debridement was performed. The degree
of damage to Bruch's membrane or preservation/removal of deposits
on Bruch's membrane following CNV removal were highly variable
within and between donor eyes. In a previous study, hES-RPE were
shown to have the potential to survive on equatorial and submacular
Bruch's membrane to a similar degree as fetal RPE after day-21
culture in RPE medium. On submacular Bruch's membrane, the survival
was poor for both cell types with hES-RPE survival impaired at
earlier times in culture than fetal RPE. In these previous studies,
hES-RPE was from frozen stock, which is a possible cause for the
difference in initial cell survival since the fetal RPE were from
fresh cultures. However, in the current study, hES-RPE were from
fresh stock, and although the nuclear densities of hES-RPE cultured
in BCEC-CM were similar to those of fetal RPE, hES-RPE in general
were very flat and not differentiated to the same degree as fetal
RPE on submacular Bruch's membrane. These results imply that
hES-RPE may take longer to acquire mature RPE features on Bruch's
membrane compared to fetal RPE, consistent with behavior observed
in cell culture. Whether fetal RPE or hES-RPE can achieve size and
differentiation features found in cell culture or in situ and
whether the cells can perform RPE functions are future studies that
must be considered.
[0232] Cultured aged adult RPE were generally larger than hES-RPE
and fetal RPE in cell culture and on Bruch's membrane. On Bruch's
membrane, most adult RPE showed well developed apical processes
even in very large flat cells, but their survival in general was
not as good as that of fetal RPE or hES-RPE. The nuclear density of
cultured adult RPE after BCEC-CM culture (10.0.+-.0.95) was not
significantly different (P=0.887) from the nuclear density of fetal
RPE after RPE medium culture (11.2.+-.1.7, FIG. 30). These results
indicate that while culture in BCEC-CM significantly enhances RPE
cell survival on aged Bruch's membrane, aged adult RPE may not be
the best choice for cell transplantation especially when compared
to the resurfacing achieved by fetal RPE and hES-RPE.
[0233] There is no other technique known that promotes such robust
RPE survival on submacular AMD Bruch's membrane (including eyes
with geographic atrophy and CNVs). 19, 20 Identification of the
critical components of this BCEC-CM and RPE function testing are
the next steps in the development of a surgically usable adjunct to
improve RPE survival and differentiation on submacular human AMD
Bruch's membrane.
TABLE-US-00009 TABLE 9 Donor information SI Table 1. Donor
information Submacular Pathology Donor Age Ethnicity/sex CM
cultured CM Vehicle cultured A. hES-RPE on AMD and non-AMD Bruch's
membrane (mean age 80.17 .+-. 3.41 yrs.) 68 CF Few mixed size
drusen Normal 74 CF Confluent soft drusen Soft drusen 80 CM Normal
Normal 83 CM RPE hyperpigmentation, mixed size RPE
hyperpigmentation, mixed drusen size drusen 84 CF Normal Normal 92
CF Confluent soft drusen Confluent soft drusen B. Fetal RPE on
non-AMD Bruch's membrane (mean age 77.8 .+-. 1.69 yrs.) 67 CM Few
mixed size drusen Few Hard drusen 75 CM Focal RPE hyperpigmentation
Normal 76 CM Normal Normal 77 CM Normal Normal 78 CM Normal Normal
79 CM Few hard drusen Few hard drusen 81 CM Few hard drusen Normal
83 CM Few hard drusen Few hard drusen 84 CF Few mixed size drusen
Few mixed size drusen C. Fetal RPE on AMD Bruch's membrane (mean
age 81.9 .+-. 2.49 yrs) 62 CM Soft drusen Soft drusen 75 CF Large
CNV Large CNV 76 CF Confluent soft drusen Confluent soft drusen 79
CF CNV, focal RPE hyperpigmentation, focal RPE hyperpigmentation
soft drusen 79 CF CNV Few hard drusen, focal RPE hyperpigmentation
79 CF Large CNV CNV 81 CF CNV, (Fuch`s dystrophy donor) Mixed size
drusen 82 CF Extrafoveal GA, soft drusen Extrafoveal GA, soft
drusen 86 CM Extensive soft drusen, small CNV Extensive soft
drusen, small CNV 86 CF CNV CNV 90 CM Large CNV Large CNV 92 CF GA
with central RPE preservation GA with large, calcified drusen 98 CM
Confluent soft drusen Confluent soft drusen D. Adult RPE on AMD and
non-AMD Bruch's membrane (mean age 75.7 .+-. 1.38 yrs.) 71 CF
Extrafoveal GA, soft drusen Unknown 73 CF Unknown (poor RPE
preservation) Unknown 73 CM Normal Normal 75 CF Normal Few hard
drusen 78 CF Normal Normal 80 CM Cluster of soft drusen Few
intermediate size drusen 80 CM Extrafoveal drusen including
calcified Few hard drusen drusen, macula normal There was no
statistically significant difference in ages of Bruch's membrane
between groups (hES-RPE vs. combined AMD and non-AMD fRPE vs. adult
RPE, One Way ANOVA P = 0.354). For AMD vs non-AMD, normal, there
was no significant difference in the ages of the two groups
(unpaired t-test, P = 0.226).
[0234] All publications cited in the specification, both patent
publications and non-patent publications, are indicative of the
level of skill of those skilled in the art to which this invention
pertains. All these publications are herein fully incorporated by
reference to the same extent as if each individual publication were
specifically and individually indicated as being incorporated by
reference.
[0235] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the following claims.
* * * * *
References