U.S. patent application number 17/486855 was filed with the patent office on 2022-04-21 for abcb5(+) stem cells for treating ocular disease.
This patent application is currently assigned to The United States of America as represented by the Department of Veterans Affairs. The applicant listed for this patent is Children's Medical Center Corporation, The United States of America as represented by the Department of Veterans Affairs, Schepens Eye Research Institute, The United States of America as represented by the Department of Veterans Affairs. Invention is credited to Markus H. Frank, Natasha Y. Frank, Paraskevi Evi Kolovou, Bruce Ksander.
Application Number | 20220118024 17/486855 |
Document ID | / |
Family ID | 1000006062438 |
Filed Date | 2022-04-21 |
View All Diagrams
United States Patent
Application |
20220118024 |
Kind Code |
A1 |
Frank; Markus H. ; et
al. |
April 21, 2022 |
ABCB5(+) STEM CELLS FOR TREATING OCULAR DISEASE
Abstract
Various aspects and embodiments of the present invention are
directed to methods of treating a subject having an ocular
condition, methods of isolating ocular stem cells, methods of
selecting and/or producing ocular grafts for transplantation, and
methods of promoting ocular cell regeneration as well as to grafts
and preparations containing isolated ocular stem cells
characterized by the expression of ABCB5 on their cell surface.
Inventors: |
Frank; Markus H.;
(Cambridge, MA) ; Frank; Natasha Y.; (Cambridge,
MA) ; Ksander; Bruce; (Boston, MA) ; Kolovou;
Paraskevi Evi; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as represented by the Department of
Veterans Affairs
Schepens Eye Research Institute
Children's Medical Center Corporation |
Washington
Boston
Boston |
DC
MA
MA |
US
US
US |
|
|
Assignee: |
The United States of America as
represented by the Department of Veterans Affairs
Washington
DC
Schepens Eye Research Institute
Boston
MA
Children's Medical Center Corporation
Boston
MA
|
Family ID: |
1000006062438 |
Appl. No.: |
17/486855 |
Filed: |
September 27, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15796920 |
Oct 30, 2017 |
11129854 |
|
|
17486855 |
|
|
|
|
14768885 |
Aug 19, 2015 |
9801912 |
|
|
PCT/US2014/017076 |
Feb 19, 2014 |
|
|
|
15796920 |
|
|
|
|
61766424 |
Feb 19, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0668 20130101;
A61L 2430/16 20130101; A61K 9/0051 20130101; C12N 5/0621 20130101;
A61L 27/3834 20130101; A61L 27/3839 20130101; A61K 35/30 20130101;
C12N 5/0623 20130101 |
International
Class: |
A61K 35/30 20060101
A61K035/30; C12N 5/079 20060101 C12N005/079; C12N 5/0775 20060101
C12N005/0775; A61K 9/00 20060101 A61K009/00; A61L 27/38 20060101
A61L027/38; C12N 5/0797 20060101 C12N005/0797 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
5R01CA113796 awarded by the National Institutes of Health and the
National Cancer Institute. The government has certain rights in the
invention.
Claims
1. A method of treating a subject having an ocular condition,
comprising administering to the subject isolated ABCB5(+) ocular
stem cells in an amount effective to regenerate ocular cells in the
subject.
2-7. (canceled)
8. The method of claim 1, wherein the isolated ABCB5(+) ocular stem
cells are administered as an ocular graft, wherein the ocular graft
comprises an artificial cornea comprised of the ABCB5(+) ocular
stem cells.
9. The method of claim 1, wherein the isolated ABCB5(+) ocular stem
cells are allogeneic stem cells.
10. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are syngeneic stem cells.
11. (canceled)
12. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are ABCB5(+) limbal stem cells.
13-30. (canceled)
31. A method of producing an ocular graft for transplantation to a
subject, comprising seeding a substrate with isolated ocular
ABCB5(+) stem cells to produce the ocular graft.
32. The method of claim 31, wherein the isolated ABCB5(+) ocular
stem cells are allogeneic stem cells.
33-38. (canceled)
39. The method of claim 31, wherein the substrate comprises fibrin
gel, amniotic membrane, aminoglycans, or a combination thereof.
40. An ocular graft enriched with isolated ABCB5(+) stem cells for
transplantation in a subject.
41. The ocular graft of claim 40, wherein the isolated ABCB5(+)
stem cells are allogeneic stem cells.
42. The ocular graft of claim 40, wherein the isolated ABCB5(+)
stem cells are syngeneic stem cells.
43. The ocular graft of claim 40, wherein the isolated ABCB5(+)
stem cells are ABCB5(+) ocular stem cells.
44-56. (canceled)
57. An in vitro substrate, comprising an artificial cornea
comprised of ABCB5(+) ocular stem cells.
58. The substrate of claim 57, wherein the ABCB5(+) ocular stem
cells are limbal stem cells.
59. The substrate of claim 57, wherein the ABCB5(+) ocular stem
cells are retinal stem cells.
60. The substrate of claim 57, wherein the ABCB5(+) ocular stem
cells are human eye cells.
61. The substrate of claim 57, wherein the ABCB5(+) ocular stem
cells are central corneal cells.
62. The substrate of claim 57, wherein at least 1.0% of the total
cell population is ABCB5(+).
63. The substrate of claim 57, wherein at least 3.0% of the total
cell population is ABCB5(+).
64. substrate of claim 57, wherein at least 5.0% of the total cell
population is ABCB5(+).
65. substrate of claim 57, wherein at least 10.0% of the total cell
population is ABCB5(+).
66. substrate of claim 57, wherein the in vitro substrate comprises
acellular collagen.
67. substrate of claim 57, wherein the in vitro substrate is in a
culture medium.
68. substrate of claim 67, wherein the culture medium comprises
salts, buffers, and amino acids.
69. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are administered in a cell population and at least 1.0%
of the total cell population is ABCB5(+).
70. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are administered in a cell population and at least 3.0%
of the total cell population is ABCB5(+).
71. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are administered in a cell population and at least 50.0%
of the total cell population is ABCB5(+).
72. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are administered in a cell population and at least 90.0%
of the total cell population is ABCB5(+).
73. The method of claim 1, wherein the isolated ABCB5(+) ocular
stem cells are administered in a cell population and at least 95.0%
of the total cell population is ABCB5(+).
74. The method of claim 31, wherein the ABCB5+ ocular stem cells
are separated from other cells with an anti-ABCB5 antibody.
75. The method of claim 31, wherein the ABCB5+ ocular stem cells
are separated from other cells with flow cytometry.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application which claims
benefit under 35 U.S.C. .sctn. 120 of U.S. application Ser. No.
14/768,885, filed on Aug. 19, 2015, which is a national stage
filing under U.S.C. .sctn. 371 of PCT International Application
PCT/US2014/017076, entitled "ABCB5(+) STEM CELLS FOR TREATING
OCULAR DISEASE" filed on Feb. 19, 2014, which claims priority under
35 U.S.C. .sctn. 119(e) of U.S. provisional application No.
61/766,424, filed Feb. 19, 2013, which are herein incorporated by
reference in their entirety.
BACKGROUND OF INVENTION
[0003] Limbal stem cells have been identified as slow-cycling,
label-retaining cells in mice. Limbal stem cells express the
nuclear transcription factor .DELTA.Np63.alpha. in humans and lack
expression of corneal epithelial differentiation markers such as
KRT12 [2,8] (FIG. 1A). Limbal stem cells generate transient
amplifying cells, which express the eye development master
regulator PAX6 [9] and, during corneal development and
regeneration, migrate out of the limbus to give rise to the
KRT12(+) central corneal epithelium [10].
SUMMARY OF INVENTION
[0004] The present invention, in some aspects, is directed
generally to the use of ABCB5(+) stem cells (e.g., human stem
cells) for the treatment of ocular conditions such as, for example,
corneal diseases and/or retinal diseases. The invention is based,
in part, on the discovery that ABCB5 is expressed in stem cells of
the eye, such ABCB5(+) stem cells are required for normal eye
development, and when administered to subjects having ocular wounds
(e.g., ocular surface wounds), these cells are capable of cell
regeneration. For example, ABCB5(+) limbal stem cells, required for
normal corneal development, are capable of corneal regeneration.
Similarly, ABCB5(+) retinal pigment epithelium (RPE) cells,
required for normal retinal development, are capable of retinal
regeneration.
[0005] Thus, in some aspects of the invention, provided herein are
methods of treating a subject having an ocular condition,
comprising administering to the subject isolated ABCB5(+) stem
cells in an amount effective to regenerate ocular cells in the
subject.
[0006] In some embodiments, the ocular condition is a corneal
disease. In some embodiments, the corneal disease is blindness due
to limbal stem cell deficiency (LSCD). "Limbal stem cell
deficiency" herein refers to severe or total, unilateral or partial
LSCD [5]. In some embodiments, isolated ABCB5(+) limbal stem cells
are administered to a subject to treat a corneal disease.
[0007] In some embodiments, the ocular condition is a retinal
disease. In some embodiments, the retinal disease is macular
degeneration. In some embodiments, the retinal disease is
retinitis. In some embodiments, isolated ABCB5(+) retinal stem
cells (e.g., ABCB5(+) RPE stem cells) are administered to a subject
to treat a corneal disease.
[0008] In some embodiments, the ocular condition is an ocular
wound.
[0009] In some embodiments, the isolated ABCB5(+) stem cells are
administered as an ocular graft. In some embodiments, the ocular
grafts contain one to about 10.sup.7 isolated ABCB5(+) stem cells.
In some embodiments, more than 10.sup.7 isolated ABCB5(+) stem
cells may be administered as an ocular graft.
[0010] In other aspects of the invention, provided herein are
methods of isolating limbal stem cells from a mixed population of
ocular cells, the methods comprising providing a mixed population
of ocular cells and isolating ABCB5(+) limbal stem cells from the
mixed population.
[0011] In yet other aspects of the invention, provided herein are
methods of identifying the number of ABCB5(+) limbal stem cells in
the ocular graft, comparing the number of ABCB5(+) limbal stem
cells to the total cell population of the graft, and based on the
comparison, selecting the ocular graft for transplantation.
[0012] In some embodiments, the methods comprise contacting cells
of the mixed population with an antibody that selectively binds to
human ABCB5.
[0013] In still other aspects of the invention, provided herein are
methods of producing ocular grafts for transplantation to a
subject, the methods comprising seeding a substrate with isolated
ABCB5(+) stem cells to produce the ocular graft.
[0014] In some embodiments, the substrate comprises fibrin gel,
amniotic membrane, aminoglycans, or a combination thereof. In some
embodiments, the substrate is an artificial cornea. In such
embodiments, the substrate, for example, an artificial cornea,
comprises acellular collagen.
[0015] In some aspects of the invention, provided herein are ocular
grafts enriched with isolated ABCB5(+) stem cells for
transplantation in a subject.
[0016] In still other aspects of the invention, provided herein are
methods of promoting ocular cell regeneration, comprising
identifying limbal stem cells as ABCB5(+) limbal stem cells and
administering to a subject in need thereof the ABCB5(+) limbal stem
cells in an amount effective to promote ocular cell
regeneration.
[0017] In further aspects of the invention, provided herein are
isolated preparations of limbal stem cells characterized by the
expression of ABCB5 on the cell surface.
[0018] In some embodiments, the isolated ABCB5(+) limbal stem cells
are administered as an ocular graft.
[0019] In some embodiments, the subject is administered one to
about 10.sup.7 isolated ABCB5(+) limbal stem cells by grafting.
[0020] In some embodiments, the isolated ABCB5(+) stem cells are
isolated ABCB5(+) human stem cells.
[0021] In some embodiments, the isolated ABCB5(+) stem cells are
allogeneic stem cells. In some embodiments, the isolated ABCB5(+)
stem cells are syngeneic stem cells.
[0022] In some embodiments, the isolated ABCB5(+) stem cells are
ABCB5(+) ocular stem cells. In some embodiments, the isolated
ABCB5(+) ocular stem cells are isolated ABCB5(+) limbal stem cells.
In some embodiments, the isolated ABCB5(+) limbal stem cells are
isolated ABCB5(+) human limbal stem cells.
[0023] In some embodiments, the isolated ABCB5(+) stem cells are
not skin stem cells (e.g., mesenchymal stem cells).
[0024] In some embodiments, the isolated ABCB5(+) stem cells are
expanded ex-vivo prior to the administering step.
[0025] In some embodiments, the subject is a mammal. In some
embodiments, the mammal is a human.
[0026] In some aspects of the invention, provided herein are kits
that include a container housing any of the foregoing grafts or
stem cell preparations and instructions for administering the graft
or preparation to a subject in need thereof.
[0027] Use of a graft or stem cell preparation of the invention for
treating an ocular condition is also provided as an aspect of the
invention.
[0028] A method for manufacturing a medicament of a stem cell
preparation of the invention for treating an ocular condition is
also provided.
[0029] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Each of the above embodiments
and aspects may be linked to any other embodiment or aspect. Also,
the phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of
"including," "comprising," or "having," "containing," "involving,"
and variations thereof herein, is meant to encompass the items
listed thereafter and equivalents thereof as well as additional
items.
BRIEF DESCRIPTION OF DRAWINGS
[0030] The accompanying drawings are not intended to be drawn to
scale. For purposes of clarity, not every component may be labeled
in every drawing. In the drawings:
[0031] FIG. 1A shows a schematic illustration of corneal structures
and the limbal stem cell niche.
[0032] FIG. 1B shows representative flow cytometric analyses of
BrdU-labeled dissociated murine corneal cells identifying the
presence of a label-retaining cell population in the limbus.
[0033] FIG. 1C shows immunofluorescence images depicting
co-expression of ABCB5 and BrdU in murine limbus.
[0034] FIG. 1D shows representative flow cytometric analyses
depicting co-expression of ABCB5 and BrdU in murine limbus. Bar
graph (right) illustrates quantitative analysis of independent
experiments (n=4).
[0035] FIG. 1E shows representative immunohistochemical analyses of
tangential limbal cross-sections from human corneas depicting ABCB5
expression (green) in the basal epithelial layer.
[0036] FIG. 1F shows representative immunohistochemical analyses of
tangential limbal cross-sections from human corneas depicting
co-expression of ABCB5 (red) with .DELTA.Np63.alpha. (green).
[0037] FIG. 1G shows representative cytometric analyses of human
limbal epithelial cells depicting co-expression of ABCB5 with
.DELTA.Np63.alpha.. Bar graphs show .DELTA.Np63.alpha. expression
on ABCB5(+) and ABCB5(-) cells (left panel), and ABCB5 expression
on .DELTA.Np63.alpha.(+) and .DELTA.Np63.alpha.(-) cells (right
panel). Data are depicted as mean.+-.s.e.m., n=3 experiments.
[0038] FIG. 1H shows dual color flow cytometry analyses of ABCB5
and KRT12 co-expression.
[0039] FIG. 1I shows representative immunohistochemical analyses of
ABCB5 expression in limbal biopsies from patients with limbal stem
cell deficiency (LSCD) performed at the time of surgery and from
their respective donors. Bar graphs show the number of ABCB5(+)
cells (green) in healthy donors and patients with LSCD (n=8
sections per patient/donor).
[0040] FIG. 2A shows a schematic of the murine Abcb5 gene locus and
protein topology. The topological structure was determined by the
TMHMM membrane topology prediction algorithm and displayed using
TOPO2 software. Amino acid residues deleted in Abcb5 knockout (KO)
(mutant) mice are highlighted in red.
[0041] FIG. 2B shows a schematic summary of the strategy employed
for generation of the Abcb5 KO mouse.
[0042] FIG. 2C, left panel, shows electrophoresis images of a
polymerase chain reaction (PCR) analysis of the genomic DNA used
for mouse genotyping, demonstrating a 113-base pair wild type (WT)
allele and a 322-base pair deleted allele. FIG. 2C, right panel,
shows Western blots of murine protein lysates with ABCB5 monoclonal
antibody (mAb) 3C2-1D12, which revealed loss of a 80 kD protein
band of predicted size in Abcb5 KO mice.
[0043] FIG. 2D shows images of a phenotypic characterization of
murine Abcb5 WT and Abcb5 KO corneas using slit lamp examination
(left panels), hematoxylin and eosin (H&E) staining (middle
panels) and 4',6-diamidino-2-phenylindole (DAPI) staining (right
panels). Bar graphs below depict the number of DAPI(+) epithelial
cells in Abcb5 KO and Abcb5 WT murine central cornea and limbus.
Data shown represent means f s.e.m, n=4 experiments.
[0044] FIG. 2E shows LC-biotin diffusion analyses and
immunofluorescence protein expression analyses of PAX6, KRT12 and
KRT14 in Abcb5 WT and Abcb5 KO mice. Bar graphs depict percent
PAX6(+) and KRT12(+) epithelial cells in Abcb5 KO and Abcb5 WT
mice. Data shown represent means.+-.s.e.m., n=6 experiments.
[0045] FIG. 2F shows H&E and DAPI staining of Abcb5 WT and
Abcb5 KO corneas 48 hours after epithelial debridement wounding.
Bar graph (bottom) represents the number of DAPI(+) cells per
section in Abcb5 WT and Abcb5 KO mice. Data shown represent
means.+-.s.e.m., n=4 experiments.
[0046] FIG. 2G shows immunofluorescence analyses of Ki67 in the
limbus and central cornea of Abcb5 WT and Abcb5 KO mice 48 hours
after epithelial debridement wounding. Bar graphs (bottom)
represent the percentage of Ki67(+) in limbus and in cornea Abcb5
KO and Abcb5 WT mice (means.+-.s.e.m., n=4 experiments,
respectively).
[0047] FIG. 2H shows immunofluorescence analyses of TUNEL staining
in the limbus and central cornea of Abcb5 WT and Abcb5 KO mice 48
hours after epithelial debridement wounding. Bar graphs (bottom)
represent the percentage of TUNEL+epithelial cells in limbus and in
cornea in Abcb5 KO and Abcb5 WT mice (means.+-.s.e.m., n=4
experiments, respectively).
[0048] FIG. 3A shows flow cytometry analyses showing loss of BrdU
label-retaining cells in Abcb5 KO and Abcb5 WT limbal epithelial
cells after an 8-week chase.
[0049] FIG. 3B shows flow cytometry analyses showing loss of BrdU
label-retaining cells in Abcb5 KO and Abcb5 WT limbal epithelial
cells after a 1-week chase (means t s.e.m., n=6 experiments).
[0050] FIG. 3C shows immunofluorescence analyses of Ki67 expression
in Abcb5 WT and Abcb5 KO mouse limbus and cornea. Bar graphs on the
right illustrate the percentages of Ki67(+) cells in Abcb5 WT and
Abcb5 KO mice in the limbus and cornea. Illustrated are means t
s.e.m. (n=3 experiments).
[0051] FIG. 3D shows a graph of mRNA expression of p53, p63, p21
and p16 in Abcb5 WIT and Abcb5 KO corneas. Bars represent relative
mRNA expression levels in Abcb5 KO mice as a percentage of mRNA
expression levels in Abcb5 WT mice (means t s.e.m., n=4
experiments).
[0052] FIG. 3E shows a schematic summary of the role of ABCB5 in
cell cycle regulation and normal corneal development and
regeneration. Abrogation of ABCB5 expression in Abcb5 KO mice
(blue) results in loss of BrdU(+) label-retaining cells and down
regulation of critical cell cycle regulators, including p63. This
leads to increased cellular proliferation as evidenced by enhanced
Ki67 expression in Abcb5 KO mice. Augmented proliferation and
inability to withdraw from the cell cycle explain the profound
differentiation deficiencies, evidenced by decreased PAX6 and KRT12
expression and increased rates of apoptosis in Abcb5 KO mice,
evidenced by enhanced TUNEL staining.
[0053] FIG. 4A shows analyses of murine syngeneic donor cell
transplants grafted onto C57BL/6 recipient mice.
[0054] FIG. 4B shows analyses of human xenogeneic donor cell
transplants grafted onto immunodeficient NSG recipient mice. The
images show tissue five weeks post transplantation performed for
the treatment of experimentally induced LSCD. In FIGS. 4A and 4B,
recipient mice received fibrin gel grafts containing no donor cells
(rows 2, respectively). ABCB5(-) cells (rows 3, respectively),
unsegregated limbal epithelial cells (rows 4, respectively), or
ABCB5(+) cells (rows 5, respectively). As a reference, normal
untreated (without induced LSCD) C57BL/6 and NSG murine corneas are
shown in rows 1 of FIG. 4A and FIG. 4B, respectively. Corneal
transparency was evaluated by slit lamp examination (FIG. 4A, 4B,
columns 1). Epithelial integrity and regeneration were evaluated by
H&E staining (columns 2-20.times. magnification; columns
3-40.times. magnification) for epithelial thickness and
stratification, by periodic acid-Schiff staining (PAS) for
detection Goblet cells associated with neovascularization (FIG. 4A,
column 4), and Krtl2 staining (green) for detection of
differentiated corneal epithelial cells (FIG. 4A, 4B, columns 5).
Nuclei are stained with DAPI (red). Bar graphs on the right show
the percentages of murine KRT12(+) cells (FIG. 4A) or human
KRT12(+) cells (FIG. 4B) in recipient corneas 5 weeks after
transplantation. The right lower panel in (FIG. 4B) shows RT-PCR
analyses of murine eyes transplanted with human cells for
evaluation of human donor cell contribution to corneal repair.
[0055] FIG. 5A shows a schematic summary of the experimental design
for BrdU pulse-chase experiments.
[0056] FIG. 5B shows representative flow cytometric analyses
depicting specific staining of BrdU label-retaining cells in limbal
epithelial cells of WT mice that did not receive BrdU (left two
panels) or WT mice that received BrdU followed by an 8 week chase
(right two panels). Limbal epithelial cells were recovered and
stained with either anti-BrdU antibody (Ab), or with an isotype
control Ab. The percentages of BrdU-positive cells within the gate
are indicated on each plot.
[0057] FIG. 6 shows a schematic illustration of tangential limbal
cross sections from human donor corneas, indicating the location of
the limbal epithelium. ABCB5(+) cells (schematically depicted as
green colored cells) were found located in the basal epithelial
layer.
[0058] FIG. 7 shows limbal biopsies from a patient with LSCD
(patient 1). Limbal biopsies were obtained from patient 1 with a
chemical burn prior to receiving a penetrating keratoplasty plus
kerato-limbal allograft from a cadaveric donor eye (donor 1).
Serial cross sections of the biopsies were stained with either
H&E, isotype control Ab or ABCB5 mAb. ABCB5 staining in the
limbal epithelium of donor 1 revealed nests of ABCB5-positive
cells, whereas ABCB5 positivity was reduced in the limbal
epithelium of patient 1. Photographs of immunofluorescent staining
are montages of sequential photos at 20.times. magnification.
[0059] FIG. 8 shows limbal biopsies from a patient with LSCD
(patient 2). Limbal biopsies were obtained from patient 2 with an
autoimmune corneal melt, peripheral ulcerative keratitis and
partial limbal stem cell deficiency prior to receiving a
kerato-limbal autograft from the patient's normal contralateral eye
(donor 2). Serial sections of the biopsies were stained with either
H&E, isotype control Ab or ABCB5 mAb. ABCB5 positivity was
present in the basal layer of the limbal epithelium of donor 2,
while a dramatically reduced epithelial layer and no ABCB5 staining
were observed in the limbus of patient 2. Photographs of
immunofluorescent staining are montages of sequential photos at
20.times. magnification.
[0060] FIG. 9 shows representative flow cytometry analyses of
either the limbal or the central corneal epithelium of Abcb5 WT and
Abcb5 KO mice. Forward scatter (FSC) and Side scatter (SSC)
indicates cellular size and granularity, respectively. Central
corneal epithelium of Abcb5 KO mice showed a reduced number of
epithelial cells compared to Abcb5 WT epithelium (left panels),
caused by a reduction in larger cells (right gates), but not
smaller cells (left gates). There was no reduction in the number of
limbal epithelial cells (right panels). Representative results of
samples pooled from four eyes are shown (n=3 experiments).
[0061] FIG. 10 shows representative flow cytometry analyses of
epithelial cells harvested from either the limbus (top) or the
central cornea (bottom) of Abcb5 WT and Abcb5 KO mice. Recovered
cells were stained with isotype control antibody, anti-Pax6
antibody or anti-Krt12 antibody. There was a reduced frequency of
PAX6(+) and KRT12(+) epithelial cells in the central cornea of
Abcb5 KO mice and a corresponding reduced frequency of PAX6(+)
cells in the limbus of Abcb5 KO mice. Red gates identify PAX6(+) or
KRT12(+) cells compared to isotype control staining. Representative
analyses of n=3 experiments are shown.
[0062] FIG. 11A shows a wound area to be debrided marked with a 2
mm trephine and the epithelium removed.
[0063] FIG. 11B shows a DAPI-stained cross section of the cornea
immediately following central epithelial debridement depicting the
wound margins and exposed central corneal stroma. Image is a
montage of sequential photos at 10.times. magnification.
[0064] FIG. 11C shows fluorescent images of corneal epithelial
wound closure monitoring at 1, 24, and 48 hours post
debridement.
[0065] FIG. 11D shows a graph of wound closure rates, which were
not significantly different between Abcb5 WT and Abcb5 KO mice
(summary of n=2 replicate experiments).
[0066] FIG. 12 shows representative DAPI-stained composite corneal
cross sections of Abcb5 WT (top) and Abcb5 KO (bottom) mice 48
hours after a corneal epithelial debridement wound, demonstrating a
reduced number of epithelial cells in Abcb5 KO mice. The white
dashed line demarcates the epithelium from stroma; the white box
indicates area shown at 20.times. magnification (montage pictures
are at 10.times. magnification): white lines demarcate the area in
which epithelial cells were counted. Epithelial cells were counted
within the standardized area in at least three consecutive
composite cross sections in three replicate mice per group in two
separate experiments (data shown in FIG. 2F).
[0067] FIG. 13 shows representative TUNEL-stained composite corneal
cross sections of Abcb5 WT (top) and Abcb5 KO (bottom) mice 48
hours after a corneal epithelial debridement wound, demonstrating
increased numbers of apoptotic cells in Abcb5 KO mice. Areas
defined by the white box are shown at 20.times. magnification
(montage pictures at 10.times. magnification). The number of
TUNEL-positive epithelial cells was counted, and the data from two
replicate experiments are summarized in FIG. 2H.
[0068] FIG. 14A shows a schematic illustration of the recovery and
separation of ABCB5(+) and ABCB5(-) limbal epithelial cells from
donor corneas followed by preparation of fibrin gels containing
donor cells.
[0069] FIG. 14B shows a schematic illustration of induction of
limbal stem cell deficiency in recipient mice and transplantation
of donor grafts.
[0070] FIG. 15A shows representative flow cytometry analyses
showing sorting gates and viability of murine donor limbal
epithelial cells. Viability is shown as the percentage of cells
excluding DAPI.
[0071] FIG. 15B shows post-sort analyses depicting the purity and
viability of ABCB5(+)-enriched and ABCB5(-)-enriched subpopulations
of limbal epithelial cells isolated from murine donors. Viability
is shown as the percentage of cells excluding DAPI.
[0072] FIG. 15C shows representative flow cytometry analyses
showing sorting gates and viability of human donor limbal
epithelial cells.
[0073] FIG. 15D shows post-sort analyses depicting the purity and
viability of ABCB5(+)-enriched and ABCB5(-)-enriched subpopulations
of limbal epithelial cells isolated from human donors. Viability is
shown as the percentage of cells excluding DAPI.
[0074] FIG. 16A shows representative H&E composite corneal
cross sections of recipient C57BL/6J mice 5 weeks after receiving
an induced limbal stem cell deficiency (LSCD) followed by
engraftment of donor fibrin gel transplants containing the
following syngeneic murine limbal epithelial cell subpopulations:
(i) no cells (negative control), (ii) ABCB5(+) cells, (iii)
ABCB5(-) cells or (iv) unsegregated cells. A normal untreated
cornea (no LSCD) served as a positive control. The positive control
displays the typical stratified corneal epithelium and iridocorneal
angle. Mice receiving transplants with no cells displayed the
typical conjunctivalization that occurs following a LSCD, i.e.,
unstratified conjunctival epithelium covers the cornea with
extensive inflammation, neovascularization, and stromal edema.
Synechia (where the iris adheres to the cornea) is typical of
intense anterior segment inflammation. In contrast, mice that
received transplants of ABCB5(+) cells, but not ABCB5(-) cells,
displayed a restored stratified corneal epithelium with no evidence
of inflammation, neovascularization, stromal edema, or
synechia.
[0075] FIG. 16B shows mice that received transplants of
unsegregated limbal epithelial cells displayed areas of stromal
edema with unstratified epithelium, while other parts of the cornea
contained normal stratified epithelial cells.
[0076] FIGS. 17A-17B show representative H&E composite corneal
cross sections of recipient immunodeficient NSG mice 5 weeks after
LSCD induction followed by transplantation of donor fibrin gel
grafts containing the following human limbal epithelial cell
subpopulations: (i) no cells (negative control), (ii) ABCB5(+)
cells, (iii) ABCB5(-) cells, and (iv) unsegregated cells. A normal
untreated NSG cornea (no LSCD) served as a positive control. The
positive control displays the typical stratified corneal epithelium
and iridocorneal angle. Mice that received transplants with no
cells displayed evidence of conjunctivalization that occurs
following a LSC deficiency. i.e., unstratified conjunctival
epithelium covers the cornea with extensive neovascularization and
synechia (anterior segment inflammation is muted in NSG mice due to
their immunodeficiency). In contrast, mice that received
transplants containing ABCB5(+) cells displayed areas of restored
stratified epithelium, whereas mice that received ABCB5(-) cell
grafts did not.
[0077] FIG. 18 shows representative immunofluorescent Krtl2
staining (green) of recipient C57BL/6J mice 5 weeks after an LSCD
induction followed by transplantation of donor fibrin gel grafts
containing the following syngeneic murine limbal epithelial cell
subpopulations: (i) no cells (negative control), (ii) ABCB5(+)
cells, (iii) ABCB5(-) cells, or (iv) unsegregated cells. Normal
untreated murine cornea (no LSCD), shown here as a positive
control, displayed high intensity of KRT12 staining. As expected,
mice that received grafts containing no cells, displayed no KRT12
expression. In contrast, mice transplanted with ABCB5(+) cells,
exhibited significantly enhanced KRT12 expression in comparison to
mice transplanted with unsegregated limbal epithelial cells. No
KRT12 expression was detected in mice transplanted with ABCB5(-)
cells. The w % bite box depicts the area shown at 40.times.
magnification. Montage images are shown at 10.times.
magnification.
DETAILED DESCRIPTION
[0078] Corneal epithelial homeostasis and regeneration are
sustained by a population of limbal stem cells (LSCs) residing in
the basal limbal epithelium of the eye [1-3]. These cells generate
new corneal cells to replace damaged ones, and loss of LSCs due to
injury or disease is a major cause of blindness worldwide [4].
Transplantation of LSCs from a healthy eye is often the only
therapeutic option available to patients with LSCD. Transplant
success depends foremost on the frequency of LSCs within grafts
[5]. However, prior to the present invention, a limbal stem cell
gene that permits prospective enrichment of this cell subset had
not been reported [5].
[0079] The present invention is based, in part, on the findings
that ATP-binding cassette, sub-family B (MDR/TAP), member 5 (ABCB5)
[6,7] marks LSCs and is required for limbal stem cell maintenance,
corneal development and repair, and that ABCB5-positive (ABCB5 (+))
LSCs prospectively isolated from donors possess the exclusive
capacity to restore the cornea upon grafting. Thus, various aspects
and embodiments of the invention are directed to methods of
treating a subject having an ocular condition, methods of isolating
ABCB5(+) stem cells of the eye, methods of selecting and/or
producing ocular grafts for transplantation, and methods of
promoting ocular cell regeneration as well as to grafts and
preparations containing isolated ocular stem cells characterized by
the expression of ABCB5 on their cell surface.
[0080] The inventors of the present invention demonstrate herein
that ABCB5 is uniformly expressed on in vivo label-retaining LSCs
in wild type mice and on .DELTA.Np63.alpha.-positive LSCs in
healthy humans. Consistent with these findings, the inventors also
demonstrate that ABCB5-positive limbal stem cell frequency is
significantly reduced in LSCD patients. ABCB5 loss of function
studies using newly generated Abcb5 knockout (KO) mice caused
depletion of quiescent LSCs due to enhanced proliferation and
apoptosis and resulted in defective corneal differentiation and
wound healing, which explains the demonstrated capacity of ABCB5(+)
LSCs to restore the cornea. Results from murine gene KO, in vivo
limbal stem cell tracing and limbal stem cell transplantation
models, and concurrent findings in phenotypic and functional
transplant analyses of human biopsy specimens, provide converging
lines of evidence that ABCB5 identifies mammalian LSCs.
Identification and prospective isolation of molecularly defined
LSCs with essential functions in corneal development and repair has
important implications for the treatment of corneal disease,
particularly corneal blindness due to LSCD.
[0081] "ABCB5(+) stem cells," as used herein, refers to cells
having the capacity to self-renew and to differentiate into mature
cells of multiple adult cell lineages. These cells are
characterized by the expression of ABCB5 on the cell surface. In
some embodiments of the invention, ABCB5(+) stem cells are limbal
stem cells. In some embodiments of the invention, ABCB5(+) stem
cells are retinal stem cells. ABCB5(+) stem cells may be obtained
from (e.g., isolated from or derived from) the basal limbal
epithelium of the eye or from the retinal pigment epithelium (RPE).
In some embodiments, ABCB5(+) stem cells are obtained from human
eye. Other ABCB5(+) stem cell types such as, for example, those
obtained from the central cornea may be used in various aspects and
embodiments of the invention.
[0082] ABCB5(+) ocular stem cells may be obtained from a subject by
isolating a sample of eye tissue, including ocular cells of the
basal limbal epithelium or RPE, and then purifying the ABCB5(+)
stem cells. It will be apparent to those of ordinary skill in the
art that a sample can be enriched for ocular stems cells having
ABCB5 in a number of ways. For example, ocular stems cells can be
selected for through binding of ABCB5 on cell surface molecules
with antibodies or other binding molecules. Ocular cells may be
obtained directly from a donor or retrieved from cryopreservative
storage. The ocular stems cells may, for instance, be isolated
using antibodies against ABCB5 and maintained in culture using
standard methodology or frozen, e.g., in liquid nitrogen, for later
use. A non-limiting example of a method that may be used in
accordance with the invention to obtain cells from the eye is
described in the Examples section and is depicted in FIG. 14A.
[0083] The present invention contemplates any suitable method of
employing ABCB5-binding molecules such as, for example, monoclonal
antibodies, polyclonal antibodies, human antibodies, chimeric
antibodies, humanized antibodies, single-chain antibodies, F(ab')2,
Fab, Fd, Fv or single-chain Fv fragments to separate ABCB5(+) stem
cells from a mixed population of ocular cells. Accordingly,
included in the present invention is a method of producing a
population of ABCB5(+) stem cells comprising the steps of providing
a cell suspension of ocular cells: contacting the cell suspension
with a monoclonal antibody, or a combination of monoclonal
antibodies, which recognize(s) an epitope, including ABCB5, on the
ABCB5(+) LSCs; and separating and recovering from the cell
suspension the cells bound by the monoclonal antibodies. The
monoclonal antibodies may be linked to a solid-phase and utilized
to capture limbal stem cells from eye tissue samples. The bound
cells may then be separated from the solid phase by known methods
depending on the nature of the antibody and solid phase.
[0084] "Monoclonal antibody," as used herein, refers to an antibody
obtained from a single clonal population of immunoglobulins that
bind to the same epitope of an antigen. Monoclonal based systems
appropriate for preparing cell populations of the invention include
magnetic bead/paramagnetic particle column utilizing antibodies for
either positive or negative selection; separation based on biotin
or streptavidin affinity; and high speed flow cytometric sorting of
immunofluorescent-stained LSCs mixed in a suspension of other
cells. Thus, the methods of the present invention include the
isolation of a population of LSCs and enhancement using monoclonal
antibodies raised against surface antigen ABCB5 (e.g., monoclonal
antibodies that selectively bind ABCB5). In some instances,
commercially available antibodies or antibody fragments that
selectively bind ABCB5 may be used in the methods disclosed herein.
Such antibodies are considered to selectively bind to ABCB5 if they
bind or are capable of binding to ABCB5 with a greater affinity
that the affinity with which the monoclonal antibodies may bind to
other antigens (i.e., antigens other than ABCB5). Such binding may
be measured or determined by standard protein-protein interaction
assays (e.g., antibody-antigen or ligand-receptor assays) such as,
for example, competitive assays, saturation assays or standard
immunoassays including, without limitation, enzyme-linked
immunosorbent assays, radioimmunoassays and radio-immuno-filter
binding assays.
[0085] The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) may be
isolated. An "isolated ABCB5(+)stem cell," as used herein, refers
to a cell that has been removed from an organism in which it was
originally found, or a descendant of such a cell. An isolated cell
also refers to a cell that is placed into conditions other than the
natural environment. Such a cell may later be introduced into a
second organism or re-introduced into the organism from which it
(or the cell or population of cells from which it descended) was
isolated. Such a cell, once manipulated according to the methods of
the invention is still considered to be an isolated cell. The term
"isolated" does not preclude the later use of the cell thereafter
in combinations or mixtures with other cells or in an in vivo
environment.
[0086] "Compositions," herein, may refer to an isolated cell
preparations or grafts, including tissue grafts and artificial
grafts (e.g., acellular collagen grafts). The compositions of the
invention, in some instances, are enriched with isolated ABCB5(+)
stem cells. A composition is considered to be enriched with
isolated ABCB5(+) stem cells if the ABCB5(+) stem cells are the
predominant cell subtype present in the preparation. For example,
an ABCB5(+) stem cell-enriched composition is a composition in
which at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99% or 100% of the cells of
the composition are ABCB5(+) stem cells (e.g., ABCB5(+) LSCs). In
some embodiments, a composition enriched with isolated ABCB5(+)
stem cells is one in which less than 50%, less than 45%, less than
40%, less than 35%, less than 30%, less than 25%, less than 20%,
less than 15%, less than 10%, less than 9%, less than 8%, less than
7%, less than 6%, less than 5%, less than 4%, less than 3%, less
than 2% or less than 1% of the cells of the composition are
ABCB5(-) cells. In some embodiments, the cells of a composition are
only ocular cells. That is, in some embodiments, a composition may
not contain non-ocular cells. In some embodiments, a composition
may not contain ABCB5(-) cells.
[0087] The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) may be
prepared as substantially pure preparations. The term
"substantially pure," as used herein, refers to a preparation that
is substantially free of cells other than ABCB5(+) stem cells
(e.g., ABCB5(+) LSCs). For example, a substantially pure
preparation of ABCB5(+) stem cells may constitute a preparation in
which at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or 100% percent of the total cells present in a
preparation are ABCB5(+) stem cells (e.g., ABCB5(+) LSCs).
[0088] In some embodiments, isolated and/or substantially pure
ABCB5(+) cell preparations may be packaged in a finished
pharmaceutical container such as an injection vial, ampoule, or
infusion bag along with any other components that may be desired,
e.g., agents for preserving cells or reducing bacterial growth. The
cell preparation may be in unit dosage form.
[0089] The ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) are useful for
treating ocular conditions. In some embodiments, the ocular
condition is an ocular wound, which may lead to ocular scarring,
which in turn may cause decreased vision or blindness. In some
embodiments, the ABCB5(+) stem cells (e.g., ABCB5(+) LSCs) may be
used to treat corneal diseases such as, for example, blindness due
to limbal stem cell deficiency (LSCD). In some embodiments, the
ABCB5(+) stem cells (e.g., ABCB5(+) LSCs and/or ABCB5(+) RPE stem
cells) may be used to treat retinal diseases such as, for example,
macular degeneration or retinitis/retinitis pigmentosa. Macular
degeneration refers to a group of conditions that includes a
deterioration of the macula causing a loss of central vision needed
for sharp, clear eyesight. It is a leading cause of vision loss and
blindness in those 65 years of age and older. Macular degeneration
may also be referred to as AMD or ARMD (age-related macular
degeneration). Retinitis refers to inflammation of the retina,
which may lead to blindness. Retinitis pigmentosa, which may be the
result of a genetic condition or an inflammatory response, refers
to a group of inherited disorders characterized by progressive
peripheral vision loss and night vision difficulties (nyctalopia)
that can lead to central vision loss.
[0090] The isolated ABCB5(+) stem cells (e.g., ABCB5(+) LSCS and/or
ABCB5(+) RPE stem cells) may be administered to a subject in need
thereof in an amount effective to regenerate ocular cells in the
subject (referred to herein as an "effective amount" of ABCB5(+)
stem cells). In some embodiments, one to about 10.sup.7 ABCB5(+)
stem cells are administered to a subject. In some embodiments, a
single isolated ABCB5(+) stem cell is administered to a subject. In
some embodiments, about 10.sup.1 to about 10.sup.7, about 10.sup.1
to about 10.sup.6, about 10.sup.1 to about 10.sup.5, about 10.sup.1
to about 10.sup.4, about 10.sup.1 to about 10.sup.3, about 10.sup.1
to about 10.sup.2 isolated ABCB5(+) stem cells are administered to
a subject. In some embodiments, about 10.sup.1, 10.sup.2, 10.sup.3,
10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7 or more isolated ABCB5(+)
stem cells are administered to a subject. In some embodiments, less
than about 10' isolated ABCB5(+) stem cells are administered to a
subject.
[0091] In some embodiments, the isolated ABCB5(+) stem cells (e.g.,
as a composition in the form of an ABCB5(+) stem cell preparation
or graft) may be administered to a subject more than once. Thus, in
some embodiments, a subject may be administered multiple doses or
grafts (e.g., 2, 3, 4 or more) of isolated ABCB5(+) stem cells over
the course of several weeks, months or years. In some embodiments,
the stem cells are administered again 3 months, 6 months, 9 months,
12 months, 18 months, 21 months or 24 months after the first
application. The number of applications and frequency of
application may depend, for example, on the degree of cellular
regeneration achieved after the first stem cell
administration/transplantation. The number and frequency of stem
cell applications may be determined by a medical professional
(e.g., surgeon, physician).
[0092] In some embodiments, a subject having an ocular condition
has an ocular wound (e.g., dead, damaged or infected ocular cells)
in, for example, the corneal epithelium. Thus, the corneal
epithelium may be wounded in a subject having an ocular condition
in accordance with the invention. It has been discovered that
ABCB5(+) limbal stem cell grafts can be used to restore the cornea.
Thus, in some embodiments, the integrity of the corneal epithelial
surface of the subject is restored following administration of an
effective amount of ABCB5(+) LSCs. Corneal regeneration may be
assessed based on, for example, corneal transparency (e.g.,
development of clear, rather than opaque, cornea) and/or visual
acuity. Methods of assessing the success of ocular cell/stem cell
transplantation (e.g., extent of cellular regeneration, visual
acuity) are known in the art, any of which may be used in
accordance with the invention. Examples of methods for assessing
success of a ocular cell/stem cell transplantation include, without
limitation, slit lamp imaging, Heidelberg retina tomography (HRT),
optical coherence tomography (OCT) and 2-photon imaging. Other
examples include, without limitation, the use of Rose Bengal
(4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) dye and
other epithelial staining solutions.
[0093] The ABCB5(+) stem cells (e.g., ABCB5(+) LSCS) may be
autologous to the subject (obtained from the same subject) or
non-autologous such as cells that are allogeneic or syngeneic to
the subject. Alternatively, the ABCB5(+) stem cells (e.g., ABCB5(+)
LSCS) may be obtained from a source that is xenogeneic to the
subject.
[0094] Allogeneic refers to cells that are genetically different
although belonging to or obtained from the same species as the
subject. Thus, an allogeneic human ABCB5(+) limbal stem cell is a
limbal stem cell obtained from a human other than the intended
recipient of the limbal stem cells. Syngeneic refers to cells that
are genetically identical or closely related and immunologically
compatible to the subject (i.e., from individuals or tissues that
have identical genotypes). Xenogeneic refers to cells derived from
or obtained from an organism of a different species than the
subject.
[0095] The ABCB5(+) stem cells (e.g., ABCB5(+) LSCS) in accordance
with the invention may be expanded ex-vivo prior to the
administering step. Thus, in some instances, ABCB5 expression
provides a basis for identifying, isolating, cloning, propagating,
and expanding ABCB5(+) stem cells (e.g., ABCB5(+) LSCS) in vitro.
The present invention contemplates any suitable method of employing
agents. e.g., isolated peptides, e.g., antibodies, that bind to
ABCB5 to separate ABCB5(+) stem cells from other cells. The
isolated ABCB5(+) stem cells may be maintained in an appropriate
culture environment using, for example, a combination of media,
supplements and reagents. Optionally, feeder cell populations or
conditioned media obtained from feeder cell populations may be used
to expand the ABCB5(+) stem cell populations.
[0096] Adhesion, attachment and matrix factors that may be used for
stem cell expansion in accordance with the invention include,
without limitation, E-cadherin, collagen, fibronectin,
superfibronectin, heparin sulfate proteoglycan, ICAM-I, laminin,
osteopontin, proteoglycan, E-selectin, L-selectin, VCAM and
vitronectin.
[0097] Bioactives and supplements that may be used for stem cell
expansion in accordance with the invention include, without
limitation, enzymes (e.g., cathepsin G, Flt-3/Fc), proteins and
peptides (e.g., activin A, albumin, angiogenin, angiopoietin, BAX
inhibiting peptide, heregulin beta-1, SMAC/Diablo), vitamins,
hormones and various other substances (e.g., L-ascorbic acid,
dexamethasone, EGF, EGF-receptor, embryonic fluid (bovine),
flt3-ligand, progesterone, retinoic acid, retinyl acetate,
thrombopoietin and TPO), antibodies, chemokines, cytokines, growth
factors and receptors.
[0098] Culture reagents that may be used for stem cell expansion in
accordance with the invention include, without limitation,
antibiotics (e.g., cycloheximide, etoposide, gentamicin, mitomycin,
penicillin-streptomycin), classical media (e.g., Claycomb Medium,
Dulbecco's Modified Eagle Medium. Iscove's Modified Dulbecco's
Medium, Minimum Essential Medium), cell freezing medium-DMSO,
Claycomb Medium without L-glutamine, Stemline.RTM. Medium
(Sigma-Aldrich, USA).
[0099] As used herein, a subject may be a mammal such as, for
example, a human, non-human primate, cow, horse, pig, sheep, goat,
dog, cat or rodent. Human ABCB5(+) stem cells (e.g., ABCB5(+) LSCs)
and human subjects are particularly important embodiments.
[0100] Compositions of the present invention may comprise stem
cells (e.g., limbal stem cells), or an isolated preparation of stem
cells, the stem cells characterized by the expression of ABCB5 on
their cell surface. A composition may comprise a preparation
enriched with isolated ABCB5(+) stem cells (e.g., ABCB5(+) LSCs),
or it may comprise a substantially pure population of ABCB5(+) stem
cells (e.g., ABCB5(+) LSCs). Compositions are meant to encompass
ocular grafts, discussed herein.
[0101] The compositions, in some embodiments, may comprises
additional bioactives and supplements to promote cell regeneration
and differentiation. Such bioactives and supplements that may be
used in accordance with the invention are describe above and
include, without limitation, various enzymes, proteins and
peptides, vitamins, antibodies, chemokines, cytokines, growth
factors and receptors. In some embodiments, the compositions may
comprise an immunosuppressant and/or an anti-vasculogenesis agent.
For example, in some embodiments, a composition may comprise
cyclosporin (e.g., CyA), which may be used to prevent and/or treat
graft rejections. In some embodiments, the compositions may
comprise bevacizumab (e.g., AVASTIN.RTM.). The use of
anti-vasculogenesis agent may be used, in some instances, to
prevent blood vessel formation, which often occurs after
transplantation and may lead to graft rejection. In some
embodiments, an immunosuppressant and/or an anti-vasculogenesis
agent is not administered as a component of a composition, but
rather is administered independently prior to or subsequent to
administration of ABCB5(+) stem cells.
[0102] In some embodiments, the compositions are formulated for
topical administration. An example of a composition formulated for
topical administration is an ocular graft. An ocular graft for
transplantation in accordance with the invention refers to a
substrate containing ACBC5(+) stem cells (e.g., ACBC5(+) LSCs) and
optionally other ocular cells and bioactive factors (e.g.,
cytokines, growth factors) that promote ocular cell regeneration,
which substrate may be transplanted to or implanted into an eye of
a subject to replace damaged or infected tissue (e.g., to treat an
ocular wound). An ocular graft may contain a mixed population of
cells including ocular cells such as, for example, corneal and/or
retinal cells. In some embodiments, an ocular graft for
transplantation is enriched with ABCB5(+) LSCs.
[0103] The cornea is the transparent front part of the eye that
covers the iris, pupil and anterior chamber. The cornea, with the
anterior chamber and lens, refracts light, with the cornea
accounting for approximately two-thirds of the eye's total optical
power. The cornea of primates has five layers: corneal epithelium
(multicellular epithelial tissue layer), Bowman's layer (condensed
layer of collagen fibers), corneal stroma (middle layer of collagen
fibers, e.g., collagen type I fibrils, and keratocytes), descemet's
membrane (thin layer from which corneal epithelium cells are
derived, composed of collagen type IV fibrils) and corneal
endothelium (simple squamous or low cuboidal layer of
mitochondria-rich cells). Compositions, including isolated
preparations and ocular grafts, in accordance with the invention
may comprise, in addition to ABCB5(+) stem cells, any one or more
of the cell subtypes of the five corneal layers. In some
embodiments, the compositions do not contain any one or more of the
cell subtypes of the five corneal layers.
[0104] The retina is the light-sensitive layer of tissue lining the
inner surface of the eye. The retina itself has several layers of
neurons interconnected by synapses, including photoreceptor cells
such as rods, cones and ganglion cells. Compositions, including
isolated preparations and ocular grafts, in accordance with the
invention may comprise, in addition to ABCB5(+) stem cells, any one
or more of the neuronal cell subtypes of the retina, including
retinal epithelial cells of the RPE. In some embodiments, the
compositions do not contain any one or more of the neuronal cell
subtypes of the retina.
[0105] The cells of a composition intended for use in
transplantation (e.g., ocular graft) may be allogeneic or
syngeneic. In some embodiments, the cells are not skin stem cells
(e.g., mesenchymal stem cells). Thus, in some embodiments, the
cells of a composition of the invention do not contain (i.e.,
exclude) ABCB5(+) mesenchymal stem cells.
[0106] In some embodiments, the compositions, including ocular
grafts, are enriched with ABCB5(+) stem cells. In some embodiments,
the ocular grafts are enriched with ABCB5(+) LSCs. In some
embodiments, the ocular grafts are enriched with ABCB5(+) RPE stem
cells. For example, an ocular graft is considered to be enriched
ABCB5(+) LSCs if the ABCB5(+) limbal stem cell is the predominant
cell subtype present in the graft. For example, an ocular graft is
enriched with ABCB5(+) LSCs if the LSCs outnumber the other cell
subtypes in the graft. In some embodiments, at least 50% of the
cells of the graft are ABCB5(+) stem cells or ABCB5(+) limbal stem
cells. For example, in some embodiments, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%
or 100% of the cells of the ocular graft are ABCB5(+) stem cells or
ABCB5(+) limbal stem cells. In some embodiments, less than 15%,
less than 10%, less than 5% or less than 1% of the cells of an
ocular graft are ABCB5(-) cells.
[0107] The compositions of the invention may comprise a substrate
such as, for example, a biocompatible material that promotes wound
healing, including biodegradable scaffolds such as, for example,
fibrin gel. Fibrin gels are typically prepared from fibrogen and
thrombin, key proteins involved in blood clotting. Other examples
of substrates that may be used in accordance with the invention
include, without limitation, amniotic membrane, aminoglycan
scaffolds, and adhesives. ABCB5(+) stem cells may be added to the
substrate to form, for example, ocular grafts for
transplantation.
[0108] Compositions of the invention may be transplanted to, for
example, the surface of the cornea or the retina Thus, in some
embodiments, the compositions are administered topically. In
instances where a stem cell graft is transplanted to the eye, the
graft may be sutured in place. In other embodiments, the stem cell
compositions are injected. In some embodiments, the compositions
are injected intravenously, intraarterially or intravascularly.
Other routes of administration are contemplated. It should be
understood that the compositions and/or ABCB5(+) stem cells of the
invention may be administered with or without a carrier. Thus, in
some embodiments, a substantially pure population of isolated
ABCB5(+) stem cells may be administered to a subject to, for
example, treat an ocular condition.
[0109] ABCB5 expression may be used to select ocular cell
preparations (e.g., grafts) for transplantation, thereby permitting
the selection of ocular cell preparations enriched with ABCB5(+)
stem cells. Such methods in accordance with the invention include
identifying the number of ABCB5(+) stem cells (e.g., ABCB5(+)
limbal stem cells) in the ocular cell preparations, comparing the
number of ABCB5(+) stem cells to the total cell population of the
cell preparations, and based on the comparison, selecting the
ocular cell preparations for transplantation. The number of
ABCB5(+) stem cells in the ocular cell preparations may be
identified using any one or more known molecules that selectively
bind to ABCB5. For example, in some embodiments, ABCB5(+) stem
cells may be identified by contacting the cells with an antibody or
other binding molecule that selectively binds to ABCB5. Viable dyes
(e.g., rhodamine or other stem cell marker dyes) may also be used
to identify ABCB5(+) stem cells. ABCB5(+) stem cells also can be
isolated based on the presence or absence of other specific markers
of interest. For example, agents can be used to recognize stem
cell-specific markers, for instance labeled antibodies that
recognize and bind to cell-surface markers or antigens on stem
cells can be used to separate and isolate ABCB5(+) stem cells using
fluorescent activated cell sorting (FACS), panning methods,
magnetic particle selection, particle sorter selection and other
methods known to persons skilled in the art, including density
separation. Typically, ocular cell preparations are selected for
transplantation if they are enriched with ABCB5(+) stem cells
(e.g., ABCB5(+) limbal stem cells). Such ABCB5(+) enriched cell
preparations increase the success of transplantation. In some
embodiments, ocular cell preparations (e.g., grafts) may be
selected for transplantation if at least 0.03% of the total cell
population is ABCB5(+). In some embodiments, ocular cell
preparations are selected for transplantation if at least 0.04%, at
least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at
least 0.09%, at least 0.10%, at least 0.15%, at least 0.20%, at
least 0.30%, at least 0.40%, at least 0.50%, at least 0.60%, at
least 0.70%, at least 0.80%, at least 0.90%, at least 1.0%, at
least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least
10.0%, at least 20.0%, at least 30.0%, at least 40.0%, at least
50.0%, at least 60.0%, at least 70.0%, at least 80%, at least 90%,
at least 95%, at least 98%, at least 99%, at least 99.9% or 100% of
the total cell population is ABCB5(+).
[0110] The ABCB5(+) stem cells of the invention may also be used to
prepare/produce artificial grafts such as, for example, artificial
corneal grafts. Such grafts may be made from acellular collagen or
other acellular biocompatible material. In some embodiments,
isolate ABCB5(+) stem cells are seeded onto an acellular matrix to
produce an artificial graft such as, for example, an artificial
cornea.
[0111] Compositions for topical administration such as, for
example, an ocular graft may be administered by any means known in
the art such as, for example, those described by Rama, J. et al
[5].
[0112] An example of a method of the invention follows. ABCB5(+)
stem cells are obtained and cultured on fibrin gel (e.g., using
lethally irradiated feeder cells, e.g., 3T3-J2 cells). A
360.degree. limbal peritomy is performed and the fibrovascular
corneal pannus carefully removed. The fibrin-cultured ABCB5(+)
epithelial sheet is placed on the prepared corneal wound bed
spanning the limbus (e.g., about 2-3 mm to reduce competition with
conjunctival ingrowth). The conjunctiva is then sutured over the
peripheral fibrin sheet with sutures (e.g., 8.0 vicryl sutures) to
protect the border of the sheet and help it to adhere on the
surface. The eyelids are kept closed (e.g., with STERI-STRIP.TM.
(3M.TM. NEXCARE.TM.)) and patched for one week.
[0113] The invention also contemplates using the isolated ABCB5(+)
stem cells (e.g., ABCB5(+) limbal stem cells or ABCB5(+) corneal
stem cells) to produce totipotent, multipotent or pluripotent stem
cells (e.g., induced pluripotent stem cells (iPSCs)), from which
other cells, tissues and/or whole animals can develop. Thus,
methods for directly reprogramming, or inducing, isolated ABCB5(+)
stem cells to become totipotent, multipotent or pluripotent stem
cells are provided in some aspects of the invention. The term
"reprogramming," as used herein, refers to a process that reverses
the developmental potential of a cell or population of cells (e.g.,
an isolated ABCB5(+) stem cell). Thus, reprogramming refers to a
process of driving a cell to a state with higher developmental
potential, i.e., backwards to a less differentiated state. The cell
to be reprogrammed can be either partially or terminally
differentiated prior to reprogramming. In some embodiments,
reprogramming encompasses a complete or partial reversion of the
differentiation state, i.e., an increase in the developmental
potential of a cell, to that of a cell having a totipotent,
multipotent or pluripotent state. In some embodiments,
reprogramming encompasses driving an isolated ABCB5(+) stem cell to
a totipotent, multipotent or pluripotent state, such that the cell
has the developmental potential of an embryonic stem cell, i.e., an
embryonic stem cell phenotype. Reprogramming also encompasses
partial reversion of the differentiation state of a cell to a state
that renders the cell more susceptible to complete reprogramming to
a totipotent, multipotent or pluripotent state when subjected to
additional manipulations.
[0114] Totipotent, multipotent or pluripotent stem cells may be
generated from ABCB5(+) stem cells (referred to herein as
"reprogrammed ABCB5(+) cells") using several reprogramming factors.
The resultant cells, which have a greater developmental potential
than the isolated ABCB5(+) stem cells, may then become the source
of stem cells for further manipulations. A "reprogramming factor"
as used herein, refers to a developmental potential altering
factor, the expression of which contributes to the reprogramming of
a cell, e.g., an isolated ABCB5(+) stem cell, to a less
differentiated or undifferentiated state, e.g., to a cell of a
pluripotent state or partially pluripotent state. Reprogramming
factors include OCT4, SOX2, KLF 4 and c-MYC (otherwise known as the
"Yamanaka factors" [32], incorporated herein by reference in its
entirety). Other reprogramming factors include, without limitation,
SOX 1, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 5, NR5A2,
LIN28, 1-MYC, n-MYC, REM2, TBX3, TERT and L1N28. Any combination of
two or more of the foregoing transcription factors may be used to
reprogram isolated ABCB5(+) stem cells. Methods of reprogramming
cells to a totipotent, multipotent or pluripotent state are
described by Stadtfeld and Hochedlinger [33], incorporated herein
by reference in its entirety.
[0115] Reprogrammed ABCB5(+)cells may be used, in some embodiments
of the invention, for basic and/or clinical applications, including
disease modeling, drug toxicity screening/drug discovery, gene
therapy and cell replacement therapy.
[0116] For example, reprogrammed ABCB5(+)cells may be used to treat
a variety of conditions (e.g., genetic conditions) including,
without limitation, sickle cell anemia, Parkinson's disease,
hemophilia A, heart disease such as ischemic heart disease,
Alzheimers disease, spinal cord injury, stroke, burns, diabetes,
osteoarthritis and rheumatoid arthritis.
[0117] In some embodiments, the reprogrammed ABCB5(+) cells may be
used in organ transplantations to provide cell types that are
genetically matched with a patient.
[0118] Other basic and clinical uses of the reprogrammed ABCB5(+)
stem cells are contemplated.
[0119] Methods for producing differentiated cells from reprogrammed
ABCB5(+) cells are also provided herein. The methods may comprise
expressing in the reprogrammed ABCB5(+) cells any one or more
differentiation factors necessary to promote differentiation into a
more mature, differentiated cell type such as, for example, a blood
cell, platelet, stromal cell, bone cell, muscle cell, skin cell,
fat cell or neural cell. As used herein, the term "differentiation
factor" refers to a developmental potential altering factor such as
a protein, or small molecule that induces a cell to differentiate
to a desired cell-type, e.g., a differentiation factor reduces the
developmental potential of a cell. Differentiation to a specific
cell type may involve simultaneous and/or successive expression of
more than one differentiation factor. The methods may further
comprise growing the reprogrammed ABCB5(+) cells under conditions
for promoting differentiation to form a differentiated cell.
[0120] Thus, reprogrammed ABCB5(+) cells can be generated from
isolated ABCB5(+) stem cells of the invention (e.g., isolated
ABCB5(+) limbal stem cells or isolated ABCB5(+) RPE stem cells),
and the reprogrammed ABCB5(+) cells can be differentiated into one
or more desired cell types. A "stem cell" as used herein is an
undifferentiated or partially differentiated cell that has the
ability to self-renew and has the developmental potential to
differentiate into multiple cell types. A "pluripotent cell" is a
cell with the developmental potential, under different conditions,
to differentiate to cell types characteristic of all three germ
cell layers, i.e., endoderm (e.g., gut tissue), mesoderm (including
blood, muscle, and vessels), and ectoderm (such as skin and nerve).
A "multipotent" cell is a cell that has the developmental potential
to differentiate into cells of one or more germ layers, but not all
three. These cells include, for instance, adult stem cells, such as
for example, hematopoietic stem cells and neural stem cells. A
"totipotent" cell is a cell that has the developmental potential to
differentiate into all the differentiated cells in an organism,
including extraembryonic tissues. Stem cells may have a propensity
for a differentiated phenotype; however, these cells can be induced
to reverse and re-express the stem cell phenotype. This process is
referred to as "dedifferentiation" or "reprogramming."
[0121] The isolated ABCB5(+) stem cells, reprogrammed ABCB5(+)
cells and differentiated cells of the invention can be manipulated
under standard conditions for these cell types. The treatment of
the cells may be performed in vitro, ex vivo or in vivo. For
instance, the cells may be present in the body or in a culture
medium. The manipulations may be performed under high or low-oxygen
conditions.
[0122] A "culture medium" contains nutrients that maintain cell
viability and support proliferation. A typical culture medium
includes: salts, buffers, amino acids, glucose or other sugar(s),
antibiotics, serum or serum replacement, and/or other components
such as peptide growth factors. Cell culture media for use in
deriving and maintaining totipotent, multipotent and pluripotent
cells are known in the art. Culture medium may also include cell
specific growth factors, such as angiogenin, bone morphogenic
protein-1, bone morphogenic protein-2, bone morphogenic protein-3,
bone morphogenic protein-4, bone morphogenic protein-5, bone
morphogenic protein-6, bone morphogenic protein-7, bone morphogenic
protein-8, bone morphogenic protein-9, bone morphogenic protein-10,
bone morphogenic protein-11, bone morphogenic protein-12, bone
morphogenic protein-13, bone morphogenic protein-14, bone
morphogenic protein-15, bone morphogenic protein receptor IA, bone
morphogenic protein receptor IB, brain derived neurotrophic factor,
ciliary neutrophic factor, ciliary neutrophic factor
receptor-alpha, cytokine-induced neutrophil chemotactic factor 1,
cytokine-induced neutrophil, chemotactic factor 2-alpha,
cytokine-induced neutrophil chemotactic factor 2-beta,
beta-endothelial cell growth factor, endothelia 1, epidermal growth
factor, epithelial-derived neutrophil attractant, fibroblast growth
factor 4, fibroblast growth factor 5, fibroblast growth factor 6
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor b, fibroblast growth factor c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor
acidic, fibroblast growth factor basic, glial cell line-derived
neutrophil factor receptor-alpha-1, glial cell line-derived
neutrophil factor receptor-alpha-2, growth related protein, growth
related protein-alpha, growth related protein-beta, growth related
protein-gamma, heparin binding epidermal growth factor, hepatocyte
growth factor, hepatocyte growth factor receptor, insulin-like
growth factor I, insulin-like growth factor receptor, insulin-like
growth factor II, insulin-like growth factor binding protein,
keratinocyte growth factor, leukemia inhibitory factor, leukemia
inhibitory factor receptor-alpha, nerve growth factor, nerve growth
factor receptor, neurotrophin-3, neurotrophin-4, placenta growth
factor, placenta growth factor 2, platelet-derived endothelial cell
growth factor, platelet derived growth factor, platelet derived
growth factor A chain, platelet derived growth factor AA, platelet
derived growth factor AB, platelet derived growth factor B chain,
platelet derived growth factor BB, platelet derived growth factor
receptor-alpha, platelet derived growth factor receptor-beta, pre-B
cell growth stimulating factor, stem cell factor, stem cell factor
receptor, transforming growth factor-alpha, transforming growth
factor-beta, transforming growth factor-beta-1, transforming growth
factor-beta-1-2, transforming growth factor-beta-2, transforming
growth factor-beta-3, transforming growth factor-beta-5, latent
transforming growth factor-beta-1, transforming growth
factor-beta-binding protein I, transforming growth
factor-beta-binding protein II, transforming growth
factor-beta-binding protein III, tumor necrosis factor receptor
type I, tumor necrosis factor receptor type II, urokinase-type
plasminogen activator receptor, vascular endothelial growth factor,
and chimeric proteins and biologically or immunologically active
fragments thereof.
[0123] The differentiation state of the cell can be assessed using
any methods known in the art for making such assessments. For
instance, the differentiation state of a cell treated according to
the methods described herein may be compared with an untreated cell
or cells treated with DNA using viral vectors to deliver DNA
resulting in the expression of the same reprogramming or
differentiation factors.
[0124] The following examples are provided to illustrate specific
instances of the practice of the present invention and are not
intended to limit the scope of the invention. As will be apparent
to one of ordinary skill in the art, the present invention will
find application in a variety of compositions and methods.
EXAMPLES
[0125] C57BL/6J, NOD.Cg-Prkdcscid Il2rgtm1Wjl/Sz) (NSG),
B6;SJL-Tg(ACTFLPe)9205Dym/J, and B6.FVB-Tg(EIIa-cre)C5379Lmgd/J
mice were purchased from Jackson Laboratory (Bar Harbor, Me.).
Abcb5 knockout (KO) mice were generated as described below. All
animals were maintained in accordance with the Institutional
Guidelines of Boston Children's Hospital and the Schepens Eye
Research Institute, Harvard Medical School. Four to twelve
weeks-old mice were used for the following experiments.
Example 1. ABCB5 is a Molecular Marker of Limbal Stem Cells
(LSCs)
[0126] To investigate whether ABCB5 is a marker of slow cycling,
label-retaining limbal stem cells in the mammalian eye, in vivo
BrdU-based `pulse and chase` experiments [2] were performed, in
which Abcb5 wild type (WT) mice were subjected over a 9-day period
to daily systemic BrdU administration in order to label
slow-cycling cells (pulse), followed by an 8-week BrdU-free period
(chase) prior to evaluation for limbal stem cell label retention
(FIG. 5A). Flow cytometric analysis of dissociated murine corneal
and limbal epithelial cells revealed BrdU label-retaining cells to
be detectable in the limbus, but not in the central cornea (FIG. 1B
and FIG. 5B). BrdU immunohistochemical staining of full thickness
murine corneas confirmed label-retaining limbal stem cells (LSCs),
consistent with previous findings [2], to be located in the basal
layer of murine limbal epithelium (FIG. 1C). Moreover,
label-retaining LSCs expressed ABCB5 (FIG. 1C). Flow cytometric
quantification confirmed ABCB5(+) cells to be predominantly
BrdU-positive (90.5.+-.0.5%, mean.+-.s.e.m.), with
ABCB5/BrdU-double positive cells comprising 1.8% of all limbal
epithelial cells 4 (FIG. 1D).
[0127] Similar to findings in mice, human ABCB5(+) cells were also
located in the basal layer of the limbal epithelium (FIG. 1E and
FIG. 6), and immunohistochemical analysis revealed that ABCB5(+)
cells co-expressed the limbal stem cell marker .DELTA.Np63.alpha.
(FIGS. 1F and 1G), absent expression of the corneal differentiation
marker KRT12 (FIG. 1H). Flow cytometry also revealed that ABCB5(+)
cells, but not ABCB5(-) cells, expressed significant levels of
.DELTA.Np63.alpha. (28.9.+-.5.7% and 0.1.+-.0.1%, respectively,
P=0.0364) (FIG. 1G) and showed that essentially all
.DELTA.Np63.alpha.(+) LSCs expressed ABCB5 (.DELTA.Np63.alpha.(+)
LSCs: 95.3.+-.4.8%. .DELTA.Np63.alpha.(-) cells: 3.6.+-.2.1%,
P=0.0032). Further, human limbal stem cell deficiency (LSCD)
patients exhibited significantly reduced ABCB5(+) frequencies
compared to healthy donors (2.8.+-.1.6% and 20.0.+-.2.6%,
respectively, P<0.0001) (FIG. 1I, FIGS. 7 and 8, Table 1).
TABLE-US-00001 TABLE 1 LSCD patient information Cause of Other
Previous Patient Gender Age LSCD Pathology surgery Procedure 1*
Male 46 Chemical Glaucoma None KLAL + PKP burn-OD suspect OD 2**
Female 31 Autoimmune Multiple 2xPKPs KLAU corneal melt; graft
Cataract PUK with failure OD surgery partial LSCD Retinal
vasculitis OD *Donor 1: cadaveric donor **Donor 2: autologous
transplant from contralateral eye Abbreviations: PKP Penetrating
keratoplasty KLAL Kerato-limbal allograft (limbal tissue was
harvested from donor eye) KLAU Kerato-limbal autograft (part of
limbal tissue was resected from uninjured contralateral eye) PUK
Peripheral ulcerative keratitis OD Right eye
[0128] The expression of ABCB5 on label-retaining limbal stem cells
in Abcb5 WT mice and .DELTA.Np63.alpha.(+) LSCs in healthy humans,
and the concurrent finding of reduced ABCB5(+) cell frequency in
clinical LSCD patients, showed that ABCB5 marks LSCs.
Example 2. ABCB5 Regulates Corneal Development and Regeneration
[0129] To investigate a potential functional role of ABCB5(+) LSCs
in corneal development and regeneration, Abcb5 KO mice carrying a
deletion of exon 10 of the murine Abcb5 gene (GenBank JQ655148)
were generated. Exon 10 the murine Abcb5 gene encodes a
functionally critical extracellular domain of the molecule
homologous to extracellular loop-associated amino acid residues
493-508 of human ABCB5 (GenBank NM_178559).
[0130] A conditional knockout targeting construct was first
generated by recombineering (i.e., recombination-mediated genetic
engineering) [25]. Briefly, a neomycin resistance cassette flanked
by two loxP sites (based on plasmid pL-452) was inserted into the
BAC clone RP23-161L22 458 base pairs upstream of exon 10 of the
murine Abcb5 gene (GenBank accession number JQ655148) (FIGS. 2A and
2B). The targeted region of the BAC clone was retrieved by gap
repair into the pL-253 plasmid. The retrieved plasmid contained
6006 base pairs upstream of exon 10 (not including the inserted neo
cassette) and 6384 base pairs downstream of exon 10. The neomycin
resistance cassette was excised by arabinose induction of Cre
recombinase to leave a single loxP site upstream of exon 10. A
neomycin resistance cassette flanked by two FRT sites and one loxP
site (based on plasmid pL-451) was inserted 460 base pairs
downstream of exon 10 to complete the targeting construct. The
targeting plasmid was verified by DNA sequencing and restriction
mapping. The linearized plasmid was transfected into TCI
(12956/SvEvTac derived) embryonic stem (ES) cells and selected in
G418 (Sigma-Aldrich, MO) and Fialuridine (Moravek Biochemicals,
CA). Resistant colonies were expanded and screened by long-range
PCR to identify targeted clones [22]. The left arm was amplified
with 5'-GTTGAGGGGAGCAGCCAGAGCAAGGTGAGAAAGGTG-3'(SEQ ID NO:1) and
5'-TTAAGGGTTATTGAATATGATCGGAATTGGGCTGCAGGAATT-3'(SEQ ID NO:2)
primers yielding a 6250 base pair PCR product (FIG. 2B). The right
arm was amplified with 5'-TGGGGCAGGACAGCAAGGGGGAGGAT-3' (SEQ ID
NO:3) and 5'-CTGGTCCCTCTCCTGTGATCTACACAGGCC-3' (SEQ ID NO:4)
primers yielding a 6384 base pair PCR product (FIG. 2B). Two
Abcb5-targeted ES clones were identified. These clones were
expanded and injected into C57BL/6 blastocysts that were then
transferred to the uterus of pseudo-pregnant females.
High-percentage chimeric male mice (Abcb5.sup.neo-loxP/wt) were
bred into a C57BL/6 background to obtain germ-line transmission.
Germ-line transmission of the Abcb5.sup.neo-loxP allele was
confirmed by PCR analysis of genomic DNA using
5'-GGAAGACAATAGCAGGCATGCTGGG-3' (SEQ ID NO:5),
5'-GGCTGGGGCAACTGAAAAGTAGCAT-3' (SEQ ID NO:6), and
5'-TTTCAGCTTCAGTTTATCACAATGTGGGTT-3' (SEQ ID NO:7) primers designed
to amplify the 385 base pair targeted allele and the 284 base pair
WT allele. Heterozygous Abcb5.sup.neo-loxP mice were then
intercrossed with hACTB-FLPe transgenic mice [26] to remove the
neomycin resistance cassette. PCR analysis of genomic DNA was
performed to confirm removal of the neomycin resistance cassette in
the genome of Abcb5.sup.loxP/wt mice using 5'-ACTT
GGTGCGGTGACTCTGAATTTTGC-3' (SEQ ID NO:8) and
5'-TAGCAACATTTCTGGCATTTTAGGCTG-3' (SEQ ID NO:9) primers designed to
amplify a 494 base pair neomycin resistance cassette-deleted allele
and a 390 base pair WT allele. Abrogation of ABCB5 protein
expression in Abcb5 KO animals was determined by Western blots of
murine tissues (FIG. 2C). Abcb5 WT and Abcb5 KO cell lysates were
immunoblotted using monoclonal ABCB5 antibody 3C2-1D12 [6,27] (5.5
.mu.g/ml) or .alpha.-Tubulin rabbit polyclonal antibody (1:5000
dilution) (Abcam, MA). After treatment with HRP-conjugated specific
secondary antibodies (1:5000 dilution) (Jackson ImmunoResearch,
PA), signals were visualized on film by enhanced
chemiluminescence.
[0131] To determine the outcome of a complete loss of ABCB5
function, exon 10 of the murine Abcb5 gene was deleted by breeding
Abcb5.sup.loxP mice with EIIa-Cre mice, which express Cre
recombinase at the zygote stage [14,15] (FIG. 2B). Deletion of the
genomic region between the two loxP sites was confirmed by PCR
analysis of genomic DNA using 5'-GGCTGGGGCAACTGAAAAGTAGCAT-3' (SEQ
ID NO:10), 5'-GCAAATGTGTACTCTGCGCTTATTTAATG-3' (SEQ ID NO: 11) and
5'-TGGTGCAGACTACAGACGTCAGTGG-3' (SEQ ID NO:12) primers designed to
amplify a 322 base pair cre-deleted allele (null) and a113 base
pair WT allele (FIG. 2C). Heterozygous Abcb5.sup.null/WT mice with
the germline deletion of exon 10 were intercrossed to produce
homozygous Abcb5.sup.null/null mutants (Abcb5 KO mice). Mice were
maintained on a 129S6/SvEvTac/C57BL/6 mixed genetic background, and
littermates were used as controls for experimental analyses.
[0132] Abcb5 KO mice were born alive and appeared indistinguishable
from their WT littermates at birth upon physical examination, with
no gross anatomical defects of Abcb5 KO corneas detectable by slit
lamp examination (FIG. 2D). However, histological analysis of
mutant corneas demonstrated profound developmental abnormalities
characterized by flattening of the corneal epithelium compared to
WT controls, with significantly reduced epithelial cell numbers in
the central cornea, but not in the limbus, as evidenced by
hematoxylin and eosin (H&E) stain,
4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry
(Central cornea: 2688.+-.399 cells and 4427.+-.346 cells,
respectively, P=0.0165; limbus: 3015.+-.433 cells and 3629.+-.94
cells, respectively, P=0.2377) (FIG. 2D and FIG. 9). Abcb5 KO
corneas also exhibited severe epithelial tight junction defects as
determined by LC biotin staining (FIG. 2E), and mutant mice showed
significantly decreased limbal and corneal PAX6 and corneal KRT12
expression as compared to WT mice (limbal PAX6: 0.3.+-.0.3% and
18.0.+-.4.6%, respectively, P=0.0181; corneal PAX6: 8.3.+-.4.6% and
42.0.+-.7.6%, respectively, P=0.0192; corneal KRT12: 6.5.+-.6.5%
and 47.7.+-.8.2%, respectively, P=0.0382) (FIG. 2E, FIG. 10),
demonstrating a novel essential role of ABCB5 in normal corneal
development.
Example 3. ABCB5 Regulates Limbal Stem Cell Quiescence
[0133] To determine whether corneal regeneration is dependent on
intact ABCB5 function, Abcb5 KO and WT mice were subjected to
central corneal epithelial debridement injury followed by
evaluation for corneal regeneration (FIGS. 11A-11D). After
anesthesia with intraperitoneal injection of Ketamine (120 mg/kg
body weight, Hospira, IL) and Xylazine (10 mg/kg body weight, Burns
Veterinary Supply, NY), followed by topical application of one drop
of 0.5% Proparacaine eye drops (Akorn, IL) into each eye, a 2 mm
diameter epithelial wound was created by demarcating an area of the
central cornea with a 2 mm trephine and removing the epithelium
within the circle with a small scalpel, leaving the basement
membrane intact. In each animal, the procedure was performed on the
right eye. Ak-Spore Ophthalmic Ointment (Bacitracin Zinc, Neomycin
Sulfate and Polymyxin B Sulfate, Akorn, IL) was applied to both
eyes immediately after wounding and then twice per day for the next
48 hours to prevent corneal infection and dryness. Analgesia was
provided by subcutaneous injections of Buprenex (Reckitt Benckiser
Pharmaceuticals, Berkshire, 30 UK) every 12 hours for 48 hours
postoperatively at the dose of 1 mg/kg. The wound healing was
monitored as described previously [29]. Animals were euthanized 48
hours post-operatively and the integrity of corneal epithelial
tight junctions was assessed using the LC-Biotin staining to method
performed as described [31]. Briefly, LC-Biotin staining solution
prepared by dissolving 1 mg/ml EZ-Link-Sulfo-NHS-LC-Biotin (Pierce,
IL) in HBSS (Hank's Balanced Salt Solution, Lonza, MD) plus 2 mM
MgCl.sub.2, and 1 mM CaCl.sub.2 was applied to wounded and
non-wounded eyes for 15 minutes at the time of euthanasia Eyes were
rinsed with PBS (Lonza, MD), enucleated and placed in Tissue-Teck
OCT (Sakura Finetek, CA) for frozen sectioning.
[0134] While no significant differences were observed in the rate
of wound closure between Abcb5 WT and Abcb5 KO mice (FIGS. 11C and
11D), histological analysis revealed severely abnormal corneal
restoration in Abcb5 KO mice, as compared to Abcb5 WT mice,
characterized by highly irregular appearance of the epithelium with
reduced number of epithelial cells (403.3.+-.29.7 and
737.2.+-.28.2, respectively, P<0.0001) (FIG. 2F and FIG. 12),
significantly increased cellular proliferation as demonstrated by
enhanced Ki67 expression (limbus: 54.0.+-.5.0% and 0.3.+-.0.2%,
respectively, P<0.0001; cornea: 41.2.+-.12.8% and 1.0.+-.0.5%,
respectively, P=0.0257) (FIG. 2G), and significantly enhanced rates
of apoptosis as demonstrated by TUNEL staining (limbus:
41.2.+-.12.8% and 1.0.+-.0.5%, respectively, P=0.001; cornea:
49.0.+-.1.0% and 0.4.+-.0.3%, respectively, P<0.0001) (FIG. 2H
and FIG. 13).
[0135] Pulse-chase BrdU-labeling (FIGS. 5A and 5B) and flow
cytometric analysis of dissociated murine limbal epithelial cells
revealed that after an early, 1-week chase period, no significant
difference existed between the numbers of BrdU-labeled epithelial
cells in Abcb5 KO-derived and Abcb5 WT-derived specimens,
indicative of equal BrdU uptake by Abcb5 KO and Abcb5 WT limbal
cells (1.9.+-.0.7% and 1.5.+-.0.4%, respectively, P=0.6971). By
contrast, after an 8-week chase period, label-retaining LSC
frequency was markedly and significantly reduced (by 89%) in Abcb5
KO mice, compared to Abcb5 WT controls (frequency: 0.1.+-.0.1% and
0.9.+-.0.3%, respectively P=0.0152) (FIGS. 3A and 3B),
demonstrating that abrogation of ABCB5 function induces cellular
proliferation of normally quiescent LSCs. Consistent with this
result, Ki67 expression, indicative of cellular proliferation, was
significantly enhanced in Abcb5 KO corneas, as compared to Abcb5 WT
control corneas (limbus: 24.0.+-.5.0% and 1.5 f 1.5%, respectively,
P.ltoreq.0.0001; cornea: 53.0.+-.16.0% vs. 11.0.+-.2.1%, P=0.0297)
(FIG. 3C). Moreover, in line with demonstrated increased
proliferation, real-time quantitative PCR (qPCR) analysis of RNA
expression revealed significant down-regulation in Abcb5 KO corneal
epithelial cells of the p53 family (p53 and p63) and the Cip/Kip
family (p21 and p27) of cell cycle regulators, which control the
G0/G1 cell cycle checkpoint and cellular quiescence, as compared WT
controls (41.6.+-.16.4% of WT p53, P=0.0377; 31.2.+-.13.8% of WT
p63, P=-0.0155; 37.2.+-.13.8% of WT p21, P=0.0197; 36.8.+-.7.0% of
WT p27, P=0.0029) (FIG. 3D). Thus, ABCB5 is required for the
maintenance of slow-cycling LSCs. Because withdrawal from the cell
cycle is a prerequisite for LSC maintenance, and hence normal
differentiation, these results provide an explanation for the
observed corneal differentiation defect in Abcb5 KO mice (FIG.
3E).
Example 4. Regenerative Role of ABCB5(+) Limbal Stem Cells in
Treatment of Limbal Stem Cell Deficiency
[0136] To investigate whether ABCB5 represents a molecular marker
for prospective enrichment of limbal stem cells within grafts to
improve transplantation outcomes, the cornea-regenerative potential
of transplanted limbal epithelial cells was examined. (FIGS. 14A
and 14B and FIGS. 15A-15C). Murine donor limbal epithelial cells
were transplanted onto the eyes of syngeneic C57BL/6J recipient
mice with an induced limbal stem cell deficiency (LSCD). Human
donor limbal epithelial cells were transplanted onto the eyes of
immunodeficient NOD.Cg-Prkdc.sup.scid Il2rg.sup.tmIWj/SzJ (NSG)
mice with an induced limbal stem cell deficiency. Four types of
donor transplants were performed: (i) ABCB5(+) limbal epithelial
cells, (ii) ABCB5(-) limbal epithelial cells, (iii) unsegregated
limbal epithelial cells, and (iv) grafts containing no cells
(fibrin gel carrier only) (500 cells in fibrin gel vehicle/graft, 1
unilateral eye graft/mouse, n=5 mice/treatment group) (Table 2).
Three days
TABLE-US-00002 TABLE 2 Number and viability of donor cells used for
transplantation ABCBS(+) cells ABCB5(-) cells (ABCB5-positive
(ABCBS-negative Unsegregated cells enriched) enriched) 500
cells/graft/mouse 500 cells/graft/mouse 500 cells/graft/mouse ABCB5
ABCB5 ABCB5 Donor Positive Negative Positive Negative Positive
Negative Mouse limbus % of 0.367 99 51 43 0 99 cells/graft viable
69 64 100 40 0 63 cells/graft (%) viable 1 319 255 86 0 312
cells/graft (number) Human limbus % of 0.03 99 59 40 0 100
cells/graft viable 93 22 99 40 0 90 cells/graft (%) viable 1 109
292 80 0 450 cells/graft (number)
prior to transplantation, murine and human donor cells were seeded
onto a fibrin carrier, which was prepared by dissolving fibrinogen
and thrombin stock solutions (TISSUCOL-Kit Immuno, Baxter, Germany)
in 1.1% NaCl and 1 mM CaCl.sub.2) to a final concentration of 10
mg/ml fibrinogen and 3 IU/ml thrombin as described [30]. On the day
of transplantation, total LSCD was induced in anesthetized
recipient mice by removing the corneal and limbal epithelium with
an Algerbrush II corneal rust ring remover with a 0.5-mm burr
(AMBLER Surgical, PA) [16]. Following induction of LSCD, recipient
mice received fibrin gel carrier-based transplants that were
secured through four sutures. Eyelids were sutured with 8-0 nylon
sutures to keep the eyes closed. Ak-Spore Ophthalmic Ointment
(Bacitracin Zinc, Neomycin Sulfate and Polymyxin B Sulfate, Akorn,
IL) was applied on both eyes immediately after wounding and then
twice per day for the next 48 hours to prevent corneal infection
and dryness. Analgesia was provided by subcutaneous injections of
5-10 mg/kg Metacam (Boehringer Ingelheim Pharmaceuticals, CT),
given preoperatively and by subcutaneous injections of 0.05-0.1
mg/kg of Buprenex (Reckitt Benckiser Pharmaceuticals, Berkshire,
UK) every 12 hours for 24 hours postoperatively. In addition, after
surgical recovery, mice were also treated with anti-inflammatory
Inflanefran Forte eye drops (Allergan, MA) for the first 5 days,
and then with 1% Avastin (Bevacizumab, Genentech, CA) eye drops
daily for 5 days. Slit lamp examination was performed weekly until
euthanasia. Eyes were enucleated postmortem and fixed in 10%
buffered formalin for methacrylate embedding (Technovit, Heraeus
Kulzer, Germany) or snap-frozen in Tissue-Teck OCT (Sakura Finetek,
CA).
[0137] Recipients of syngeneic murine Abcb5(-) limbal cell grafts
or vehicle-only negative controls displayed opaque corneas,
epithelial conjunctivalization with infiltrating goblet cells, and
absence of differentiated KRT12(+) cells (0%, respectively) when
analyzed 5-weeks post transplantation, consistent with persistent
LSCD (FIG. 4A, FIGS. 16A-16B). Recipients of syngeneic grafts
containing unsegregated limbal cells displayed partial corneal
restoration with detectable differentiated KRT12(+) cells in the
central cornea (17% of cells, significantly enhanced compared to
Abcb5(-) or vehicle-only treatment regimens, P<0.01), but
exhibited persistence of LSCD-characteristic goblet cells and
epithelial conjunctivalization (FIG. 4A, FIGS. 16A-16B). By
contrast, syngeneic ABCB5(+) limbal cell grafts resulted in the
development of clear corneas with normal histology in recipient
mice, gave rise to higher numbers of differentiated KRT12(+)
corneal epithelial cells (47% of cells, significantly increased
compared unsegregated or ABCB5(-) limbal cell treatment regimens or
compared vehicle-only controls, P<0.001) and prevented goblet
cell formation or epithelial conjunctivalization (FIG. 4A, FIGS.
16A-16B).
[0138] Immuno-compromised NSG recipients of freshly isolated human
ABCB5(-) limbal cell grafts or vehicle-only negative controls also
displayed epithelial conjunctivalization and absence of
differentiated KRT12(+) cells (0%, respectively) 5-weeks post
transplantation, consistent with persistent LSCD (FIG. 4B, FIGS. 17
and 18). Immuno-compromised NSG recipients of freshly isolated
human unsegregated limbal cell grafts, similar to findings in
murine unsegregated limbal cell transplantation experiments,
displayed partial corneal restoration with detectability of
differentiated KRT12(+) cells in the central cornea (12% of cells,
significantly enhanced vs. ABCB5(-) or vehicle-only treatment
regimens, P<0.01), but exhibited persistence of LSCD
characteristic epithelial conjunctivalization (FIG. 4B, FIGS.
17A-17B and 18). Strikingly, only freshly isolated human ABCB5(+)
limbal cell grafts resulted in the development of clear corneas
with normal histology in recipient NSG mice with presence of a
stratified epithelial layer containing high numbers of KRT12+ cells
(31% of cells, significantly increased compared to vehicle-only or
compared to ABCB5(-) or unsegregated limbal cell treatment
regimens, P<0.001) and absence of LSCD characteristic epithelial
conjunctivalization (FIG. 4B, FIGS. 17A-17B).
[0139] In order to confirm that human donor cells had caused
corneal restoration in this xenotransplantation model, regenerated
corneal tissue was assayed by RT-PCR for expression of
human-specific .beta.2 microglobulin (.beta.2M), an identifier of
all cells of human origin, and for expression of human-specific
PAX6 and KRT12 as markers of corneal differentiation. Only corneal
epithelium of recipients grafted with human ABCB5(+) or
unsegregated human limbal cells contained human-specific, .beta.2M,
PAX6 and KRT12 transcripts, whereas vehicle-only-grafted control
eyes that did not exhibit corneal restoration did not, confirming
human specificity of the RT-PCR assay system (FIG. 4B). Moreover,
despite similar viability in ABCB5(-) compared to unsegregated or
ABCB5(+) cell grafts (Table 2, FIGS. 15A-15C), ABCB5(-)
cell-grafted eyes were deficient in human-specific 2M, PAX6 or
KRT12 transcript expression (FIG. 4B), indicating that long-term
engraftment capacity is exclusively contained within the human
ABCB5(+) limbal cell population.
[0140] The Examples provided herein demonstrate that ABCB5(+) cell
frequency is reduced in limbal stem cell deficiency (LSCD), that
ABCB5-positivity preferentially characterizes slow-cycling and
.DELTA.Np63.alpha.-positive populations enriched for limbal stem
cells (LSCs), and that prospectively isolated ABCB5(+) limbal cells
are exclusively capable of reversing LSCD, indicating that
ABCB5-positivity defines LSCs. These findings are further supported
by data demonstrating that ABCB5 loss of function in Abcb5 gene
knockout (KO) mice causes LSCD and impairs LSC-dependent corneal
development and regeneration, through abrogation of LSC
self-renewal capacity. These results have several important
implications.
[0141] First, successful enrichment of human LSCs has the potential
to decisively advance the field of LSCD therapy, because long-term
clinical success has been shown to depend on limbal stem cell
frequency within grafts[5] and because, prior to the present
invention, no marker for prospective limbal stem cell enrichment
has been available. Indeed, these Examples show that prospective
limbal stem cell enrichment within grafts can significantly enhance
LSCD therapeutic success. ABCB5 expression on the limbal stem cell
surface permits monoclonal antibody-based cell sorting strategies
and significant limbal stem cell enrichment as demonstrated herein,
unlike intracellularly expressed .DELTA.Np63.alpha. or alternative
candidate limbal stem cell markers [17] that have not been
successfully employed for prospective isolation of LSCs capable of
LSCD reversal. This underscores the promise of ABCB5 as a potential
marker for limbal stem cell isolation also for clinical limbal stem
cell transplantation.
[0142] Second, the data provided herein reveal a novel in vivo
physiological role of ABCB5 in the maintenance of stem cell
quiescence. Specifically, abrogation of ABCB5 function in newly
created Abcb5 KO mice resulted in loss of slow-cycling LSC with
inhibited expression of molecules regulating G0/G1 cell cycle
progression, including the limbal stem cell marker
.DELTA.Np63.alpha.. This explains the observed co-expression of
ABCB5 with .DELTA.Np63.alpha. by normally quiescent LSCs. and
provides an explanation for induction of limbal stem cell
proliferation and apoptosis associated with reduction of
differentiated cells observed in Abcb5 KO corneas, because the
ability of a cell to withdraw from the cell cycle is critical for
both stem cell pool preservation and normal differentiation.
Additional Materials and Methods:
[0143] BrdU pulse and chase experiments. Four-week old Abcb5 KO
mice and their Abcb5 WT littermates were subjected to daily
intraperitoneal injections of 50 mg/kg Bromodeoxyuridine (BrdU, BD
Pharmingen, CA) for 9 consecutive days (FIG. 5A). Corneal and
limbal epithelial cells isolated from Abcb5 WT and Abcb5 KO mice
sacrificed at either one week or eight weeks after receiving the
last BrdU injection were analyzed by flow cytometry and
immunofluorescence. Limbal and central corneal epithelial cells
from age-matched Abcb5 WT and Abcb5 KO littermates were used as
experimental controls. Flow cytometry and immunohistochemistry
staining were used to determine the frequency of BrdU-positive and
BrdU-negative cells within epithelia of the limbus and central
cornea.
[0144] Human and murine corneal cell isolation. Cadaveric human
corneoscleral tissues derived from consented donors were obtained
from Heartland Lions Eye Banks (Kansas City, Mo.), Bascom Palmer
Eye Institute (Miami, Fla.), and Carver College of Medicine (Iowa
City, Iowa). After removal of the scleral rim, iris and trabecular
meshwork, the limbus and central cornea were dissected under a
microscope. Limbal and central corneal tissues were subsequently
incubated with 2.4 units/ml Dispase II (Roche Diagnostics, IN) at
37.degree. C. for 1 hour, followed by incubation with 0.5M EDTA
(Invitrogen, CA) at 37.degree. C. for ten 5-minute cycles to
recover the epithelial cells [22,23]. Murine limbal and corneal
epithelial cells were obtained from Abcb5 KO and Abcb5 WT mice as
follows. Immediately after euthanasia by CO.sup.2 narcosis and
subsequent eye enucleation, limbal and central corneal tissues were
removed with micro scissors under a dissecting microscope, placed
in low Ca.sup.2+ Keratinocyte Serum Free Medium (KSFM, Invitrogen.
CA) and centrifuged for 5 min at 250 g at 4.degree. C. After
removal of the supernatant, tissue pellets were digested in 0.5%
Trypsin solution (Lonza, MD) [24]. For transplantation experiments,
ABCB5(+) and ABCB5(-) limbal epithelial cells were isolated by
Fluorescence Activated Cell Sorting (FACS) using ABCB5 monoclonal
antibody (mAb) labeling [18]. Briefly, either human or murine
limbal epithelial cells were labeled with primary ABCB5 mAb (20
.mu.g/.mu.l) for 30 minutes at 4.degree. C., washed to remove
excess antibody, followed by a 30 minute incubation with secondary
anti-mouse FITC conjugated IgG. The ABCB5(+) and ABCB5(-) sorting
gates were established on a Modified Digital Vantage cell sorter
(Becton Dickinson and MGH Pathology Flow Cytometry Core, Simches
Research Building, Boston) as displayed in FIGS. 15A-15C.
[0145] Only viable cells were selected for sorting by excluding all
DAPI(+) cells (1 .mu.g/ml DAPI, Sigma-Aldrich, MO, added
immediately prior to sorting) as identified using a 70 MW UV laser
for excitation. The purity and viability of ABCB5(+) and ABCB5(-)
sorted cells were established in representative post sort analyses
in which samples were re-analyzed (FIGS. 15A-15C). ABCB5(+) cell
purification resulted in a 255-fold increase for murine ABCB5(+)
limbal cells (0.37% positivity before and 51% positivity after
sorting, Table 2) and a 292-fold increase for human ABCB5(+) limbal
cells (0.03% positivity before and 59% positivity after sorting,
Table 2). ABCB5(-) cell enrichment resulted in complete absence of
ABCB5(+) cells in both mouse and human samples (Table 2).
[0146] Flow cytometric analysis. Dual-color flow cytometry was used
to determine whether human ABCB5(+) limbal epithelial cells
co-expressed .DELTA.Np63.alpha. or KRT12 and whether murine
ABCB5(+) limbal epithelial cells co-expressed PAX6 and KRT12, and
was performed as described previously 1181. For human and murine
ABCB5 and KRT12 co-expression analysis, cells were first incubated
with mouse anti-ABCB5 mAb, counterstained with goat anti-mouse FITC
IgG, followed by incubation with goat polyclonal anti-KRT12
antibody and counterstaining with Dylight 649 donkey anti-goat IgG.
For human ABCB5 and .DELTA.Np63.alpha. co-expression and murine
ABCB5 and PAX6 co-expression analysis, cells were incubated with
mouse anti-ABCB5 mAb, counterstained with goat anti-mouse FITC IgG,
permeabilized in BD Cytofix/Cytoperm Buffer (BD Biosciences, CA),
stained with either .DELTA.Np63.alpha. or PAX6 Abs, and
counterstained with goat anti-rabbit Alexa 647 IgG. Washing steps
with staining buffer or BD Perm/Wash Buffer (BD Biosciences, CA)
were performed between each step. Dual-color flow cytometry was
performed by acquisition of fluorescence emission at the Fl1 (FITC)
and Fl4 (Alexa 647 and/or Dylight 649) spectra on a Becton
Dickinson FACScan (Becton Dickinson, NJ), as described [18]. Murine
ABCB5 and BrdU co-expression analysis was performed using the FITC
BrdU Flow Kit (BD Biosciences, CA), according to the manufacturer's
instructions. Statistical differences between expression levels of
the above-listed markers by ABCB5(+) and ABCB5(-) cells were
determined using the unpaired t test. A two-sided P value of
P<0.05 was considered significant.
[0147] RT-PCR and quantitative real time PCR For cell cycle gene
expression analyses, total RNA was isolated from Abcb5 KO and Abcb5
WT corneas using a RT.sup.2 qPCR Grade RNA isolation kit and then
reverse-transcribed using a RT.sup.2 First Strand Kit for reverse
transcriptase-PCR according to the manufacturer's protocol
(SABiosciences, CA). Samples were assayed using SYBR Green qPCR
Master Mixes (SABiosciences, CA), murine cell cycle arrays (catalog
number PAMM-020Z, SABiosciences. CA) and kinetic PCR (ABI 7700
Sequence Detector: Applied Biosystems, CA), as described [28]. All
quantifications were normalized to the endogenous control genes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and .beta.-actin,
to account for variability in the initial concentration and quality
of the total RNA, and efficiency of the reverse transcription
reaction. Statistical differences in gene expression levels between
Abcb5 KO and Abcb5 WT mice were determined using the one sample t
test. A two-sided P value of P<0.05 was considered significant.
For detection of human-specific gene transcripts, total RNA was
isolated from transplanted murine eyes and non-injured murine or
human control corneas using the RNAeasy Plus isolation kit (Qiagen,
CA) and then transcribed using the High Fidelity RT kit (Applied
Biosystems, CA). PCR was performed using Taq 2X Master Mix (New
England Biolabs, MA) and the following gene-specific primers: human
.beta.2-microglobulin (B2M, NM_004048): Forward
5'-GTGTCTGGGTTTCATCCATC-3' (SEQ ID NO:13), Reverse
5'-AATGCGGCATCTTCAACCTC-3' (SEQ ID NO: 14); human paired box 6
(PAX6, NM_000280.3): Forward 5'-CAGCGCTCTGCCGCCTAT-3' (SEQ ID
NO:15), Reverse 5'-CATGACCAACACAGATCAAACATCC-3' (SEQ ID NO:16);
human keratin 12 (KRT12, NM_000223.3): Forward
5'-GAAGCCGAGGGCGATTACTG-3' (SEQ ID NO:17), Reverse
5'-GTGCTTGTGATTTGGAGTCTGTCAC-3' (SEQ ID NO:18); and murine
.beta.-actin (Actb, NM_007393): Forward 5'-TCCTAGCACCATGAAGATC-3'
(SEQ ID NO:19). Reverse 5'-AAACGCAGCTCAGTAACAG-3' (SEQ ID
NO:20).
[0148] Histopathology and immunohistochemical staining. To recover
intact mouse ocular tissue, the whole decapitated mouse head was
fixed in 4% paraformaldehyde (PFA) overnight, then eyes were
enucleated with the lids attached, incubated in 30% sucrose in
1.times. phosphate buffered saline (PBS) overnight at 4.degree. C.,
embedded in Tissue-Tek OCT compound (Sakura Finetek USA, CA) and
snap-frozen. Representative cryostat sections from each tissue
block were stained with hematoxylin and eosin (H&E). For
immunofluorescence staining, cryostat sections (10 .mu.m) were
fixed in cold methanol for 10 minutes, blocked in 10% secondary
serum+2% bovine serum albumin (BSA) in 1.times.PBS for 1 hour,
incubated with the primary antibody (or isotype control), followed
by the appropriate secondary antibody for 1 hour at room
temperature. Following several washes, the slides were then
cover-slipped in hard-set mounting media with 4',
6-diamidino-2-phenylindole (DAPI). BrdU staining was performed
using the BrdU In-situ Kit (BD Pharmingen, CA) followed by staining
with rabbit ABCB5 antibody at 1:250 dilution (NBP1-50547, Novus,
CO). TUNEL staining was performed using the In Situ Cell Death Kit
(Roche, IN) and DAPI (Invitrogen, MA) was used to stain all
nucleated cells. All tissue sections were analyzed using a Nikon
Eclipse E800 immunofluorescence microscope. Composite corneal
photographs were assembled using Photoshop (Adobe) to overlay and
match sequential images. Stitching was done by reducing the added
photograph to 50% transparency, matching images, and returning the
composite photograph to 0% transparency. The average number of
epithelial cells per cornea (FIG. 2D) was determined by counting
the number of DAPI-positive cells within the area defined by a 2 mm
trephine in a composite photograph of a complete corneal section.
At least three composite corneal sections were analyzed per mouse,
and five mice were analyzed per group in four replicate
experiments. The percentages of epithelial cells expressing Ki67
(FIGS. 2I and 3C), TUNEL (FIG. 2I) and KRT12 (FIGS. 4A and 4B) were
determined by counting the number of positive cells among the total
number of DAPI-positive corneal epithelial cells using the
techniques described herein. Comparisons between the Abcb5 WT and
Abcb5 KO mice were performed using the unpaired t test. The results
of transplantation experiments were compared using One-way ANOVA
followed by Bonferroni post tests. Differences with P<0.05 were
considered statistically significant.
[0149] Antibodies. The following primary antibodies were used in
flow cytometry experiments: rabbit polyclonal
anti-.DELTA.Np63.alpha. antibody (cloneH-129, Santa Cruz, Calif.),
mouse monoclonal anti-ABCB5 antibody (clone 3C2-2D12) [6], goat
polyclonal anti-cytokeratin antibody (clone L15, Santa Cruz,
Calif.), rabbit polyclonal anti-PAX6 antibody (Covance, CA), rabbit
polyclonal anti-ABCB5 antibody (Novus Biologicals, CO), rabbit
polyclonal IgG isotype control antibody (Abcam, MA), mouse IgG1k
isotype control antibody (BD Biosciences, CA), and goat IgG isotype
control antibody (Santa Cruz, Calif.). The secondary antibodies
were goat anti-mouse FITC (Sigma-Aldrich, MO), Alexa 647 goat
anti-rabbit IgG (Invitrogen, NY) and Dylight 649 donkey anti-goat
(Jackson ImmunoResearch, PA). For human histopathology and
immunohistochemical analyses, the following primary antibodies were
used: mouse monoclonal anti-ABCB5 (clone 3C2-1D12) [6] and rabbit
antibody against .DELTA.Np63.alpha. at 1:75 dilution (sc8344, Santa
Cruz, Calif.) followed by the appropriate secondary antibodies
obtained from Jackson ImmunoResearch, PA: FITC-donkey anti-rabbit
at 1:75 dilution or Alexa Fluor 594-goat anti-mouse at 1:250
dilution. In all cases, isotype-matched antibodies rabbit IgG
(550875, BD Pharmingen, CA) and mouse IgG1kappa isotype control
antibody (BD Biosciences. CA) served as negative controls. For
histopathology and immunohistochemical analyses mouse tissues were
stained with the following primary antibodies: rabbit anti-ABCB5
antibody at 1:250 dilution (NBP1-50547, Novus, CO), rabbit
anti-Pax6 at 1:300 dilution (PRB278P, Covance, CA), goat
anti-cytokeratin 12 (L15) at 1:50 dilution (sc17101, Santa Cruz,
Calif.), rabbit anti-cytokeratin 14 (AF64) at 1:1000 dilution
(PRB-155P, Covance, CA), rabbit anti-Ki67 at 1:200 dilution
(ab66155, Abcam, MA), followed by the appropriate secondary
antibodies obtained from Jackson ImmunoResearch, PA:donkey
anti-goat Alexa Fluor 488 at 1:250 dilution (705-545-003), donkey
anti-rabbit Alexa Fluor 594 at 1:20 dilution (711-585-152), goat
anti-rabbit DyLight 549 at 1:250 dilution (111-504-144), or
Cy3-donkey anti-rabbit at 1:250 dilution (711-165-152). In all
cases, isotyped matched antibodies (rabbit IgG (550875, BD
Pharmingen, CA) and goat IgG (sc2028, Santa Cruz, Calif.) served as
negative controls.
[0150] The concurrently filed Sequence Listing, filed as a text
file, is incorporated by reference herein.
REFERENCES
[0151] Each of the references listed below is incorporated by
reference herein in its entirety. [0152] 1. Davanger, M. &
Evensen, A. Role of the pericorneal papillary structure in renewal
of corneal epithelium. Nature 229, 560-1 (1971). [0153] 2.
Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T. & Lavker, R.
M. Existence of slow cycling limbal epithelial basal cells that can
be preferentially stimulated to proliferate: implications on
epithelial stem cells. Cell 57, 201-9 (1989). [0154] 3. Majo, F.,
Rochat, A., Nicolas, M., Jaoude, G. A. & Barrandon, Y.
Oligopotent stem cells are distributed throughout the mammalian
ocular surface. Nature 456, 250-4 (2008). [0155] 4. Dua, H. S.,
Joseph, A., Shanmuganathan, V. A. & Jones, R. E. Stem cell
differentiation and the effects of deficiency. Eye (Lond) 17,
877-85 (2003). [0156] 5. Rama, P. et al. Limbal stem-cell therapy
and long-term corneal regeneration. N Engl J Med 363, 147-55
(2010). [0157] 6. Frank. N. Y. et al. Regulation of progenitor cell
fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette
transporter. J Biol Chem 278, 47156-65 (2003). [0158] 7. Schatton,
T. et al. Identification of cells initiating human melanomas.
Nature 451, 345-9 (2008). [0159] 8. Pellegrini, G. et al. p63
identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98,
3156-61 (2001). [0160] 9. Li, W. et al. Down-regulation of Pax6 is
associated with abnormal differentiation of corneal epithelial
cells in severe ocular surface diseases. J Pathol 214, 114-22
(2008). [0161] 10. Sun, T. T. & Lavker, R. M. Corneal
epithelial stem cells: past, present, and future. J Investig
Dermatol Symp Proc 9, 202-7 (2004). [0162] 11. Liu, C. Y. et al.
Characterization and chromosomal localization of the
cornea-specific murine keratin gene Krt1.12. J Biol Chem 269,
24627-36 (1994). [0163] 12. Luo, Y. et al. Side population cells
from human melanoma tumors reveal diverse mechanisms for
chemoresistance. J Invest Dermatol 132, 2440-50. [0164] 13. Wilson,
B. J. et al. ABCB5 identifies a therapy-refractory tumor cell
population in colorectal cancer patients. Cancer Res 71, 5307-16
(2011). [0165] 14. Hutcheson, D. A. & Kardon, G. Genetic
manipulations reveal dynamic cell and gene functions: Creating a
new view of myogenesis. Cell Cycle 8, 3675-8 (2009). [0166] 15.
Lakso, M. et al. Efficient in vivo manipulation of mouse genomic
sequences at the zygote stage. Proc Natl Acad Sci U S A 93, 5860-5
(1996). [0167] 16. Meyer-Blazejewska, E. A. et al. From hair to
cornea: toward the therapeutic use of hair follicle-derived stem
cells in the treatment of limbal stem cell deficiency. Stem Cells
29, 57-66 (2011). [0168] 17. Watanabe, K. et al. Human limbal
epithelium contains side population cells expressing the
ATP-binding cassette transporter ABCG2. FEBS Lett 565, 6-10 (2004).
[0169] 18. Frank, N. Y. et al. ABCB5-mediated doxorubicin transport
and chemoresistance in human malignant melanoma. Cancer Res 65,
4320-33 (2005). [0170] 19. Cheung, S. T., Cheung, P. F., Cheng, C.
K., Wong, N. C. & Fan, S. T. Granulin-epithelin precursor and
ATP-dependent binding cassette (ABC)B5 regulate liver cancer cell
chemoresistance. Gastroenterology 140, 344-55 (2011). [0171] 20.
Yang, M. et al. Expression of ABCB5 gene in hematological
malignances and its significance. Leuk Lymphoma 53, 1211-5 (2012).
[0172] 21. Lehne, G. et al. Upregulation of stem cell genes in
multidrug resistant K562 leukemia cells. Leuk Res 33, 1379-85
(2009). [0173] 22. Pellegrini, G. et al. Location and clonal
analysis of stem cells and their differentiated progeny in the
human ocular surface. J Cell Biol 145, 769-82 (1999). [0174] 23.
Meyer-Blazejewska, E. A. et al. Preservation of the limbal stem
cell phenotype by appropriate culture techniques. Invest Ophthalmol
Vis Sci 51, 765-74 (2010). [0175] 24. Krulova, M. et al. A rapid
separation of two distinct populations of mouse corneal epithelial
cells with limbal stem cell characteristics by centrifugation on
percoll gradient. Invest Ophthalmol Vis Sci 49, 3903-8 (2008).
[0176] 25. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly
efficient recombineering-based method for generating conditional
knockout mutations. Genome Res 13, 476-84 (2003). [0177] 26.
Rodriguez, C. I. et al High-efficiency deleter mice show that FLPe
is an alternative to Cre-loxP. Nat Genet 25, 139-40 (2000). [0178]
27. Frank, N. Y. et al. VEGFR-1 expressed by malignant
melanoma-initiating cells is required for tumor growth. Cancer Res
71, 1474-85 (2011). [0179] 28. Frank, N. Y. et al. Regulation of
myogenic progenitor proliferation in human fetal skeletal muscle by
BMP4 and its antagonist Gremlin. J Cell Biol 175, 99-110 (2006).
[0180] 29. Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M. &
Stepp, M. A. A mouse model for the study of recurrent corneal
epithelial erosions: alpha9beta1 integrin implicated in progression
of the disease. Invest Ophthalmol Vis Sci 45, 1775-88 (2004).
[0181] 30. Pellegrini, G. et al. The control of epidermal stem
cells (holoclones) in the treatment of massive full-thickness burns
with autologous keratinocytes cultured on fibrin. Transplantation
68, 868-79 (1999). [0182] 31. Klocke, J. et al. Spontaneous
bacterial keratitis in DC36 knockout mice. Invest Ophthalmol Vis
Sci 52(1), 256-63 (2011) (including Supplemental Appendix). [0183]
32. Okita, K, et al. Generation of germline competent induced
pluripotent stem cells. Nature 448: 313-317 (2007). [0184] 33.
Stadfeld, M. and Hochedlinger, K. Induced pluripotency: history,
mechanisms, and applications, Genes and Development 24, 2239-2263
(2010).
[0185] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
EQUIVALENTS
[0186] While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. It is, therefore, to be
understood that the foregoing embodiments are presented by way of
example only and that, within the scope of the appended claims and
equivalents thereto, inventive embodiments may be practiced
otherwise than as specifically described and claimed. Inventive
embodiments of the present disclosure are directed to each
individual feature, system, article, material, kit, and/or method
described herein. In addition, any combination of two or more such
features, systems, articles, materials, kits, and/or methods, if
such features, systems, articles, materials, kits, and/or methods
are not mutually inconsistent, is included within the inventive
scope of the present disclosure.
[0187] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0188] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0189] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0190] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, w % ben separating items in a list,
"or" or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0191] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently.
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0192] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0193] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0194] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
* * * * *