U.S. patent application number 16/367045 was filed with the patent office on 2019-10-24 for photoreceptors and photoreceptor progenitors produced from pluripotent stem cells.
This patent application is currently assigned to Astellas Institute for Regenerative Medicine. The applicant listed for this patent is Astellas Institute for Regenerative Medicine. Invention is credited to Robert P. Lanza, Shi-Jiang Lu, Wei Wang.
Application Number | 20190321414 16/367045 |
Document ID | / |
Family ID | 51537881 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190321414 |
Kind Code |
A1 |
Lanza; Robert P. ; et
al. |
October 24, 2019 |
PHOTORECEPTORS AND PHOTORECEPTOR PROGENITORS PRODUCED FROM
PLURIPOTENT STEM CELLS
Abstract
Methods are provided for the production of photoreceptor cells
and photoreceptor progenitor cells from pluripotent stem cells.
Additionally provided are compositions of photoreceptor cells and
photoreceptor cells, as well as methods for the therapeutic use
thereof. Exemplary methods may produce substantially pure cultures
of photoreceptor cells and/or photoreceptor cells.
Inventors: |
Lanza; Robert P.; (Clinton,
MA) ; Lu; Shi-Jiang; (Shrewsbury, MA) ; Wang;
Wei; (Rochester, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Institute for Regenerative Medicine |
Marlborough |
MA |
US |
|
|
Assignee: |
Astellas Institute for Regenerative
Medicine
Marlborough
MA
|
Family ID: |
51537881 |
Appl. No.: |
16/367045 |
Filed: |
March 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14214598 |
Mar 14, 2014 |
10307444 |
|
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16367045 |
|
|
|
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61793168 |
Mar 15, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/30 20130101;
C12N 2500/33 20130101; C12N 2501/105 20130101; A61P 27/02 20180101;
C12N 5/0623 20130101; A61P 27/06 20180101; C12N 5/0606 20130101;
C12N 2501/385 20130101; A61P 9/10 20180101; C12N 2501/155 20130101;
C12N 2501/33 20130101; C12N 5/0696 20130101; C12N 5/062 20130101;
C12N 2501/13 20130101; C12N 2501/235 20130101; C12N 2506/02
20130101; A61K 35/545 20130101 |
International
Class: |
A61K 35/545 20060101
A61K035/545; C12N 5/074 20060101 C12N005/074; C12N 5/0793 20060101
C12N005/0793; C12N 5/0735 20060101 C12N005/0735; C12N 5/0797
20060101 C12N005/0797; A61K 35/30 20060101 A61K035/30 |
Claims
1. A method of producing eye field progenitor cells, comprising (a)
culturing pluripotent stem cells in a retinal induction culture
medium.
2. The method of claim 1, wherein said retinal induction culture
medium comprises at least one component selected from the group
consisting of insulin, DMEM/F12, DMEM/high glucose, DMEM/knock-out,
D-glucose, antibiotics, N2 supplement, B27 supplement,
non-essential amino acids, MEM non-essential amino acids,
glutamine, GlutaMAX.TM., Noggin, BMP signaling inhibitor, and any
combination thereof.
3-5. (canceled)
6. The method of claim 2, wherein said retinal induction culture
medium comprises about 450 mg/ml D-glucose or between about 400 and
about 500 mg/ml D-glucose, one or both of streptomycin and
penicillin, optionally in concentrations of about 100 unit/ml of
penicillin and 100 .mu.g/ml of streptomycin, N2 supplement in a
concentration of about 0.1 to 5% or about 1%, B27 supplement in a
concentration of about 0.05-2.0% or about 0.2%, non-essential amino
acids or MEM non-essential amino acids in a concentration of about
0.1 mM, or Noggin at a concentration of between about 5-100 ng/ml
or about 10-100 ng/ml or about 50 ng/ml.
7-18. (canceled)
19. The method of claim 1, wherein said pluripotent stem cells
comprise human ES cells or human iPS cells.
20. The method of claim 1, wherein said pluripotent stem cells are
cultured under feeder-free and/or xeno-free conditions prior to
being cultured in said retinal induction culture medium comprising
insulin, or cultured on a substrate comprising Matrigel.TM. and
optionally in mTESR1 medium.
21-22. (canceled)
23. The method of claim 1, further comprising (b) culturing the
cells in a neural differentiation medium.
24. The method of claim 23, wherein said neural differentiation
medium comprises at least one component selected from the group
consisting of Neurobasal medium, D-glucose, antibiotics, N2
supplement, B27 supplement, non-essential amino acids, MEM
non-essential amino acids, glutamine, GlutaMAX.TM., Noggin, BMP
signaling inhibitor, and any combination thereof.
25. The method of claim 24, wherein said neural differentiation
medium comprises about 450 mg/ml D-glucose or between about 400 and
about 500 mg/ml D-glucose, one or both of streptomycin and
penicillin, optionally in concentrations of about 100 unit/ml of
penicillin and 100 .mu.g/ml of streptomycin, N2 supplement in a
concentration of about 0.1 to 5% or about 2%, B27 supplement in a
concentration of about 0.05-5.0%, about 0.05-2.0% or about 2%,
non-essential amino acids or MEM non-essential amino acids in a
concentration of about 0.1 mM, or Noggin at a concentration of
between about 10-100 ng/ml or about 50 ng/ml.
26-37. (canceled)
38. The method of claim 23, wherein said cells are cultured in said
neural differentiation medium for about 10-60 days or about 15-35
days or about 24 days.
39-43. (canceled)
44. A composition comprising eye field progenitor cells produced
according to the method of claim 1.
45. A composition comprising eye field progenitor cells, wherein
said eye field progenitor cells comprise at least 50%, at least
75%, at least 85%, at least 95%, at least 99% or about 100% of the
cells in said composition.
46-49. (canceled)
50. The composition of claim 45, wherein said eye field progenitor
cells are cryopreserved.
51-52. (canceled)
53. A method of producing retinal neural progenitor cells or
photoreceptor progenitor cells, comprising (a) culturing eye field
progenitor cells in a neural differentiation medium.
54.-88. (canceled)
89. A composition comprising retinal neural progenitor cells
produced according to the method of claim 53.
90. A composition comprising retinal neural progenitor cells,
wherein said retinal neural progenitor cells comprise at least 50%,
at least 75%, at least 85%, at least 95%, at least 99% or about
100% of the cells in said composition.
91-94. (canceled)
95. The composition of claim 89 or 90, wherein said retinal neural
progenitor cells are cryopreserved.
96-97. (canceled)
98. A composition comprising photoreceptor progenitor cells
produced according to the method of claim 53.
99. A composition comprising photoreceptor progenitor cells,
wherein said photoreceptor progenitor cells comprise at least 50%,
at least 75%, at least 85%, at least 95%, at least 99% or about
100% of the cells in said composition.
100-102. (canceled)
103. The composition of claim 98, wherein said photoreceptor
progenitor cells are cryopreserved.
104. A method of treatment of an individual in need thereof,
comprising administering a composition of claim 98 to said
individual.
105. (canceled)
106. A method of producing photoreceptor cells, comprising (a)
culturing photoreceptor progenitor cells in a photoreceptor
differentiation medium.
107. The method of claim 106, wherein said photoreceptor
differentiation medium comprises at least one component selected
from the group consisting of Neurobasal medium, D-glucose,
antibiotics, N2 supplement, B27 supplement, non-essential amino
acids, MEM non-essential amino acids, glutamine, GlutaMAX.TM.,
forskolin, BDNF, CNTF, LIF, DATP, and any combination thereof.
108. The method of claim 106, wherein said photoreceptor
differentiation medium comprises about 450 mg/ml D-glucose or
between about 400 and about 500 mg/ml D-glucose, one or both of
penicillin and streptomycin, optionally in concentrations of about
100 unit/ml of penicillin and optionally about 100 .mu.g/ml of
streptomycin, N2 supplement in a concentration of about 0.1 to 5%
or about 2%, B27 supplement in a concentration of about 0.05-5.0%,
about 0.05-2.0% or about 2%, non-essential amino acids or MEM
non-essential amino acids are present in a concentration of about
0.1 mM, forskolin in a concentration between about 1-100 uM or
about 5 .mu.M, BDNF in a concentration between about 1-100 ng/ml or
about 10 ng/ml, CNTF in a concentration between about 1-100 ng/ml
or about 10 ng/ml, LIF in a concentration between about 5-50 ng/ml
or about 10 ng/ml, or DATP in a concentration between about 1-100
.mu.M or about 10 .mu.M.
109-126. (canceled)
127. The method of claim 106, wherein said photoreceptor progenitor
cells are differentiated from retinal neural progenitor cells,
which are optionally human.
128. A composition comprising photoreceptor cells produced
according to the method of claim 106.
129-131. (canceled)
132. A method of treatment of an individual in need thereof,
comprising administering a composition of claim 128 to said
individual.
133. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/214,598, filed Mar. 14, 2014, which claims the benefit under
35 U.S.C. .sctn. 119(e) of U.S. Provisional Application Ser. No.
61/793,168, entitled "PHOTORECEPTORS AND PHOTORECEPTOR PROGENITORS
PRODUCED FROM PLURIPOTENT STEM CELLS," filed on Mar. 15, 2013, the
entire contents of each of which are incorporated herein by
reference.
BACKGROUND
[0002] Retinal diseases often result in blindness due to loss of
post-mitotic neuronal cells. Among the retinal diseases are rod or
cone dystrophies, retinal degeneration, retinitis pigmentosa,
diabetic retinopathy, macular degeneration, Leber congenital
amaurosis and Stargardt disease. In most retinal degenerations,
cell loss is primarily in the outer nuclear layer which includes
rod and cone photoreceptors. With the loss of post-mitotic neuronal
cell populations, an exogenous source of new cells as a replacement
for photoreceptor cells is needed.
[0003] A potential replacement source of photoreceptor cells
includes stem cells. Early studies incorporated the use of mouse
cells, mouse stem cells or heterogeneous populations of retinal
progenitor cells as a possible source of cells for replacement of
lost photoreceptors. These early studies described transplantation
of photoreceptor precursor cells from postnatal day 1 mouse retina
(Maclaren et al. Nature 444(9):203-207, 2006), in vitro generation
of retinal precursor cells from mouse embryonic stem cells (Ikeda
et al. Proc. Natl. Acad. Sci. 102(32):11331-11336, 2005),
generation of retinal progenitor cells from postnatal day 1 mouse
retinas (Klassen et al. Invest. Ophthal. Vis. Sci.
45(11):4167-4175, 2004), implantation of bone marrow mesenchymal
stem cells in an RCS rat model of retinal degeneration (Inoue et
al. Exp. Eye Res. 8(2):234-241, 2007), production of retinal
progenitor cells, including ganglion cells, amacrine cells,
photoreceptors wherein 0.01% of the total cells expressed S-opsin
or rhodopsin, bipolar cells and horizontal cells, from the H1 human
embryonic stem cell line (Lamba et al. Proc. Natl. Acad. Sci.
10(34):12769-12774, 2006) and induction of induced pluripotent stem
cells (iPS) from human fibroblasts to produce retinal progenitor
cells (Lamba et al. PLoS ONE 5(1):e8763.
doi:10.1371/journal.pone.0008763). None of these approaches
produced a homogeneous population of photoreceptor progenitor cells
or photoreceptor cells for implantation. None of these approaches
produced a homogeneous population of photoreceptor progenitor cells
or photoreceptor cells that showed in vivo rod or cone function
(e.g., detectable by conferring improvements in visual acuity).
Supplies of donor-derived tissue from which photoreceptors and
photoreceptor progenitors may be isolated (such as cadavers, fetal
tissue, and live animals) are limited. Stem cells can be propagated
and expanded in vitro indefinitely, providing a potentially
inexhaustible source of non-donor derived cells for human therapy.
Differentiation of stem cells into a homogeneous population of
photoreceptor progenitors or photoreceptors may provide an abundant
supply of non-donor derived cells for implantation and treatment of
retinal diseases.
BRIEF SUMMARY
[0004] In one aspect, the disclosure provides a method of producing
eye field progenitor cells, comprising (a) culturing pluripotent
stem cells in a retinal induction culture medium. Said pluripotent
stem cells may be human.
[0005] Said retinal induction culture medium may comprise insulin.
Said insulin may be human. Said insulin may be present in a
concentration of about 5-50 .mu.g/ml human insulin or about 25
.mu.g/ml.
[0006] Said retinal induction culture medium may comprise DMEM/F12,
DMEM/high glucose, or DMEM/knock-out. Said retinal induction
culture medium may comprise about 450 mg/ml D-glucose or between
about 400 and about 500 mg/ml D-glucose.
[0007] The retinal induction culture medium may comprise one or
more antibiotics. Said antibiotics may include one or both of
penicillin and streptomycin, optionally in concentrations of about
100 unit/ml of penicillin and optionally about 100 .mu.g/ml of
streptomycin.
[0008] The retinal induction culture medium may comprise N2
supplement. Said N2 supplement may be present in a concentration of
about 0.1 to 5% or about 1%.
[0009] The retinal induction culture medium may comprise B27
supplement. Said B27 supplement may be present in a concentration
of about 0.05-2.0% or about 0.2%.
[0010] The retinal induction culture medium may comprise
non-essential amino acids or MEM non-essential amino acids or
glutamine or GlutaMAX.TM.. Said non-essential amino acids or MEM
non-essential amino acids may be present in a concentration of
about 0.1 mM
[0011] The retinal induction culture medium may comprise a BMP
signaling inhibitor. Said BMP signaling inhibitor may be selected
from the group consisting of: Noggin polypeptide, dorsomorphin,
LDN-193189, and any combination thereof.
[0012] The retinal induction culture medium may comprise Noggin.
Said Noggin may be present at a concentration of between about
5-100 ng/ml or about 10-100 ng/ml or about 50 ng/ml.
[0013] Said pluripotent stem cells may comprise human ES cells or
human iPS cells. Said pluripotent stem cells may be cultured under
feeder-free and/or xeno-free conditions prior to being cultured in
said retinal induction culture medium comprising insulin, or a
cultured on a substrate comprising Matrigel.TM. and optionally in
mTESR1 medium.
[0014] Said retinal induction culture medium may be replaced with
fresh retinal induction culture medium daily. Said culturing in
step (a) may be continued for about 1-10 days or about 2-7 days, or
about 5-6 days.
[0015] The method may further comprise (b) culturing the cells in a
neural differentiation medium. Said neural differentiation medium
may comprise Neurobasal medium. Said neural differentiation medium
may comprise about 450 mg/ml D-glucose or between about 400 and
about 500 mg/ml D-glucose.
[0016] The neural differentiation medium may comprise one or more
antibiotics. Said antibiotics may include one or both of penicillin
and streptomycin, optionally in concentrations of about 100 unit/ml
of penicillin and optionally about 100 .mu.g/ml of
streptomycin.
[0017] The neural differentiation medium may comprise N2
supplement. Said N2 supplement may be present in a concentration of
about 0.1 to 5% or about 2%.
[0018] The neural differentiation medium may comprise B27
supplement. Said B27 supplement may be present in a concentration
of about 0.05-5.0%, about 0.05-2.0% or about 2%.
[0019] The neural differentiation medium may comprise non-essential
amino acids or MEM non-essential amino acids or glutamine or
GlutaMAX.TM.. Said non-essential amino acids or MEM non-essential
amino acids may be present in a concentration of about 0.1 mM
[0020] The neural differentiation culture medium may comprise a BMP
signaling inhibitor. Said BMP signaling inhibitor may be selected
from the group consisting of: Noggin polypeptide, dorsomorphin,
LDN-193189, and any combination thereof.
[0021] The neural differentiation culture medium may comprise
Noggin. Said Noggin may be present at a concentration of between
about 10-100 ng/ml or about 50 ng/ml.
[0022] Said cells may be cultured in said neural differentiation
medium for about 10-60 days or about 15-35 days or about 24
days.
[0023] Said eye field progenitor cells may comprise at least 50%,
at least 75%, at least 85%, at least 95%, at least 99% or about
100% of the cells in said culture. Said eye field progenitor cells
express one or both of the PAX6 and RX1 markers, and thus may be
PAX6(+) and/or RX1(+). Said eye field progenitor cells may be one
or more of SIX3(+), SIX6(+), LHX2(+), TBX3(+), and/or Nestin(+).
Said eye field progenitor cells may be one or more of SOX2(+) and
OCT4(-) and NANOG(-).
[0024] The method may further comprise differentiating said eye
field progenitor cells into retinal neural progenitor cells.
[0025] Said eye field progenitor cells may be human.
[0026] In another aspect, the disclosure provides a composition
comprising eye field progenitor cells produced using a method as
described herein, e.g., as described in the preceding paragraphs.
In another aspect, the disclosure provides a composition comprising
eye field progenitor cells, which are optionally human.
[0027] Said eye field progenitor cells may comprise at least 50%,
at least 75%, at least 85%, at least 95%, at least 99% or about
100% of the cells in said culture. Said eye field progenitor cells
express one or both of the PAX6 and RX1 markers, and thus may be
PAX6(+) and/or RX1(+). Said eye field progenitor cells may be one
or more of SIX3(+), SIX6(+), LHX2(+), TBX3(+), and/or Nestin(+).
Said eye field progenitor cells may be one or more of SOX2(+) and
OCT4(-) and NANOG(-).
[0028] Said eye field progenitor cells may be cryopreserved.
[0029] In another aspect, the disclosure provides a method of
treatment of an individual in need thereof, comprising
administering a composition comprising eye field progenitor cells
(e.g., a composition as described herein or a composition produced
using a method as described herein) to said individual. Said
composition may be administered to the eye, subretinal space, or
intravenously.
[0030] In another aspect, the disclosure provides a method of
producing retinal neural progenitor cells or photoreceptor
progenitor cells, comprising (a) culturing eye field progenitor
cells in a neural differentiation medium.
[0031] Said neural differentiation medium may comprise Neurobasal
medium. Said neural differentiation medium may comprise about 450
mg/ml D-glucose or between about 400 and about 500 mg/ml
D-glucose.
[0032] The neural differentiation medium may comprise one or more
antibiotics. Said antibiotics may include one or both of penicillin
and streptomycin, optionally in concentrations of about 100 unit/ml
of penicillin and optionally about 100 .mu.g/ml of
streptomycin.
[0033] The neural differentiation medium may comprise N2
supplement. Said N2 supplement may be present in a concentration of
about 0.1 to 5% or about 2%.
[0034] The neural differentiation medium may comprise B27
supplement. Said B27 supplement may be present in a concentration
of about 0.05-5.0%, about 0.05-2.0% or about 2%.
[0035] The neural differentiation medium may comprise non-essential
amino acids or MEM non-essential amino acids or glutamine or
GlutaMAX.TM.. Said non-essential amino acids or MEM non-essential
amino acids may be present in a concentration of about 0.1 mM
[0036] The neural differentiation medium optionally does not
contain exogenously added Noggin. The neural differentiation
culture medium optionally does not comprises an exogenously added
BMP signaling inhibitor.
[0037] Step (a) may comprise (i) culturing eye field progenitor
cells until the cells form spheres form, and (ii) plating the
spheres under adherent conditions.
[0038] Step (i) may comprise culturing the cells on low-adherent
plates. Step (i) may comprise culturing the cells in a hanging
drop. The culture of step (i) may be formed by mechanically or
enzymatically breaking cultured cells into a single cell
suspension. Step (i) may be continued for 1-10, 3-8, or about 5
days.
[0039] Step (ii) may comprise plating the spheres on Matrigel.TM..
Step (ii) may comprise plating the spheres on laminin or collagen.
Step (ii) may be continued until said culture is confluent.
[0040] Steps (i) and (ii) may be repeated in an alternating
fashion.
[0041] Said cells may be cultured in said neural differentiation
medium for about 10-60 days or about 15-35 days or about 25
days.
[0042] Said retinal neural progenitor cells may differentiate from
said eye field progenitor cells and may be present in increasing
numbers in said culture.
[0043] Said retinal neural progenitor cells may comprise at least
50%, at least 75%, at least 85%, at least 95%, at least 99% or
about 100% of the cells in said culture. Said retinal neural
progenitor cells may express one or both of the PAX6 and CHX10
markers, and thus may be PAX6(+) and/or CHX10(+). Said retinal
neural progenitor cells may be SOX2-. Said retinal neural
progenitor cells may be Tuj1(+) or Tuj1(-).
[0044] Said cells may be cultured in said neural differentiation
medium for about 10-330 days or about 15-300 days or about 10-100
days or about 15-100 days or about 100 days.
[0045] Said photoreceptor progenitor cells differentiate from said
retinal neural progenitor cells and may be present in increasing
numbers in said culture.
[0046] Said photoreceptor progenitor cells may comprise at least
50%, at least 75%, at least 85%, at least 95%, at least 99% or
about 100% of the cells in said culture. Said photoreceptor
progenitor cells may be PAX6(+) and/or CHX10(-). Said photoreceptor
progenitor cells may express one or more of the Nr2e3, Tr.beta.2,
Mash1, ROR.beta. and/or NRL markers, and thus may be Nr2e3(+),
Tr.beta.2(+), Mash1(+), ROR.beta.(+) and/or NRL(+).
[0047] Said cells may be cultured in said neural differentiation
medium for at least about 130 days, at least about 160 days, at
least about 190 days, or longer, whereby said photoreceptor
progenitor cells exhibit decreased or absent ability to
differentiate into cones while retaining the ability to form
rods.
[0048] The method may further comprise differentiating said
photoreceptor progenitor cells into photoreceptors.
[0049] Said eye field progenitor cells may be differentiated from a
pluripotent cell, ES cell, or iPS cell, which pluripotent cell, ES
cell, or iPS cell may optionally be human.
[0050] In another aspect, said retinal neural progenitor cells may
be human.
[0051] In another aspect, the disclosure provides a composition
comprising retinal neural progenitor cells produced according to
any method described herein, e.g., the methods described in the
preceding paragraphs. In another aspect, the disclosure provides a
composition comprising retinal neural progenitor cells, which are
optionally human.
[0052] Said retinal neural progenitor cells may comprise at least
50%, at least 75%, at least 85%, at least 95%, at least 99% or
about 100% of the cells in said culture.
[0053] Said retinal neural progenitor cells may express one or both
of the PAX6 and CHX10 markers, and thus may be PAX6(+) and/or
CHX10(+). Said retinal neural progenitor cells may be SOX2(-). Said
retinal neural progenitor cells may be Tuj1(+) or Tuj1(-).
[0054] Said retinal neural progenitor cells may be
cryopreserved.
[0055] In another aspect, the disclosure provides a method of
treatment of an individual in need thereof, comprising
administering a composition comprising retinal neural progenitor
cells, e.g., a composition described herein or a composition
produced according to a method described herein, to said
individual. Said composition may be administered to the eye,
subretinal space, or intravenously. Said photoreceptor progenitor
cells may be human.
[0056] In another aspect, the disclosure provides a composition
comprising photoreceptor progenitor cells produced according to a
method described herein, e.g., a method according to the preceding
paragraphs. In another aspect, the disclosure provides a
composition comprising photoreceptor progenitor cells, which are
optionally human.
[0057] Said photoreceptor progenitor cells may comprise at least
50%, at least 75%, at least 85%, at least 95%, at least 99% or
about 100% of the cells in said culture. Said photoreceptor
progenitor cells may be PAX6(+) and/or CHX10(-). Said photoreceptor
progenitor cells express one or more of the Nr2e3, Tr.beta.2,
Mash1, ROR.beta. and/or NRL markers, and thus may be Nr2e3(+),
Tr.beta.2(+), Mash1(+), ROR.beta.(+) and/or NRL(+).
[0058] Said photoreceptor progenitor cells may be
cryopreserved.
[0059] In another aspect, the disclosure provides a method of
treatment of an individual in need thereof, comprising
administering a composition comprising photoreceptor progenitor
cells, e.g., a composition as described herein e.g., in the
preceding paragraphs, or a composition produced according to the
methods described herein e.g., in the preceding paragraphs, to said
individual. Said composition may be administered to the eye,
subretinal space, or intravenously.
[0060] In another aspect, the disclosure provides a method of
producing photoreceptor cells, comprising (a) culturing
photoreceptor progenitor cells in a photoreceptor differentiation
medium.
[0061] Said photoreceptor differentiation medium may comprise
Neurobasal medium. Said photoreceptor differentiation medium may
comprise about 450 mg/ml D-glucose or between about 400 and about
500 mg/ml D-glucose.
[0062] The photoreceptor differentiation medium may comprise one or
more antibiotics. Said antibiotics may include one or both of
penicillin and streptomycin, optionally in concentrations of about
100 unit/ml of penicillin and optionally about 100 .mu.g/ml of
streptomycin.
[0063] The photoreceptor differentiation medium may comprise N2
supplement. Said N2 supplement may be present in a concentration of
about 0.1 to 5% or about 2%.
[0064] The photoreceptor differentiation medium may comprise B27
supplement. Said B27 supplement may be present in a concentration
of about 0.05-5.0%, about 0.05-2.0% or about 2%.
[0065] The photoreceptor differentiation medium may comprise
non-essential amino acids or MEM non-essential amino acids or
glutamine or GlutaMAX.TM.. Said non-essential amino acids or MEM
non-essential amino acids may be present in a concentration of
about 0.1 mM
[0066] Said photoreceptor differentiation medium may comprise
forskolin. Said forskolin may be present in the photoreceptor
differentiation medium at a concentration between about 1-100 .mu.M
or about 5 .mu.M.
[0067] Said photoreceptor differentiation medium may comprise BDNF.
Said BDNF may be present in the photoreceptor differentiation
medium at a concentration between about 1-100 ng/ml or about 10
ng/ml.
[0068] Said photoreceptor differentiation medium may comprise CNTF.
Said CNTF may be present in the photoreceptor differentiation
medium at a concentration between about 1-100 ng/ml or about 10
ng/ml.
[0069] Said photoreceptor differentiation medium may comprise LIF.
Said LIF may be present in the photoreceptor differentiation medium
at a concentration between about 5-50 ng/ml or about 10 ng/ml.
[0070] Said photoreceptor differentiation medium may comprise DATP.
Said DATP may be present in the photoreceptor differentiation
medium at a concentration between about 1-100 .mu.M or about 10
.mu.M.
[0071] Said photoreceptor progenitor cells may be differentiated
from retinal neural progenitor cells, which are optionally human.
Said photoreceptor cells may be human.
[0072] In another aspect, the disclosure provides a composition
comprising photoreceptor cells produced according to a method as
described herein, e.g., in the preceding paragraphs, which are
optionally human. Said photoreceptors may be PAX6(-). Said
photoreceptor cells may comprise at least 50%, at least 75%, at
least 85%, at least 95%, at least 99% or about 100% of the cells in
said culture. Said photoreceptor cells may be cryopreserved.
[0073] In another aspect, the disclosure provides a method of
treatment of an individual in need thereof, comprising
administering a composition comprising photoreceptor cells, e.g., a
composition as described herein such as in the preceding paragraphs
or a composition produced by a method as described herein e.g., in
the preceding paragraphs, to said individual. Said composition may
be administered to the eye, subretinal space, or intravenously.
[0074] In another embodiment, the invention is directed to a
substantially pure preparation of photoreceptor progenitor cells
(PRPCs) or photoreceptor cells (PRs) of human origin, preferably
non-donor derived photoreceptor progenitor cells or photoreceptor
cells, originating from cells not grown on a mouse fibroblast
feeder platform. For example, the preparation may be 85%-95% pure.
In an embodiment, the invention is directed to a method of
preparing the substantially pure preparation of PRPCs or PRs of
human origin which omits the need for cells derived from a mouse
fibroblast feeder platform. Replacing a feeder system with the
methods of the present invention produces a greater homogeneity of
photoreceptors cells, e.g., at 75%-100% or 85%-95%. The
differentiation of the feeder-free stem cells can also occur in the
absence of the introduction of exogenous inducing factors, which is
a substantial improvement over the prior art. The optional addition
of Noggin, however, can accelerate differentiation of the stem
cells, even though it is not necessary for differentiation to
occur. The resultant photoreceptor progenitor cells are uniquely
characterized immunocytochemically as PAX6 positive and CHX10
negative.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0076] FIG. 1: Real-time PCR analysis of transcripts of eye field
transcription factors in cells differentiated under different
conditions.
[0077] FIGS. 2A-2C: Morphology of differentiating cells. (FIG. 2A)
At day 1 after cell differentiation, cells at the colony margin
were column-shaped (arrow). (FIG. 2B) At day 10 after
differentiation, the edge cells became big and flat (arrow head)
and the central cells were small and compact (arrow). (FIG. 2C)
Rosette like structures formed at day 21.
[0078] FIGS. 3A-3E: Cells cultured at 21 days after initiation of
differentiation expressed eye-field transcription factors. (FIG.
3A) Co-expression of PAX6 (green) and RX1 (red), as is apparent
from the color version of the Figure. (FIG. 3B) 93% of cells
co-expressed PAX6 and RX1 as shown by dual-color flow cytometric
analysis. (FIG. 3C) Cells expressed Nestin (red). (FIG. 3D) Cells
expressed SOX2 (red). In both (C) and (D), DAPI (blue) labels cell
nuclei. (FIG. 3E) RT-PCR analysis of transcripts of eye field
transcription factors: RX1, PAX6, LHX2, SIX3, SIX6, TBX3 and
SOX2.
[0079] FIGS. 4A-4C: Cells cultured at 30 days after initiation of
differentiation expressed retinal neural progenitor markers. (FIG.
4A) Morphology of cells. After plating on Matrigel.TM., neurons
migrated out from cell aggregates (arrow). A few epithelial-like
cells (arrow head) are observed around cell aggregates. (FIG. 4B)
Upper panel, phase contrast image of migrating neurons; Lower
panel, migrating neurons expressed Tuj1 (red). (FIG. 4C) Cells
co-expressed PAX6 (red) and CHX10 (green), as is apparent from the
color version of the Figure.
[0080] FIGS. 5A-5D: Cells cultured at 3 months after initiation of
differentiation. (FIG. 5A) Morphology of cells. (FIG. 5B) Cells
express PAX6 but not CHX10, as is apparent from the color version
of the Figure which shows red staining of some of the cells but no
green staining. (FIG. 5C) The expression of Recoverin was
restricted to the cytoplasm of the cell body, as is apparent from
the color version of the Figure. (FIG. 5D) Real-time RT-PCT
analysis of transcripts of Rhodopsin, Opsin, and Recoverin in
retinal neural progenitors (RNPs) and photoreceptor progenitor
cells (indicated as PhRPs).
[0081] FIGS. 6A-6D: Differentiated cells express photoreceptor cell
markers. Cells expressed (FIG. 6A) Rhodopsin (red), (FIG. 6B)
Rhodopsin (red) and Recoverin (green), (FIG. 6C) Opsin (green), and
(FIG. 6D) phosphodiesterase 6A alpha subunit (PDE6a) (red). DAPI
(blue) labels cell nuclei. Expression of these markers is apparent
from the color version of the Figure.
[0082] FIG. 7: Schematic diagram of animal studies in
ELOVL4-transgenic mice.
[0083] FIG. 8: Scotopic ERG intensity-response function recorded at
one month after subretinal cell injection. Stimulus intensity
curves for scotopic a-waves (upper panel) and b-waves (lower panel)
from ELOVL4-TG2 mice administered PBS (black line) or photoreceptor
progenitor cells (indicated as PhRPs, grey line). p<0.001 (vs.
PBS).
[0084] FIG. 9: Scotopic ERG intensity-response function recorded at
one month after systemic cell injection. Stimulus intensity curves
for scotopic a-waves (upper panel) and b-waves (lower panel) from
ELOVL4-TG2 mice administered PBS, photoreceptor progenitor cells
(indicated as PhRPs) or retinoic acid treated photoreceptor
progenitor cells (PhRPs-RA). Blank represents untreated mice. #,
p<0.01 (vs. PBS).
[0085] FIGS. 10A-10B. Photoreceptor progenitor cell systemic
injection restores rod function between one month and two months
after cell transplantation. Scotopic ERG amplitude of a-waves (FIG.
10A) and b-waves (FIG. 10B) at one and two month after cell
injection from ELOVL4-TG2 mice administered PBS (PBS) or retinoic
acid treated photoreceptor progenitor cells (PhRPs-RA).
[0086] FIG. 10C: Scotopic ERG intensity-response function recorded
at two months after systemic cell injection. Stimulus intensity
curves for scotopic a-waves (upper panel) and b-waves (lower panel)
from ELOVL4-TG2 mice administered PBS or retinoic acid treated
photoreceptor progenitor cells (PhRPs-RA). Blank represents
untreated mice. Baseline is the level recorded at 4 weeks.
p<0.001 (vs. PBS).
[0087] FIG. 11: Photopic ERG amplitude of a-waves (upper panel) and
b-waves (lower panel) at one and two month after cell injection
from ELOVL4-TG2 mice administered with PBS or retinoic acid treated
Photoreceptor progenitors (PhRPs-RA). p<0.001 (vs. PBS 2
month).
[0088] FIG. 12: Whole central retina thickness measured by OCT at
one and two months after cell injection from untreated ELOVL4-TG2
mice (blank) and mice administered PBS, photoreceptor progenitor
cells (PhRPs), or retinoic acid treated photoreceptor progenitor
cells (PhRPs-RA).
[0089] FIGS. 13A-13B: (FIG. 13A) Representative images of retina HE
staining at two months after cell injection from ELOVL4-TG2 mice
administered PBS (Left panel) and retinoic acid treated
photoreceptor progenitor cells (Right panel). ONL, outer nuclear
layer. INL, internal nuclear layer. (FIG. 13B), Quantification of
the thickness of ONL of retina at two month after cell injection
from untreated ELOVL4-TG2 mice (blank) and mice administered PBS or
retinoic acid treated Photoreceptor progenitors (PhRPs-RA).
[0090] FIG. 14: Schematic diagram of animal studies in RCS
rats.
[0091] FIGS. 15A-15C: Preservation of host photoreceptor cells
after transplantation of human ES cell-derived photoreceptor
progenitor cells. Retinal sections at P90 stained with DAPI: (FIG.
15A) Outer nuclear layer (ONL) is reduced to 0-1 layer in control
rats. (FIG. 15B) Rescued ONL cells in RCS rat after intravenous
cell injection, which is 2-4 cells deep. (FIG. 15C) Rescued ONL
cells in RCS rat receiving intravitreal cell injection, which is
3-5 cells deep. INL, inner nuclear layer; GL, ganglion cell
layer.
[0092] FIGS. 16A-16C: Preservation of host rod photoreceptor cell
outer segment (OS) after transplantation of human ES cell-derived
photoreceptor progenitor cells. Retinal sections at P90 stained for
Rhodopsin (green). (FIG. 16A) Complete loss of rod OS in control
rats (arrow). (FIG. 16B) Expression of Rhodopsin in the OS of host
rod photoreceptor cells in RCS rat retina after intravenous
injection of photoreceptor progenitor cells (arrow). (FIG. 16C)
Expression of Rhodopsin in the OS of host rod photoreceptor cells
in RCS rat retina after intravitreal transplantation of
photoreceptor progenitor cells (arrow). Expression of Rhodopsin is
apparent in the color version of the Figure.
[0093] FIGS. 17A-17C: Preservation of host cone photoreceptor cell
outer segment (OS) after transplantation of human ES cell-derived
photoreceptor progenitor cells. Retinal sections at P90 stained for
Opsin (green). (FIG. 17A) Complete loss of cone OS in control rats
(arrow). (FIG. 17B) Expression of Opsin in the OS of host cone
photoreceptor cells in RCS rat retina after intravenous injection
of photoreceptor progenitor cells (arrow). (FIG. 17C) Expression of
Opsin in the OS of host cone photoreceptor cells in RCS rat retina
after intravitreal transplantation of photoreceptor progenitors
(arrow). Expression of Opsin is apparent in the color version of
the Figure.
[0094] FIGS. 18A-18B: Human ES cell-derived photoreceptor
progenitor cells transplanted into the vitreous of RCS rats
differentiated into mature rod photoreceptor cells. Retinal
sections at P90 stained for rhodopsin (green in FIG. 18A),
Recoverin (green in FIG. 18B). Human cells were labeled with
anti-HuNU antibody (red). DAPI labeled all nuclei. Expression of
rhodopsin and recoverin and staining with DAPI is apparent in the
color version of the Figure.
[0095] FIG. 19: Illustrates the overall method used for
photoreceptor development in Examples 1-2 and further illustrates
the media used at each step of the process.
[0096] FIG. 20: Illustrates the timing of photoreceptor cell and
photoreceptor progenitor cell development in Examples 1-2.
[0097] FIGS. 21A-21E: Show the components of culture media and
media supplements used in the Examples.
[0098] FIG. 22: Illustrates the gene expression pattern of ESC, eye
field progenitor cells, retinal neural progenitor cells,
photoreceptor progenitor cells, and photoreceptor cells during in
vitro differentiation from human pluripotent stem cells.
DETAILED DESCRIPTION
Definitions
[0099] As defined here, singular forms are provided for
illustrative purposes, but may also apply to plural versions of the
phrase. The following definitions are meant to supplement
conventional definitions of the terms as they would be understood
by persons of ordinary skill.
[0100] "Substantially pure preparation of photoreceptor progenitor
cells (PRPCs)." As used herein, this phrase refers to a preparation
of cells (e.g., a composition comprising cells) wherein the cells
are at least 75% pure or preferably at least 85% pure, at least 95%
pure, or are about 85% to 95% pure. For example, the level of
purity may be quantified by determining the proportion of cells in
the preparation that express one or more markers, such as those
markers of PRPCs (including those markers identified in this
application or others known in the art), relative to the total
number of cells in the preparation, e.g., by detecting cells that
do or do not express said one or more markers. Optionally
expression of markers indicative of non-PRPC cells may also be
detected, thereby facilitating detection and/or quantitation of
said cells. Exemplary methods that may be utilized to include,
without limitation, Fluorescence Activated Cell Sorting (FACS),
immunohistochemistry, in situ hybridization, and other suitable
methods known in the art. Optionally the determination of purity
may be performed disregarding non-viable cells present in the
preparation.
[0101] "Substantially pure preparation of photoreceptor cells (PRs)
of human origin." As used herein, this phrase refers to a
preparation of cells (e.g., a composition comprising cells) wherein
the cells are at least 75% pure or preferably at least 85% pure, at
least 95% pure, or are about 85% to 95% pure. For example, the
level of purity may be quantified by determining the proportion of
cells in the preparation that express one or more markers, such as
those markers of PRs (including those markers identified in this
application or others known in the art), relative to the total
number of cells in the preparation, e.g., by detecting cells that
do or do not express said one or more markers. Optionally
expression of markers indicative of non-PR cells may also be
detected, thereby facilitating detection and/or quantitation of
said cells. Exemplary methods that may be utilized to include,
without limitation, Fluorescence Activated Cell Sorting (FACS),
immunohistochemistry, in situ hybridization, and other suitable
methods known in the art. Optionally the determination of purity
may be performed disregarding non-viable cells present in the
preparation.
[0102] "Embryoid bodies" refers to clumps or clusters of
pluripotent cells (e.g., iPSC or ESC) which may be formed by
culturing pluripotent cells under non-attached conditions, e.g., on
a low-adherent substrate or in a "hanging drop." In these cultures,
pluripotent cells can form clumps or clusters of cells denominated
as embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000
February; 6(2):88-95, which is hereby incorporated by reference in
its entirety. Typically, embryoid bodies initially form as solid
clumps or clusters of pluripotent cells, and over time some of the
embryoid bodies come to include fluid filled cavities, the latter
former being referred to in the literature as "simple" EBs and the
latter as "cystic" embryoid bodies.
[0103] The term "embryonic stem cell" (ES cell or ESC) is used
herein as it is used in the art. This term includes cells derived
from the inner cell mass of human blastocysts or morulae, including
those that have been serially passaged as cell lines. The ES cells
may be derived from fertilization of an egg cell with sperm, as
well as using DNA, nuclear transfer, parthenogenesis, or by means
to generate ES cells with homozygosity in the HLA region. ES cells
are also cells derived from a zygote, blastomeres, or
blastocyst-staged mammalian embryo produced by the fusion of a
sperm and egg cell, nuclear transfer, parthenogenesis,
androgenesis, or the reprogramming of chromatin and subsequent
incorporation of the reprogrammed chromatin into a plasma membrane
to produce a cell. Embryonic stem cells, regardless of their source
or the particular method used to produce them, can be identified
based on (i) the ability to differentiate into cells of all three
germ layers, (ii) expression of at least OCT4 and alkaline
phosphatase, and (iii) ability to produce teratomas when
transplanted into immunodeficient animals. Embryonic stem cells
that may be used in embodiments of the present invention include,
but are not limited to, human ES cells ("ESC" or "hES cells") such
as MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14 and ACT30 embryonic
stem cells. Additional exemplary cell lines include NED1, NED2,
NED3, NED4, NED5, and NED7. See also NIH Human Embryonic Stem Cell
Registry. An exemplary human embryonic stem cell line that may be
used is MA09 cells. The isolation and preparation of MA09 cells was
previously described in Klimanskaya, et al. (2006) "Human Embryonic
Stem Cell lines Derived from Single Blastomeres." Nature 444:
481-485. The human ES cells used in accordance with exemplary
embodiments of the present invention may be derived and maintained
in accordance with GMP standards.
[0104] As used herein, the term "pluripotent stem cells" includes
embryonic stem cells, embryo-derived stem cells, and induced
pluripotent stem cells, regardless of the method by which the
pluripotent stem cells are derived. Pluripotent stem cells are
defined functionally as stem cells that are: (a) capable of
inducing teratomas when transplanted in immunodeficient (SCID)
mice; (b) capable of differentiating to cell types of all three
germ layers (e.g., can differentiate to ectodermal, mesodermal, and
endodermal cell types); and (c) express one or more markers of
embryonic stem cells (e.g., express OCT4, alkaline phosphatase.
SSEA-3 surface antigen, SSEA-4 surface antigen, NANOG, TRA-1-60,
TRA-1-81, SOX2, REX1, etc). In certain embodiments, pluripotent
stem cells express one or more markers selected from the group
consisting of: OCT4, alkaline phosphatase, SSEA-3, SSEA-4,
TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be
generated using, for example, methods known in the art. Exemplary
pluripotent stem cells include embryonic stem cells derived from
the ICM of blastocyst stage embryos, as well as embryonic stem
cells derived from one or more blastomeres of a cleavage stage or
morula stage embryo (optionally without destroying the remainder of
the embryo). Such embryonic stem cells can be generated from
embryonic material produced by fertilization or by asexual means,
including somatic cell nuclear transfer (SCNT), parthenogenesis,
and androgenesis. Further exemplary pluripotent stem cells include
induced pluripotent stem cells (iPSCs) generated by reprogramming a
somatic cell by expressing a combination of factors (herein
referred to as reprogramming factors). The iPSCs can be generated
using fetal, postnatal, newborn, juvenile, or adult somatic
cells.
[0105] In certain embodiments, factors that can be used to
reprogram somatic cells to pluripotent stem cells include, for
example, a combination of OCT4 (sometimes referred to as OCT3/4),
SOX2, c-Myc, and Klf4. In other embodiments, factors that can be
used to reprogram somatic cells to pluripotent stem cells include,
for example, a combination of OCT4, SOX2, NANOG, and LIN28. In
certain embodiments, at least two reprogramming factors are
expressed in a somatic cell to successfully reprogram the somatic
cell. In other embodiments, at least three reprogramming factors
are expressed in a somatic cell to successfully reprogram the
somatic cell. In other embodiments, at least four reprogramming
factors are expressed in a somatic cell to successfully reprogram
the somatic cell. In other embodiments, additional reprogramming
factors are identified and used alone or in combination with one or
more known reprogramming factors to reprogram a somatic cell to a
pluripotent stem cell. Induced pluripotent stem cells are defined
functionally and include cells that are reprogrammed using any of a
variety of methods (integrative vectors, non-integrative vectors,
chemical means, etc). Pluripotent stem cells may be genetically
modified or otherwise modified to increase longevity, potency,
homing, to prevent or reduce alloimmune responses or to deliver a
desired factor in cells that are differentiated from such
pluripotent cells (for example, photoreceptors, photoreceptor
progenitor cells, rods, cones, etc. and other cell types described
herein, e.g., in the examples).
[0106] "Induced pluripotent stem cells" (iPS cells or iPSC) can be
produced by protein transduction of reprogramming factors in a
somatic cell. In certain embodiments, at least two reprogramming
proteins are transduced into a somatic cell to successfully
reprogram the somatic cell. In other embodiments, at least three
reprogramming proteins are transduced into a somatic cell to
successfully reprogram the somatic cell. In other embodiments, at
least four reprogramming proteins are transduced into a somatic
cell to successfully reprogram the somatic cell.
[0107] The pluripotent stem cells can be from any species.
Embryonic stem cells have been successfully derived in, for
example, mice, multiple species of non-human primates, and humans,
and embryonic stem-like cells have been generated from numerous
additional species. Thus, one of skill in the art can generate
embryonic stem cells and embryo-derived stem cells from any
species, including but not limited to, human, non-human primates,
rodents (mice, rats), ungulates (cows, sheep, etc), dogs (domestic
and wild dogs), cats (domestic and wild cats such as lions, tigers,
cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats,
elephants, panda (including giant panda), pigs, raccoon, horse,
zebra, marine mammals (dolphin, whales, etc.) and the like. In
certain embodiments, the species is an endangered species. In
certain embodiments, the species is a currently extinct
species.
[0108] Similarly, iPS cells can be from any species. These iPS
cells have been successfully generated using mouse and human cells.
Furthermore, iPS cells have been successfully generated using
embryonic, fetal, newborn, and adult tissue. Accordingly, one can
readily generate iPS cells using a donor cell from any species.
Thus, one can generate iPS cells from any species, including but
not limited to, human, non-human primates, rodents (mice, rats),
ungulates (cows, sheep, etc), dogs (domestic and wild dogs), cats
(domestic and wild cats such as lions, tigers, cheetahs), rabbits,
hamsters, goats, elephants, panda (including giant panda), pigs,
raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and
the like. In certain embodiments, the species is an endangered
species. In certain embodiments, the species is a currently extinct
species.
[0109] Induced pluripotent stem cells can be generated using, as a
starting point, virtually any somatic cell of any developmental
stage. For example, the cell can be from an embryo, fetus, neonate,
juvenile, or adult donor. Exemplary somatic cells that can be used
include fibroblasts, such as dermal fibroblasts obtained by a skin
sample or biopsy, synoviocytes from synovial tissue, foreskin
cells, cheek cells, or lung fibroblasts. Although skin and cheek
provide a readily available and easily attainable source of
appropriate cells, virtually any cell can be used. In certain
embodiments, the somatic cell is not a fibroblast.
[0110] The induced pluripotent stem cell may be produced by
expressing or inducing the expression of one or more reprogramming
factors in a somatic cell. The somatic cell may be a fibroblast,
such as a dermal fibroblast, synovial fibroblast, or lung
fibroblast, or a non-fibroblastic somatic cell. The somatic cell
may be reprogrammed through causing expression of (such as through
viral transduction, integrating or non-integrating vectors, etc.)
and/or contact with (e.g., using protein transduction domains,
electroporation, microinjection, cationic amphiphiles, fusion with
lipid bilayers containing, detergent permeabilization, etc.) at
least 1, 2, 3, 4, 5 reprogramming factors. The reprogramming
factors may be selected from OCT3/4, SOX2, NANOG, LIN28, C-MYC, and
KLF4. Expression of the reprogramming factors may be induced by
contacting the somatic cells with at least one agent, such as a
small organic molecule agents, that induce expression of
reprogramming factors.
[0111] Further exemplary pluripotent stem cells include induced
pluripotent stem cells generated by reprogramming a somatic cell by
expressing or inducing expression of a combination of factors
("reprogramming factors"). iPS cells may be obtained from a cell
bank. The making of iPS cells may be an initial step in the
production of differentiated cells. iPS cells may be specifically
generated using material from a particular patient or matched donor
with the goal of generating tissue-matched PHRPS or photoreceptor
cells. iPSCs can be produced from cells that are not substantially
immunogenic in an intended recipient, e.g., produced from
autologous cells or from cells histocompatible to an intended
recipient.
[0112] The somatic cell may also be reprogrammed using a
combinatorial approach wherein the reprogramming factor is
expressed (e.g., using a viral vector, plasmid, and the like) and
the expression of the reprogramming factor is induced (e.g., using
a small organic molecule.) For example, reprogramming factors may
be expressed in the somatic cell by infection using a viral vector,
such as a retroviral vector or a lentiviral vector. Also,
reprogramming factors may be expressed in the somatic cell using a
non-integrative vector, such as an episomal plasmid. See, e.g., Yu
et al., Science. 2009 May 8; 324(5928):797-801, which is hereby
incorporated by reference in its entirety. When reprogramming
factors are expressed using non-integrative vectors, the factors
may be expressed in the cells using electroporation, transfection,
or transformation of the somatic cells with the vectors. For
example, in mouse cells, expression of four factors (OCT3/4, SOX2,
C-MYC, and KLF4) using integrative viral vectors is sufficient to
reprogram a somatic cell. In human cells, expression of four
factors (OCT3/4, SOX2, NANOG, and LIN28) using integrative viral
vectors is sufficient to reprogram a somatic cell.
[0113] Once the reprogramming factors are expressed in the cells,
the cells may be cultured. Over time, cells with ES characteristics
appear in the culture dish. The cells may be chosen and subcultured
based on, for example, ES morphology, or based on expression of a
selectable or detectable marker. The cells may be cultured to
produce a culture of cells that resemble ES cells--these are
putative iPS cells.
[0114] To confirm the pluripotency of the iPS cells, the cells may
be tested in one or more assays of pluripotency. For example, the
cells may be tested for expression of ES cell markers; the cells
may be evaluated for ability to produce teratomas when transplanted
into SCID mice; the cells may be evaluated for ability to
differentiate to produce cell types of all three germ layers. Once
a pluripotent iPSC is obtained it may be used to produce cell types
disclosed herein, e.g., photoreceptor progenitor cells,
photoreceptor cells, rods, cones, etc. and other cell types
described herein, e.g., in the examples.
[0115] "Stem cell" is used here to refer to a pluripotent cell
which can proliferate and/or differentiate into a mature cell and
is optionally of human origin.
[0116] "Adult stem cell" refers to a multipotent cell isolated from
adult tissue and can include bone marrow stem cells, cord blood
stem cells and adipose stem cells and is of human origin.
[0117] "Retina" refers to the neural cells of the eye, which are
layered into three nuclear layers comprised of photoreceptors,
horizontal cells, bipolar cells, amacrine cells, Muller glial cells
and ganglion cells.
[0118] "Progenitor cell" refers to a cell that remains mitotic and
can produce more progenitor cells or precursor cells or can
differentiate to an end fate cell lineage.
[0119] "Precursor cell" refers to a cell capable of differentiating
to an end fate cell lineage.
[0120] In embodiments of the invention, an "eye field progenitor
cell" is differentiated from embryonic stem cells or induced
pluripotent stem cells and expresses the markers PAX6 and RX1RX1.
In embodiments of the invention, a "retinal neural progenitor cell"
refers to a cell differentiated from embryonic stem cells or
induced pluripotent stem cells, that expresses the cell markers
PAX6 and CHX10. In embodiments of the invention, "photoreceptor
progenitor" refers to cells differentiated from embryonic stem
cells or induced pluripotent stem cells and that expresses the
marker PAX6 while not expressing the marker CHX10 (i.e. CHX10(-)).
These cells transiently express CHX10 at retinal neural progenitor
stage, but the CHX10 expression is turned off when cells
differentiate into the photoreceptor progenitor stage. Also,
"photoreceptor" may refer to post-mitotic cells differentiated from
embryonic stem cells or induced pluripotent stem cells and that
expresses the cell marker rhodopsin or any of the three cone
opsins, and optionally express the rod or cone cGMP
phosphodiesterase. The photoreceptors may also express the marker
recoverin, which is found in photoreceptors. The photoreceptors may
be rod and/or cone photoreceptors.
[0121] "Signs" of disease, as used herein, refers broadly to any
abnormality indicative of disease, discoverable on examination of
the patient; an objective indication of disease, in contrast to a
symptom, which is a subjective indication of disease.
[0122] "Symptoms" of disease as used herein, refers broadly to any
morbid phenomenon or departure from the normal in structure,
function, or sensation, experienced by the patient and indicative
of disease.
[0123] "Therapy," "therapeutic," "treating," "treat" or
"treatment", as used herein, refers broadly to treating a disease,
arresting or reducing the development of the disease or its
clinical symptoms, and/or relieving the disease, causing regression
of the disease or its clinical symptoms. Therapy encompasses
prophylaxis, prevention, treatment, cure, remedy, reduction,
alleviation, and/or providing relief from a disease, signs, and/or
symptoms of a disease. Therapy encompasses an alleviation of signs
and/or symptoms in patients with ongoing disease signs and/or
symptoms. Therapy also encompasses "prophylaxis" and "prevention".
Prophylaxis includes preventing disease occurring subsequent to
treatment of a disease in a patient or reducing the incidence or
severity of the disease in a patient. The term "reduced", for
purpose of therapy, refers broadly to the clinical significant
reduction in signs and/or symptoms. Therapy includes treating
relapses or recurrent signs and/or symptoms. Therapy encompasses
but is not limited to precluding the appearance of signs and/or
symptoms anytime as well as reducing existing signs and/or symptoms
and eliminating existing signs and/or symptoms. Therapy includes
treating chronic disease ("maintenance") and acute disease. For
example, treatment includes treating or preventing relapses or the
recurrence of signs and/or symptoms.
[0124] Cell Markers: Exemplary cell markers that may be assessed
for expression include the following: PAX6, RX1, SIX3, SIX6, LHX2,
TBX3, SOX2, CHX10, Nestin, TRbeta2, NR2E3, NRL, MASH1, ROR.beta.,
Recoverin, Opsin, Rhodopsin, rod and cone cGMP Phosphodiesterase,
which may be assessed at the protein and/or mRNA (see Fischer A J,
Reh T A, Dev Neurosci. 2001; 23(4-5):268-76; Baumer et al.,
Development. 2003 July; 130(13):2903-15, Swaroop et al., Nat Rev
Neurosci. 2010 August; 11(8):563-76, Agathocleous and Harris, Annu.
Rev. Cell Dev. Biol. 2009. 25:45-69, each of which is hereby
incorporated by reference in its entirety). Said marker identifiers
are generally used as in the literature and in the art, particular
in the fields of art in related to the contexts in which those gene
identifiers are recited herein, which may include literature
related to photoreceptors, rods, cones, photoreceptor
differentiation, photoreceptor progenitors, neural differentiation,
neural stem cells, pluripotent stem cells, and other fields as
indicated by context. Additionally, the markers are generally
human, e.g., except where the context indicates otherwise. The cell
markers can be identified using conventional immunocytochemical
methods or conventional PCR methods which techniques are well known
to those of ordinary skill in the art.
[0125] Cell Culture Media:
[0126] In embodiments of the invention, the cells are stored,
proliferated or differentiated in various cell culture media.
Retinal induction medium is utilized for the stem cell production
into Eye Field Progenitor Cells. The retinal induction medium may
comprise D-glucose, penicillin, streptomycin, N2 supplement (e.g.
0.1-5%), B27 supplement (e.g., 0.005 to 0.2%), MEM Non-essential
amino acids solution and optionally including insulin and/or
Noggin, and may be in a DMEM/F12 (Invitrogen) or similar base
medium. For example, the Retinal induction medium may include at
least insulin. Additionally, the insulin concentration may be
varied or increased which may promote cell survival and/or yield of
differentiated cells. For example, the insulin concentration may be
varied across a range and survival and/or differentiation monitored
in order to identify an insulin concentration with improves either
or both of these attributes. The addition of Noggin is believed not
to be necessary but was observed to increase the expression of eye
field transcription factors.
[0127] Noggin is a secreted BMP inhibitor that reportedly binds
BMP2, BMP4, and BMP7 with high affinity to block TGF.beta. family
activity. SB431542 is a small molecule that reportedly inhibits
TGF.beta./Activin/Nodal by blocking phosphorylation of ACTRIB,
TGF.beta.R1, and ACTRIC receptors. SB431542 is thought to
destabilize the Activin- and Nanog-mediated pluripotency network as
well as suppress BMP induced trophoblast, mesoderm, and endodermal
cell fates by blocking endogenous Activin and BMP signals. It is
expected that agents having one or more of the aforementioned
activities could replace or augment the functions of one or both of
Noggin and SB431542, e.g., as they are used in the context of the
disclosed methods. For example, applicants envision that the
protein Noggin and/or the small molecule SB4312542 could be
replaced or augmented by one or more inhibitors that affect any or
all of the following three target areas: 1) preventing the binding
of the ligand to the receptor; 2) blocking activation of receptor
(e.g., dorsomorphin), and 3) inhibition of SMAD intracellular
proteins/transcription factors. Exemplary potentially suitable
factors include the natural secreted BMP inhibitors Chordin (which
blocks BMP4) and Follistatin (which blocks Activin), as well as
analogs or mimetics thereof. Additional exemplary factors that may
mimic the effect of Noggin include use of dominant negative
receptors or blocking antibodies that would sequester BMP2, BMP4,
and/or BMP7. Additionally, with respect to blocking receptor
phosphorylation, dorsomorphin (or Compound C) has been reported to
have similar effects on stem cells. Inhibition of SMAD proteins may
also be effected using soluble inhibitors such as SIS3
(6,7-Dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo [2,3-b]
pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinoline,
Specific Inhibitor of Smad3, SIS3), overexpression of one or more
of the inhibitor SMADs (e.g., SMAD6, SMAD7, SMAD10) or RNAi for one
of the receptor SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8/9).
Another combination of factors expected to be suitable for
generating neural progenitors comprises a cocktail of Leukemia
Inhibitory Factor (LIF), GSK3 inhibitor (CHIR 99021), Compound E
(.gamma. secretase inhibitor XXI) and the TGF.beta. inhibitor
SB431542 which has been previously shown to be efficacious for
generating neural crest stem cells (Li et al., Proc Natl Acad Sci
USA. 2011 May 17; 108(20):8299-304). Additional exemplary factors
may include derivatives of SB431542, e.g., molecules that include
one or more added or different substituents, analogous functional
groups, etc. and that have a similar inhibitory effect on one or
more SMAD proteins. Suitable factors or combinations of factors may
be identified, for example, by contacting pluripotent cells with
said factor(s) and monitoring for adoption of eye field progenitor
cell phenotypes, such as characteristic gene expression (including
expression of the markers described herein, expression of a
reporter gene coupled to an eye field progenitor cell promoter, or
the like) or the ability to form a cell type disclosed herein such
as retinal neural progenitor cells, photoreceptor progenitors, rod
progenitors, cones, and/or rods.
[0128] Preferably the cells are treated with or cultured in a
retinal induction medium prior to culture with a neural
differentiation medium. A neural differentiation medium is utilized
for Eye Field Progenitor Cell production into Retinal Neural
Progenitor Cells. The neural differentiation medium may comprise
D-glucose, penicillin, streptomycin, GlutaMAX.TM., N2 supplement,
B27 supplement, MEM Non-essential amino acids solution and
optionally including Noggin. The neural differentiation medium may
also be utilized for Retinal Neural Progenitor Cell production into
Photoreceptor Progenitor Cells but without the inclusion of Noggin.
The use of a neural differentiation medium, optionally supplemented
with retinoic acid and taurine, followed by utilization of a
photoreceptor differentiation medium (Invitrogen) which optionally
may comprise D-glucose, penicillin, streptomycin, GlutaMAX.TM., N2
supplement, B27 supplement with the addition of Forskolin, BDNF,
CNTF, LIF and DATP is utilized for Photoreceptor Progenitor Cells
production into Photoreceptor Cells. For example the photoreceptor
differentiation medium may comprise thyroid hormone, e.g., in an
amount that is present in the foregoing medium, or in a different
or greater amount. For example said medium may comprise exogenously
added thyroid hormone. In exemplary embodiments the photoreceptor
differentiation medium may comprise one, two, or all three BDNF,
CNTF and DATP, e.g., BDNF, CNTF, DATP, BDNF and CNTF, CNTF and
DATP, BDNF and DATP, or all three of BDNF, CNTF and DATP, which
medium may optionally comprise Neurobasal Medium and/or may
optionally comprise thyroid hormone.
[0129] The neural differentiation medium constituents are as
follows:
[0130] N2: 1% (1 ml of N2/100 ml)
[0131] B27: 2% (2 ml of b27/100 ml)
[0132] Noggin: 50 ng/ml
[0133] Noggin is not needed after cells have all become eye field
progenitors.
[0134] Embryonic Stem Cells (ESCs) or Adult Stem Cells or Induced
Pluripotent Stem Cells (iPS):
[0135] The ESCs, or Adult Stem Cells or iPS cells utilized herein
may be propagated on a feeder-free system, such as in Matrigel.TM.
(a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells) or another matrix. Additionally, or alternatively,
said pluripotent cells may be cultured on a matrix which may be
selected from the group consisting of laminin, fibronectin,
vitronectin, proteoglycan, entactin, collagen, collagen I, collagen
IV, collagen VIII, heparan sulfate, Matrigel.TM. (a soluble
preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells),
CellStart, a human basement membrane extract, and any combination
thereof. Said matrix may comprise, consist of, or consist
essentially of Matrigel.TM. (a soluble preparation from
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). The stem cells do
not form embryoid bodies in culture, which is an improvement over
the prior art. The cells differentiate into eye field progenitor
cells in the absence of exogenous factors. In an embodiment, ESCs
differentiate into eye field progenitor cells in the presence of
Noggin.
[0136] Eye Field Progenitor Cells (EFPCs):
[0137] The EFPCs differentiate from ESCs, Adult stem cells or
induced pluripotent cells (iPCs) into cells that are PAX6(+) and
RX1(+). The EFPCs can also be SIX3(+), SIX6(+), LHX2(+), TBX3(+)
Nestin(+) and/or SOX2(+) and OCT4(-) and Nanog (-). The
differentiation into EFPCs occurs in a retinal induction medium
which may comprise DMEM/F12, D-glucose, penicillin, streptomycin,
N2 supplement, B27 supplement, MEM non-essential amino acid and
insulin. On day 5, when cells reach confluence, cells are changed
to neural differentiation medium. Preferably the step of producing
EFPCs is performed prior to culturing pluripotent cells in the
neural differentiation medium described below, as it has been
observed that such culture conditions may adversely affect
pluripotent cell viability.
[0138] Retinal Neural Progenitor Cells (RNPCs):
[0139] The RNPCs differentiate from the EFPCs in the absence of
exogenous factors. The RNPCs are PAX6(+) and CHX10(+). The cells at
this state may be Tuj1+ or Tuj1-. Optionally the method may include
enriching or purifying Tuj1+ or Tuj1- cells at this stage, and/or
purifying or removing strongly Tuj1+ cells and/or purifying or
removing strongly Tuj1- cells (e.g., cells lacking even low level
detectable expression thereof) and proceeding with the subsequent
method steps with one or the other of these populations. In an
embodiment, Noggin is added to accelerate the differentiation from
EFPCs to RNPCs The differentiation into RNPCs occurs in a neural
differentiation media which may comprise Neurobasal Medium
(Invitrogen), D-glucose, penicillin, streptomycin, GlutaMAX.TM., N2
supplement, B27 supplement and MEM non-essential amino acid
solution. Noggin may be added at a final concentration of 5-100
.mu.g/ml.
[0140] Photoreceptor Progenitor Cells (PhRPCs):
[0141] The PhRPCs may be differentiated from the RNPCs in the
absence of Noggin and in neural differentiation medium). The PRPCs
are PAX6(+) and CHX10(-). In embodiment, 60%, 70%, 80%, 85%, 90%,
or 95% of the PRPCs are PAX6(+) and CHX10(-) The PRPCs can also be
Nr2e3(+), Tr.beta.2(+), Mash1(+), ROR.beta.(+) and/or NRL(+). The
presence of CHX10 would suggest a bipolar cell lineage, but in the
present method, the PRPCs have differentiated to a photoreceptor
lineage, and therefore do not possess CHX10 at this stage. The
cells may be grown as spheres or neurospheres (e.g., on low
attachment plates or optionally on hanging drop cultures, in a
low-gravity environment, or other suitable culture condition).
[0142] Photoreceptors (PRs):
[0143] The PRs may differentiate from the PhRPCs in a two-step
differentiation process 1) Adding neural differentiation medium
with retinoic acid and taurine for 2 weeks and 2) addition of the
photoreceptor differentiation medium.--see Example 2.
[0144] The PRs may be rhodopsin(+), recoverin(+), PE6a(+) or
opsin(+). The opsin may be any of the cone opsins. The PRs may be
bipotential for cones or rods. Exemplary photoreceptors produced by
this method may be PAX6-, which may be in contrast to some
previously described purported photoreceptor cells. As described
below in exemplary embodiments there is a 2 step differentiation
process 1) adding ND medium and retinoic acid and taurine for 2
weeks and 2) use of the photoreceptor differentiation medium, which
methods are further exemplified in the working examples below.
[0145] In exemplary embodiments the method may produce 40-60
million EFPCs, 60-90 million RNPCs, or 0.5-1 billion PhRPCs per
starting 1 million pluripotent cells.
[0146] In an exemplary embodiment, the cells may be transplanted
into a rat in need thereof, e.g., an RCS rat, or other animal model
of disease (e.g. for night blindness or for color blindness), and
the resulting effect on visual function may be detected by the
Optomotor response test, ERG, luminance threshold recording and/or
the visual center blood flow assay.
[0147] Pharmaceutical Preparations
[0148] The PRPCs or photoreceptor cells may be formulated with a
pharmaceutically acceptable carrier. For example, PRPCs or
photoreceptor cells may be administered alone or as a component of
a pharmaceutical formulation. The subject compounds may be
formulated for administration in any convenient way for use in
medicine. Pharmaceutical preparations suitable for administration
may comprise the PRPCs or photoreceptor cells, in combination with
one or more pharmaceutically acceptable sterile isotonic aqueous or
nonaqueous solutions (e.g., balanced salt solution (BSS)),
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes or suspending or thickening agents.
Exemplary pharmaceutical preparations comprises the PRPCs or
photoreceptor cells in combination with ALCON.RTM. BSS PLUS.RTM. (a
balanced salt solution containing, in each mL, sodium chloride 7.14
mg, potassium chloride 0.38 mg, calcium chloride dihydrate 0.154
mg, magnesium chloride hexahydrate 0.2 mg, dibasic sodium phosphate
0.42 mg, sodium bicarbonate 2.1 mg, dextrose 0.92 mg, glutathione
disulfide (oxidized glutathione) 0.184 mg, hydrochloric acid and/or
sodium hydroxide (to adjust pH to approximately 7.4) in water).
[0149] When administered, the pharmaceutical preparations for use
in this disclosure may be in a pyrogen-free, physiologically
acceptable form.
[0150] The preparation comprising PRPCS photoreceptor cells used in
the methods described herein may be transplanted in a suspension,
gel, colloid, slurry, or mixture. Further, the preparation may
desirably be encapsulated or injected in a viscous form into the
vitreous humor for delivery to the site of retinal or choroidal
damage. Also, at the time of injection, cryopreserved PRPCS
photoreceptor cells may be resuspended with commercially available
balanced salt solution to achieve the desired osmolality and
concentration for administration by subretinal injection. The
preparation may be administered to an area of the pericentral
macula that was not completely lost to disease, which may promote
attachment and/or survival of the administered cells.
[0151] The PRPCS or photoreceptor cells of the disclosure may be
delivered in a pharmaceutically acceptable ophthalmic formulation
by intraocular injection. When administering the formulation by
intravitreal injection, for example, the solution may be
concentrated so that minimized volumes may be delivered.
Concentrations for injections may be at any amount that is
effective and non-toxic, depending upon the factors described
herein. The pharmaceutical preparations of PRPCS or photoreceptor
cells for treatment of a patient may be formulated at doses of at
least about 10.sup.4 cells/mL. The PRPCS or photoreceptor cell
preparations for treatment of a patient are formulated at doses of
at least about 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 107,
10.sup.8, 10.sup.9, or 10.sup.10 PRPCS or photoreceptor cells/mL.
For example, the PRPCS or photoreceptor cells may be formulated in
a pharmaceutically acceptable carrier or excipient.
[0152] The pharmaceutical preparations of PRPCS or photoreceptor
cells described herein may comprise at least about 1,000; 2,000;
3,000; 4,000; 5,000; 6,000; 7,000; 8,000; or 9,000 PRPCS or
photoreceptor cells. The pharmaceutical preparations of PRPCS or
photoreceptor cells may comprise at least about 1.times.10.sup.4,
2.times.10.sup.4, 3.times.10.sup.4, 4.times.10.sup.4,
5.times.10.sup.4, 6.times.10.sup.4, 7.times.10.sup.4,
8.times.10.sup.4, 9.times.10.sup.4, 1.times.10.sup.5,
2.times.10.sup.5' 3.times.10.sup.5, 4.times.10.sup.5,
5.times.10.sup.5, 6.times.10.sup.5, 7.times.10.sup.5,
8.times.10.sup.5, 9.times.10.sup.5, 1.times.10.sup.6,
2.times.10.sup.6, 3.times.10.sup.6, 4.times.10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 1.times.10.sup.7,
2.times.10.sup.7, 3.times.10.sup.7, 4.times.10.sup.7,
5.times.10.sup.7, 6.times.10.sup.7, 7.times.10.sup.7,
8.times.10.sup.7, 9.times.10.sup.7, 1.times.10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 4.times.10.sup.8,
5.times.10.sup.8, 6.times.10.sup.8, 7.times.10.sup.8,
8.times.10.sup.8, 9.times.10.sup.8, 1.times.10.sup.9,
2.times.10.sup.9, 3.times.10.sup.9, 4.times.10.sup.9,
5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 1.times.10.sup.10,
2.times.10.sup.10, 3.times.10.sup.10, 4.times.10.sup.10,
5.times.10.sup.10, 6.times.10.sup.10, 7.times.10.sup.10,
8.times.10.sup.10, or 9.times.10.sup.10 PRPCS or photoreceptor
cells. The pharmaceutical preparations of PRPCS or photoreceptor
cells may comprise at least about
1.times.10.sup.2-1.times.10.sup.3,
1.times.10.sup.2-1.times.10.sup.4,
1.times.10.sup.4-1.times.10.sup.5, or
1.times.10.sup.3-1.times.10.sup.6 PRPCS or photoreceptor cells. The
pharmaceutical preparations of PRPCS or photoreceptor cells may
comprise at least about 10,000, 20,000, 25,000, 50,000, 75,000,
100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, or
200,000 PRPCS or photoreceptor cells. For example, the
pharmaceutical preparation of PRPCS or photoreceptor cells may
comprise at least about 20,000-200,000 PRPCS or photoreceptor cells
in a volume at least about 50-200 .mu.L. Further, the
pharmaceutical preparation of PRPCS or photoreceptor cells may
comprise about 50,000 PRPCS or photoreceptor is in a volume of 150
.mu.L, about 200,000 PRPCS or photoreceptor cells in a volume of
150 .mu.L, or at least about 180,000 PRPCS or photoreceptor cells
in a volume at least about 150 pt.
[0153] In the aforesaid pharmaceutical preparations and
compositions, the number of PRPCS or photoreceptor cells or
concentration of PRPCS or photoreceptor cells may be determined by
counting viable cells and excluding non-viable cells. For example,
non-viable PRPCS or photoreceptor may be detected by failure to
exclude a vital dye (such as Trypan Blue), or using a functional
assay (such as the ability to adhere to a culture substrate,
phagocytosis, etc.). Additionally, the number of PRPCS or
photoreceptor cells or concentration of PRPCS or photoreceptor
cells may be determined by counting cells that express one or more
PRPCS or photoreceptor cell markers and/or excluding cells that
express one or more markers indicative of a cell type other than
PRPCS or photoreceptor.
[0154] The PRPCS or photoreceptor cells may be formulated for
delivery in a pharmaceutically acceptable ophthalmic vehicle, such
that the preparation is maintained in contact with the ocular
surface for a sufficient time period to allow the cells to
penetrate the affected regions of the eye, as for example, the
anterior chamber, posterior chamber, vitreous body, aqueous humor,
vitreous humor, cornea, iris/ciliary, lens, choroid, retina,
sclera, suprachoridal space, conjunctiva, subconjunctival space,
episcleral space, intracorneal space, epicorneal space, pars plana,
surgically-induced avascular regions, or the macula.
[0155] The PRPCS or photoreceptor cells may be contained in a sheet
of cells. For example, a sheet of cells comprising PRPCS or
photoreceptor cells may be prepared by culturing PRPCS or
photoreceptor cells on a substrate from which an intact sheet of
cells can be released, e.g., a thermoresponsive polymer such as a
thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted
surface, upon which cells adhere and proliferate at the culture
temperature, and then upon a temperature shift, the surface
characteristics are altered causing release the cultured sheet of
cells (e.g., by cooling to below the lower critical solution
temperature (LCST) (see da Silva et al., Trends Biotechnol. 2007
December; 25(12):577-83; Hsiue et al., Transplantation. 2006 Feb.
15; 81(3):473-6; Ide, T. et al. (2006); Biomaterials 27, 607-614,
Sumide, T. et al. (2005), FASEB J. 20, 392-394; Nishida, K. et al.
(2004), Transplantation 77, 379-385; and Nishida, K. et al. (2004),
N. Engl. J. Med. 351, 1187-1196 each of which is incorporated by
reference herein in its entirety). The sheet of cells may be
adherent to a substrate suitable for transplantation, such as a
substrate that may dissolve in vivo when the sheet is transplanted
into a host organism, e.g., prepared by culturing the cells on a
substrate suitable for transplantation, or releasing the cells from
another substrate (such as a thermoresponsive polymer) onto a
substrate suitable for transplantation. An exemplary substrate
potentially suitable for transplantation may comprise gelatin (see
Hsiue et al., supra). Alternative substrates that may be suitable
for transplantation include fibrin-based matrixes and others. The
sheet of cells may be used in the manufacture of a medicament for
the prevention or treatment of a disease of retinal degeneration.
The sheet of PRPCS or photoreceptor cells may be formulated for
introduction into the eye of a subject in need thereof. For
example, the sheet of cells may be introduced into an eye in need
thereof by subfoveal membranectomy with transplantation the sheet
of PRPCS or photoreceptor cells, or may be used for the manufacture
of a medicament for transplantation after subfoveal
membranectomy.
[0156] The volume of preparation administered according to the
methods described herein may be dependent on factors such as the
mode of administration, number of PRPCS or photoreceptor cells, age
and weight of the patient, and type and severity of the disease
being treated. If administered by injection, the volume of a
pharmaceutical preparations of PRPCS or photoreceptor cells of the
disclosure may be from at least about 1, 1.5, 2, 2.5, 3, 4, or 5
mL. The volume may be at least about 1-2 mL. For example, if
administered by injection, the volume of a pharmaceutical
preparation of PRPCS or photoreceptor cells of the disclosure may
be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 .mu.t
(microliters). For example, the volume of a preparation of the
disclosure may be from at least about 10-50, 20-50, 25-50, or 1-200
.mu.L. The volume of a preparation of the disclosure may be at
least about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, or 200 .mu.L, or higher.
[0157] For example, the preparation may comprise at least about
1.times.103, 2.times.103, 3.times.103, 4.times.103, 5.times.103,
6.times.103, 7.times.103, 8.times.103, 9.times.103, 1.times.104,
2.times.104, 3.times.104, 4.times.104, 5.times.104, 6.times.104,
7.times.104, 8.times.104, or 9.times.104 PRPCS or photoreceptor
cells per .mu.L. The preparation may comprise 2000 PRPCS or PRs per
.mu.L, for example, 100,000 PRPCS or photoreceptor cells per 50
.mu.L or 180,000 PRPCS or photoreceptor cells per 90 .mu.L.
[0158] The method of treating retinal degeneration may further
comprise administration of an immunosuppressant. Immunosuppressants
that may be used include but are not limited to anti-lymphocyte
globulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG)
polyclonal antibody, azathioprine, BASILIXIMAB.RTM. (anti-IL-2Ra
receptor antibody), cyclosporin (cyclosporin A), DACLIZUMAB.RTM.
(anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid,
RITUXIMAB.RTM. (anti-CD20 antibody), sirolimus, and tacrolimus. The
immunosuppressants may be dosed at least about 1, 2, 4, 5, 6, 7, 8,
9, or 10 mg/kg. When immunosuppressants are used, they may be
administered systemically or locally, and they may be administered
prior to, concomitantly with, or following administration of the
PRPCS or photoreceptor cells. Immunosuppressive therapy may
continue for weeks, months, years, or indefinitely following
administration of PRPCS or photoreceptor cells. For example, the
patient may be administered 5 mg/kg cyclosporin for 6 weeks
following administration of the PRPCS or photoreceptor cells.
[0159] The method of treatment of retinal degeneration may comprise
the administration of a single dose of PRPCS or photoreceptor
cells. Also, the methods of treatment described herein may comprise
a course of therapy where PRPCS or photoreceptor cells are
administered multiple times over some period. Exemplary courses of
treatment may comprise weekly, biweekly, monthly, quarterly,
biannually, or yearly treatments. Alternatively, treatment may
proceed in phases whereby multiple doses are administered initially
(e.g., daily doses for the first week), and subsequently fewer and
less frequent doses are needed.
[0160] If administered by intraocular injection, the PRPCS or
photoreceptor cells may be delivered one or more times periodically
throughout the life of a patient. For example, the PRPCS or
photoreceptor cells may be delivered once per year, once every 6-12
months, once every 3-6 months, once every 1-3 months, or once every
1-4 weeks. Alternatively, more frequent administration may be
desirable for certain conditions or disorders. If administered by
an implant or device, the PRPCS or photoreceptor cells may be
administered one time, or one or more times periodically throughout
the lifetime of the patient, as necessary for the particular
patient and disorder or condition being treated. Similarly
contemplated is a therapeutic regimen that changes over time. For
example, more frequent treatment may be needed at the outset (e.g.,
daily or weekly treatment). Over time, as the patient's condition
improves, less frequent treatment or even no further treatment may
be needed.
[0161] The methods described herein may further comprise the step
of monitoring the efficacy of treatment or prevention by measuring
electroretinogram responses, optomotor acuity threshold, or
luminance threshold in the subject. The method may also comprise
monitoring the efficacy of treatment or prevention by monitoring
immunogenicity of the cells or migration of the cells in the
eye.
[0162] The PRPCs or PRs may be used in the manufacture of a
medicament to treat retinal degeneration. The disclosure also
encompasses the use of the preparation comprising PRPCs or PRs in
the treatment of blindness. For example, the preparations
comprising human PRPCs or PRs may be used to treat retinal
degeneration associated with a number of vision-altering ailments
that result in photoreceptor damage and blindness, such as,
diabetic retinopathy, macular degeneration (including age related
macular degeneration, e.g., wet age related macular degeneration
and dry age related macular degeneration), retinitis pigmentosa,
and Stargardt's Disease (fundus flavimaculatus), night blindness
and color blindness. The preparation may comprise at least about
5,000-500,000 PRPCs or PRs (e.g., 100,00 PRPCs or PRs) which may be
administered to the retina to treat retinal degeneration associated
with a number of vision-altering ailments that result in
photoreceptor damage and blindness, such as, diabetic retinopathy,
macular degeneration (including age related macular degeneration),
retinitis pigmentosa, and Stargardt's Disease (fundus
flavimaculatus).
[0163] The PRPCs or PRs provided herein may be PRPCs or PRs. Note,
however, that the human cells may be used in human patients, as
well as in animal models or animal patients. For example, the human
cells may be tested in mouse, rat, cat, dog, or non-human primate
models of retinal degeneration. Additionally, the human cells may
be used therapeutically to treat animals in need thereof, such as
in veterinary medicine.
[0164] The following are examples to illustrate the invention and
should not be viewed as limiting the scope of the invention.
EXAMPLES
Example 1: Generation of Photoreceptor Progenitor Cells
[0165] Human embryonic stem cells were cultured under feeder free
conditions in mTESR1 media (Stem Cell Technology) on a Matrigel.TM.
(a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells, BD Biosciences) surface. Upon 80-90% confluence,
cells were passaged or frozen. Passaging of stem cells was
performed using enzymatic (dispase) or non-enzymatic (EDTA-based
cell dissociation buffer, Invitrogen) techniques.
[0166] Direct differentiation methods were used for generation of
eye field progenitor cells, retinal neural progenitor cells,
photoreceptor progenitor cells and retinal photoreceptor cells.
Formation of embryoid bodies was not required.
[0167] The overall method used for photoreceptor development in
these examples is schematically illustrated in FIG. 19, which
further illustrates the media used at each step of the process.
[0168] Based on staining data it was determined that the cells
become EFPC between day 7-day 30 (indicated by staining done at day
20 which confirmed this cell identity), they become RNPC between
day 21-day 45 (indicated by staining done at day 30), and they
become PhRPC between 1-4 month (based on staining done at day
90).
[0169] Additionally it was estimated that the timing at which
different cell types arose using the methods described in Example 1
were as follows:
[0170] Eye Field Progenitors (EFPC): 7-30 days/65%-98% purity
[0171] Retinal Neural Progenitors (RNPC): 21-45 days/70%-95%
purity
[0172] Photoreceptor Progenitors (PhRPs) capable of becoming both
rod photoreceptors and cone photoreceptors: 1-4 months/85%-95%
purity
[0173] Photoreceptor Progenitors (PhRPs) thought to have lost or
experienced reduction in their capability of becoming rod
photoreceptors (but not cone photoreceptors): 5-12 months/85%-95%
purity.
[0174] Day 0: Cell differentiation of human pluripotent stem cells
was induced at 15-20% confluence. Culture media was changed to
retinal induction (RI) medium: DMEM/F12 supplied with 450 mg/ml
D-glucose, 100 unit/ml of penicillin, 100 .mu.g/ml of streptomycin,
1% (or optionally 0.1 to 5%) N2 supplement (components listed in
FIGS. 21A-21E, Invitrogen), 0.2% (or optionally 0.05-2.0%) B27
supplement, 0.1 mM MEM Non-essential amino acids solution, 25
.mu.g/ml (or optionally 5-50 .mu.g/ml) human insulin was added to
the RI medium. The Smad inhibitor Noggin was also included and
increased the expression of eye field transcription factors when
included at a concentration of 10-100 ng/ml or preferably 50 ng/ml.
As shown in FIG. 1, inclusion of different factors including 50
ng/ml Noggin, 5 ng/ml Dkk1, 5 ng/ml IGF-1, or a combination of 5
ng/ml Noggin, 5 ng/ml Dkk1, and 5 ng/ml IGF-1 affected the level of
expression of eye field transcription factors in differentiated eye
field progenitor cells at day 21. Among those conditions, inclusion
of 50 ng/ml Noggin greatly induced the expression of eye field
progenitor markers.
[0175] The RI medium composition included the following:
[0176] N2: 1% (1 ml of N2 per 100 ml media)
[0177] B27: 0.2% (0.2 ml of b27 per 100 ml)
[0178] Human insulin: 20 .mu.g/ml (in addition to the 5 .mu.g/ml
insulin supplied by N2). The final concentration of insulin was 25
.mu.g/ml.
[0179] Noggin: 50 ng/ml final concentration.
[0180] Day 1-Day 4: A complete media change was done on every day.
Though this frequency is preferred, it is thought that changing the
medium less often, e.g., every 2-3 days, may be suitable
particularly if a larger volume of media is used. Cell colonies
continued to grow in the RI media with insulin and Noggin in the
same concentrations as in the previous step. After 1 day exposure
to RI media, cells located at the colony margin were elongated and
column-shaped, as shown in FIG. 2A.
[0181] Day 5: Cell cultures became 80-90% confluent on day 5. Media
was changed to neural differentiation (ND) medium: Neurobasal
Medium (components listed in FIGS. 21A-21E, Invitrogen) supplied
with 450 mg/ml D-glucose, 100 unit/ml of penicillin, 100 .mu.g/ml
of streptomycin, 1.times.GlutaMAX.TM. (a stabilized form dipeptide
from L-glutamine, L-alanyl-L-glutamine), 1% (or optionally 0.1 to
5%) N2 supplement (a chemically defined, serum-free supplement
based on Bottenstein's N-1 formulation comprising 1 mM Human
Transferrin (Holo), 0.0861 mM Insulin Recombinant Full Chain, 0.002
Progesterone, 10.01 mM Putrescine, and 0.00301 mM Selenite,
Invitrogen), 2% (or optionally 0.05-2.0%) B27 supplement
(components listed in FIGS. 21A-21E), 0.1 mM MEM Non-essential
amino acids solution. Noggin was also added to the ND media at the
final concentration of 50 ng/ml (or optionally 10-100 ng/ml).
[0182] Day 6-Day 20: Cells were maintained in the ND medium. Half
the amount of medium was changed every 2 days. Cell colonies
continued to grow in the ND medium. The edge cells become flat and
large, while the central cells were smaller and formed compact cell
clusters (FIG. 2B). Around Day 14, cells located at the center of
colonies began to form Rosette-like structures (FIG. 2C). At day
21, over 90% of the cells co-expressed PAX6 and RX1 (FIG. 3A-3B) as
revealed by immunostaining and flow cytometry. By immunostaining,
cells were positive for Nestin and SOX2 (FIG. 3C-3D). Cells were
negative for an ES cell marker (specifically OCT4) and a retinal
neural progenitor marker (specifically CHX10). By RT-PCR, cells
expressed eye field transcription factors: PAX6, RX1, LHX2, SIX3,
SIX6, TBX3 and SOX2 (FIG. 3E). These results indicate that the
cells were eye-field progenitor cells.
[0183] The cells become eye field progenitors after they are
cultured with neural differentiation media, from about day
7-8(about 2-3 days culture in ND media). At these time points
detectable PAX6/rx1 double positive cells arise. After about day 14
(between days 14-30), high purity (>90%) of eye field progenitor
are generated.
[0184] Between about days 7-30 "Eye Field Progenitor Cells" or
"EFPCs" are formed.
[0185] Day 21-Day24: At Day 21, cells were lifted off from the
growth surface and mechanically fragmented into clusters in ND
medium without Noggin. Cell clusters were transferred to 100 mm
ultra-low attachment culture dishes. Cell clusters rounded up and
formed individual spheres (solid clusters) in the suspension
culture. At Day 23, half of the culture medium was replaced.
[0186] At Day 25, spheres were collected and dead cells and debris
were removed by washing the spheres with the ND media. Cell spheres
were plated onto Matrigel.TM. coated glass chamber slide (for
immunostaining) or tissue culture dishes in the ND medium. Spheres
attached within 12 hours. They continued to grow and show neuronal
phenotypes, specifically exhibiting cell aggregates within the
spheres that extended axon-like neurites with some cells migrating
out from aggregates (FIG. 4A). There were few big epithelial-like
cells which could be eliminated during cell passage (see "Month
2-Month 3" below). The cultures were maintained with half of the
culture medium changed every two days until the cell cultures
become confluent It was observed that balls of spheres attached to
the plate.
[0187] At Day 30, the migrating cells were positive for Tuj1, which
labels immature and mature neurons (FIG. 4B). Cells in the
aggregates were negative for Tuj1. Over 95% cells (including cells
in the aggregates or migrating out from aggregates) co-expressed
PAX6 and CHX10 suggesting that they had become retinal neural
progenitors (FIG. 4C).
[0188] Month 2-Month 3: Growth and passaging cells in the ND media.
The cells from the previous step were passaged when they became
confluent. A two-step successive passaging technique was used to
produce high-purity neural cultures by eliminating the majority of
non-neuronal phenotype cells. The first step: neural sphere
culture. Cells were enzymatically (e.g., using Accutase) or
mechanically dissociated into a mixture of single cells and cell
clusters. Cells were transferred to ultra-low attachment dishes in
ND medium. All cells with neuronal phenotype form neural spheres in
the suspension culture. On day 3, half of the medium was changed
and the cells were maintained until day 5. The second step:
adherent culture. Neural spheres were collected on day 5 and dead
cells and debris were removed by washing the spheres with ND
medium. Spheres were plated on Matrigel.TM.-coated tissue culture
dishes until confluent. The first and second steps were alternated
and the cells were so maintained until the end of the third
month.
[0189] At the end of the 3rd month, the cells showed neural
phenotype. Specifically, the cells formed neurites in culture (FIG.
5A). They were capable of proliferation. They expressed PAX6 but
were negative for CHX10 as assessed by immunostaining (FIG. 5B). By
immunostaining, the cells were positive for Recoverin, which was
expressed in the cytoplasm of the cell body (FIG. 5C). The cells
also expressed Rhodopsin, Opsin and Recoverin mRNA (FIG. 5D).
Real-time PCR analyses revealed that the expressions of
transcription factors controlling rod and/or cone photoreceptor
differentiation are highly expressed (Table 1). These results
indicate that the cells were photoreceptor progenitors.
Additionally, at this time-point it was through based on
observations that all or essentially all of the cells in the
culture are photoreceptor progenitors.
TABLE-US-00001 TABLE 1 Quantitative RT-PCT analyses of
transcription factors controlling photoreceptor differentiation and
regeneration. Transcription Factors Rod/Cone Fold change (vs. ESC)
TR.beta.2 Cone 3.5-5 NR2E3 Rod 7-11 NRL Rod 4-8 MASH1 Rod 1000-1200
CRX Rod, Cone -- ROR.beta. Rod, Cone 40-60 OTX2 Rod, Cone --
[0190] Month 4-Month 9/or longer: In vitro expansion of
photoreceptor progenitors. In some experiments the cells were
further expanded using the two-step successive passaging technique
described above ("Month 2-Month 3"). However, it was observed that
over time the cells lose their capability to differentiate into
cone photoreceptors (though they retain the ability to
differentiate into rod photoreceptors). Specifically, after
photoreceptor progenitors were maintained by the two-step
successive passaging technique for 9 months in culture and then
induced to differentiate, they only produced cells that expressed
rod photoreceptor markers and not cells that expressed cone
photoreceptor markers. This property could potentially be put to
advantageous use, as progenitor cells that preferentially produce
rod photoreceptors may be useful in the treatment of diseases
wherein rod formation is desirable, or as a reagent for the study
of factors involved in photoreceptor progenitor fate
determination.
Example 2: Differentiation of Photoreceptor Progenitor Cells: Cell
Treatment with Retinoic Acid and Taurine
[0191] Attached photoreceptor progenitors were treated with
retinoic acid in the following conditions for two weeks: ND medium
supplied with 100 ng/ml (or optionally 10-1000 ng/ml) retinoic acid
and 100 .mu.M (or optionally 20-500 .mu.M) taurine. Half of the
culture medium was changed every 2 days.
[0192] Differentiate cells in Photoreceptor differentiation media:
The medium was changed to Photoreceptor Differentiation Medium
comprising Neurobasal Medium (Invitrogen) supplied with 450 mg/ml
D-glucose, 100 unit/ml of penicillin, 100 .mu.g/ml of streptomycin,
lx GlutaMAX.TM., 1% N2 supplement (Invitrogen), 2% B27 supplement
(formula number 080085-SA), with the addition of 5 .mu.M (or
optionally 1-100 .mu.M) Forskolin, 10 ng/ml (or optionally 1-100
ng/ml) BDNF, 10 ng/ml (or optionally 1-100 ng/ml) CNTF, 10 ng/ml
(or optionally 5-50 ng/ml) LIF and 10 .mu.M (or optionally 1-100
.mu.M) DATP. Half of the medium was changed every 2 days.
Specifically the amounts of each factor were as follows: Forskolin
(5 .mu.M), BDNF (10 ng/ml), CNTF (10 ng/ml), LIF (10 ng/ml) and
DATP (10 .mu.M). LIF was determined not to be necessary and can be
left out.
[0193] At two weeks after initiating cell differentiation, the
expressions of Rhodopsin, Opsin (green/red), Recoverin and
phosphodiesterase 6A alpha subunit (PDE6a) were detected in the
cytoplasm of the cell body and neurites (FIG. 6A-6D). These gene
expression results indicate that these are photoreceptor cells.
Example 3: Cryopreservation of Human ESC-Derived Retinal Neural
Progenitors
[0194] Retinal neural progenitors of the invention, photoreceptor
progenitors of the invention and retinoic acid treated
photoreceptor progenitors of the invention can be frozen down in an
animal-free cryopreservation buffer, such as Cryostor CS10, or
another cryopreservation buffer such as 90% FBS and 10% DMSO. With
respect to the photoreceptor progenitors, it was observed that
freezing cells as neurospheres was beneficial, which may be due to
the benefits of cell-cell contact. Preferably the neurospheres were
frozen down at a size that was not too large, such as 50-250
cells.
Example 4: Animal Studies in Stargardt Macular Dystrophy Animal
Model
[0195] Animal studies were carried out in a Stargardt macular
dystrophy animal model, ELOVL4 transgenic 2 (TG2) mice (FIG.
7).
[0196] Photoreceptor progenitors (produced as described in Example
1) and separately, retinoic acid and taurine treated photoreceptor
progenitors (i.e., immature photoreceptor cells, produced as
described in Example 2) were dissociated into single cells using
Accutase. Cells were re-suspended in PBS buffer.
[0197] 28 days-old TG2 mice received an injection of 1 .mu.l of
cell suspension containing 5.times.10.sup.5 cells into the
subretinal space or 150 .mu.l of cell suspension containing
1.times.10.sup.6 cells into the tail vein. All mice underwent
baseline ERG and OCT tests before cell injection.
[0198] Mice were fed with water supplied with Cyclosporin A (USP
modified).
[0199] At one month after cell injection, mice that received a
subretinal injection of photoreceptor progenitors showed a
significant improvement of the rod photoreceptor function revealed
by a significant increase of the scotopic ERG amplitude of both the
a- and b-wave (FIG. 8). Mice that received a tail vein injection of
photoreceptor progenitors and retinoic acid and taurine-treated
photoreceptor progenitors showed a significant improvement of the
Rod photoreceptor function revealed by a significant increase of
the scotopic ERG amplitude of both a- and b-wave (FIG. 9).
[0200] At two months after cell injection, mice that received a
tail vein injection of retinoic acid treated photoreceptor
progenitors showed a further improvement of the rod photoreceptor
function revealed by a further increase of the amplitude of both a-
and b-wave of scotopic ERG responsive curve (FIG. 10A-10C). The
function of cone photoreceptors was significantly improved as
revealed by a significant increase of the photopic ERG amplitude of
both a- and b-wave (FIG. 11).
[0201] At two months after injection, mice that received a tail
vein injection of immature photoreceptor cells treated with
retinoic acid and taurine showed a significant increase of whole
retina thickness revealed by OCT (FIG. 12).
[0202] At two months after cell transplantation, there was a
significant preservation of photoreceptor neurons in the ONL of
retina in mice that received retinoic acid and taurine-treated
photoreceptor progenitors (FIG. 13).
Example 5: Animal Models of Achromatopsia (Color Blindness) and
Improving Night Vision
[0203] Cells produced according to the methods described in Example
1 or Example 2 are tested in mouse, sheep, and/or dog models of
Achromatopsia (color blindness). The following models are used:
[0204] Mouse: (1) the cpfl5 mouse: a naturally occurring mouse
model of achromatopsia with a CNGA3 mutation; (2) CNGA3 knockout
mice; (3) GNAT2cpfl3 mice: mutation related to GNAT2; (4)
PDE6C-cpfl1: mutation related to pde6c.
[0205] Sheep: Awassi sheep lambs: mutation in CNGA3
[0206] Dog: Two natural occurring canines for mutation in CNGA3
have been identified: the autosomal recessive canine cone
degeneration in the Alaskan malamute and the German shorthaired
pointer.
[0207] Photoreceptor progenitors (produced as described in Example
1) are dissociated into single cells using accutase. Cells and are
re-suspended in PBS buffer. The animals receive injections of
2.times.10.sup.5 cells or more into the vitreous cavity or
5.times.10.sup.6 cells or more into a tail vein (e.g., the tail
vein). Control animals receive an injection with PBS buffer. After
one or two months or at other time points, the animals are given
optomotor responsiveness tests to check visual function in order to
detect possible improvements thereto. Additionally, histological
analysis is performed to determine whether there is any significant
preservation of photoreceptor neurons or growth of photoreceptor
neurons, and additionally to detect whether cells transplanted into
the vitreous cavity showed good survival after injection, and
whether the cells differentiated into rod or cone photoreceptor
cells expressing markers thereof.
Example 6: Animal Studies in a Photoreceptor Degeneration Rat
Model, Royal College of Surgeons (RCS) Rat
[0208] Photoreceptor progenitors (produced as described in Example
1) were dissociated into single cells using accutase. Cells were
re-suspended in PBS buffer.
[0209] On postnatal day 30, RCS rats received injections of
2.times.10.sup.5 cells into the vitreous cavity or 5.times.10.sup.6
cells into the tail vein. Control rats received an injection with
PBS buffer.
[0210] RCS rats were fed with water supplied with Cyclosporin A
(USP modified).
[0211] At one month and two months after cell injection, rats were
given optomotor responsive tests to check visual function. There
was no significant improvement in visual function in treated rats
(data not shown).
[0212] The resulting effect on visual function may be detected by
the Optomotor response test, ERG, luminance threshold recording
and/or visual center blood flow assay.
[0213] At two months after cell injection, Histology revealed a
significant preservation of photoreceptor neurons in the ONL of
retina in RCS rats administered with cell treatment (FIG. 15).
[0214] Preservation of rod and cone photoreceptor outer segment
revealed by immunostaining of Rhodopsin (rod) and Opsin (cone) was
observed in cell treated groups (both intravitreal and tail vein
injection, FIG. 16 and FIG. 17).
[0215] Cells transplanted into the vitreous cavity showed good
survival at 2 months after injection, then further differentiated
into rod photoreceptor cells expressing rod photoreceptor markers
(FIG. 18).
* * * * *