U.S. patent application number 11/063335 was filed with the patent office on 2006-09-28 for pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof.
This patent application is currently assigned to Reliance Life Sciences Pvt. Ltd.. Invention is credited to Khan Firdos Alam, Khanna Aparna, Subhadra Devi Kashyap, Pai Rajarshi, Geeta Ravindran, Tipnis Shabri, Satish Mahadeorao Totey.
Application Number | 20060216821 11/063335 |
Document ID | / |
Family ID | 40666905 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060216821 |
Kind Code |
A1 |
Totey; Satish Mahadeorao ;
et al. |
September 28, 2006 |
Pluripotent embryonic-like stem cells derived from corneal limbus,
methods of isolation and uses thereof
Abstract
The present disclosure describes mammalian pluripotent
embryonic-like stem cells (ELSCs) isolated from corneal limbal
tissue, a non-embryonic tissue. The ELSCs of the present disclosure
are capable of proliferating in an in vitro culture, maintain the
potential to differentiate into cells of endoderm, mesoderm, and
ectoderm lineage in culture, and are capable of forming
embryoid-like bodies when placed in suspension culture. Thus, these
cells possess multi-lineage differentiation potential and
self-renewing capability. ELSCs may be a promising therapeutic
tool, and may provide new therapeutic alternatives for various
diseases, conditions, and injuries.
Inventors: |
Totey; Satish Mahadeorao;
(Wadala, IN) ; Kashyap; Subhadra Devi; (Navi,
IN) ; Alam; Khan Firdos; (Navi, IN) ;
Rajarshi; Pai; (Navi, IN) ; Aparna; Khanna;
(Navi, IN) ; Shabri; Tipnis; (Bhandup (E), IN)
; Ravindran; Geeta; (Vikhroli (W), IN) |
Correspondence
Address: |
VINSON & ELKINS, L.L.P.
1001 FANNIN STREET
2300 FIRST CITY TOWER
HOUSTON
TX
77002-6760
US
|
Assignee: |
Reliance Life Sciences Pvt.
Ltd.
|
Family ID: |
40666905 |
Appl. No.: |
11/063335 |
Filed: |
February 22, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60621476 |
Oct 22, 2004 |
|
|
|
Current U.S.
Class: |
435/368 |
Current CPC
Class: |
C12N 2506/08 20130101;
C12N 2501/237 20130101; C12N 2500/30 20130101; C12N 2500/25
20130101; C12N 2500/62 20130101; C12N 2501/01 20130101; C12N 5/0621
20130101; C12N 5/0607 20130101; C12N 2533/90 20130101; C12N
2501/119 20130101; C12N 2501/11 20130101; C12N 2501/235 20130101;
C12N 2501/115 20130101; C12N 2533/52 20130101; C12N 2501/2306
20130101; C12N 2533/54 20130101; C12N 2533/32 20130101 |
Class at
Publication: |
435/368 |
International
Class: |
C12N 5/08 20060101
C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 2004 |
IN |
240/MUM/2004 |
Claims
1. Isolated mammalian pluripotent embryonic-like stem cells (ELSCs)
which (i) are capable of proliferating in an in vitro culture, (ii)
maintain the potential to differentiate into cells of endoderm,
mesoderm or ectoderm lineage in culture, and (iii) are capable of
forming embryoid-like bodies when placed in suspension culture.
2. The isolated stem cells of claim 1, wherein the mammalian ELSCs
are human ELSCs.
3. The isolated stem cells of claim 1, wherein the ELSCs are
derived from corneoscleral limbus tissue.
4. The isolated stem cells of claim 3, wherein the corneoscleral
limbus tissue is human tissue.
5. The isolated stem cells of claim 1, wherein the ELSCs remain
substantially undifferentiated after 20 passages in culture.
6. The isolated stem cells of claim 1, wherein the ELSCs remain
substantially undifferentiated after 100 passages in culture.
7. Isolated mammalian pluripotent embryonic-like stem cells (ELSCs)
which (i) are isolated from corneoscleral limbus, (ii) are capable
of proliferating in an in vitro culture, and (iii) maintain the
potential to differentiate into lineage-committed endodermal,
ectodermal, or mesodermal cells.
8. The isolated stem cells of claim 7, wherein the mammalian ELSCs
are human ELSCs.
9. The isolated stem cells of claim 7, wherein the ELSCs are
capable of forming embryoid-like bodies when placed in suspension
culture.
10. The isolated stem cells of claim 7, wherein the ELSCs remain
substantially undifferentiated after 20 passages in culture.
11. The isolated stem cells of claim 7, wherein the ELSCs remain
substantially undifferentiated after 100 passages in culture.
12. A method of isolating a population of mammalian pluripotent
embryonic-like stem cells (ELSCs), comprising the steps of: (a)
isolating corneal limbal tissue from a donor; (b) culturing the
corneal limbal tissue to expand corneal limbal cells in culture;
and (c) isolating a population of pluripotent ELSCs from the
cultured corneal limbal cells by sorting the corneal limbal cells
to select for one or more undifferentiated cell-specific surface
markers.
13. The method of claim 12, wherein the mammalian ELSCs are human
ELSCs.
14. The method of claim 12, wherein the donor is human.
15. The method of claim 12, wherein the corneal limbal tissue is
cultured on an extracellular matrix.
16. The method of claim 15, wherein the extracellular matrix is
mammalian amniotic membrane.
17. The method of claim 15, further comprising the step of
dissociating the cultured corneal limbal cells from the
extracellular matrix prior to isolating the pluripotent ELSCs.
18. The method of claim 12, wherein the corneal limbal tissue is
cultured in culture media supplemented with one or more soluble
factors selected from the group consisting of dimethyl sulphoxide,
recombinant human epidermal growth factor, insulin, sodium
selenite, transferrin, basic fibroblast growth factor, and leukemia
inhibitory factor.
19. The method of claim 12, wherein the corneal limbal tissue is
cultured until the corneal limbal cells become confluent.
20. The method of claim 12, wherein the corneal limbal cells are
sorted using magnetic-affinity cell sorting (MACS).
21. The method of claim 12, wherein the corneal limbal cells are
sorted using fluorescence-activated cell sorting (FACS).
22. The method of claim 12, wherein the undifferentiated
cell-specific surface markers are selected from the group
consisting of SSEA-4, SSEA-3, CD73, CD105, CD31, CD54, and
CD117.
23. The method of claim 12, wherein the undifferentiated
cell-specific surface marker selected for is SSEA-4.
24. The method of claim 12, further comprising culturing the
isolated population of pluripotent ELSCs to produce an
embryonic-like stem cell line.
25. The method of claim 24, wherein the pluripotent ELSCs are
cultured in culture media supplemented with one or more soluble
factors selected from the group consisting of dimethyl sulphoxide,
recombinant human epidermal growth factor, insulin, sodium
selenite, transferrin, basic fibroblast growth factor, and leukemia
inhibitory factor.
26. The method of claim 12, wherein the isolated population of
pluripotent ELSCs comprises at least about 70% ELSCs.
27. The method of claim 12, wherein the isolated pluripotent ELSCs
are capable of forming embryoid-like bodies when placed in
suspension culture.
28. The method of claim 12, wherein the pluripotent ELSCs remain
substantially undifferentiated after 20 passages in culture.
29. The method of claim 12, wherein the pluripotent ELSCs remain
substantially undifferentiated after 100 passages in culture.
30. The method of claim 13, further comprising the step of
differentiating the human ELSCs into endodermal lineage-committed
cells.
31. The method of claim 13, further comprising the step of
differentiating the human ELSCs into mesodermal lineage-committed
cells.
32. The method of claim 13, further comprising the step of
differentiating the human ELSCs into ectodermal lineage-committed
cells.
Description
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Not applicable.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present disclosure relates to purified preparations of
mammalian pluripotent stem cells, preferably human pluripotent stem
cells, derived from corneal limbus tissue. In preferred
embodiments, the pluripotent limbal stem cell lines are
self-renewing and have the ability to differentiate into tissues
derived from all three embryonic germ layers (endoderm, mesoderm
and ectoderm). Methods for isolating pluripotent limbal stem cell
lines and methods of their use are also disclosed.
[0004] 2. Description of Related Art
[0005] In early development, the ultimate source of all tissues in
a mammalian embryo or fetus are stem cells. In the embryonic stage
embryonic stem cells (ES cells) are totipotent and therefore
capable of developing into all the cells of a complete organism.
Cellular development occurs through several phases, including
cellular proliferation, lineage commitment, and lineage
progression, resulting in the formation of differentiated cell
types. There are three main lineages that are derived from
embryonic germ layers: ectoderm, mesoderm and endoderm. The
ectoderm germ layer forms the epidermis of the skin, sense organs,
nervous system, and spinal cord. The mesoderm germ layer forms
smooth muscle, connective tissues, blood vessels, heart, blood
cells and bone marrow, reproductive organ, excretory system,
striated muscles, and skeletal muscles. Finally, the endoderm germ
layer forms epithelial linings of respiratory and gastrointestinal
tract, pharynx, esophagus, stomach, intestine, and other associated
organs. ES cells are referred to as pluripotent stem cells because
they can differentiate into almost all cell types in an adult
organism.
[0006] During the last decade there has been ongoing research on
the isolation and use of ES cells and cell lines, which in addition
to having the ability to develop into most of the specialized cells
in the human body also have the capacity to divide and proliferate
indefinitely in culture. ES cells are often referred to as
pluripotent stem cells because they are not fixed in their
developmental potentialities and can differentiate into many
different cell types in vitro. Cultured ES cells that are highly
pluripotent can form clumps of cells in suspension culture referred
to as embryoid bodies. ES cells were isolated from humans
relatively recently (Thomson et al., (1998) Science 282:1145-1147;
Gearhart, (1998) Science 282:1061-62). In embryoid bodies derived
from human ES cells it is possible to discern differentiated cells
bearing markers of a wide variety of cell types.
[0007] The isolation of human ES cells offers the promise of a
remarkable array of novel therapeutics. Biologic therapies derived
from such cells through tissue regeneration and repairs, as well as
through targeted delivery of genetic material, are expected to be
effective in the treatment of a wide range of medical conditions.
However, despite the enormous potential of these materials, serious
ethical issues related to the use of human pluripotent stem cells
derived from human embryos or from fetal tissue obtained from
terminated pregnancies make stem cell research and treatments
problematic. In addition, technical issues associated with the use
of ES cells are problematic. Tissues or cells derived from ES cells
are not ideal for use in medical treatments because generally the
ES cells will not be derived from the patient who will ultimately
be receiving the treatment. Use of autologous tissues is preferred
for stem-cell-based therapies in order to avoid tissue rejection
problems.
[0008] I. Adult Stem Cells
[0009] The problems associated with human ES cells led many
researchers to turn their attention to adult tissues as a possible
source of undifferentiated stem cells with properties similar to
those of ES cells or germ cells derived from fetal tissue. It was
known that after birth and throughout adulthood a small number of
specialized stem cells are retained in an organism for the
replacement of cells and the regeneration of tissues. Indeed, adult
stem cells (also referred to as "tissue-specific stem cells") have
been found in very small numbers in various tissues of the adult
body, including bone marrow, (Weissman, (2000) Science
287:1442-1446), neural tissue (Gage, (2000) Science 287:1433-1438),
gastrointestinal tissue (Potten, (1998) Phil. Trans. R. Soc. Lond.
B. 353:821-830), epidermal tissue (Watt, (1997) Phil. Trans. R.
Soc. Lond. B. 353:831), hepatic tissue (Alison and Sarraf, (1998)
J. Hepatol. 29:678-683), and mesenchymal tissue. (Pittenger et al.,
(1999) Science 284:143-147).
[0010] Nevertheless, while some potential sources of adult stem
cells have been identified, to date adult stem cells have not been
found to be an adequate replacement for ES cells. First, adult stem
cells can be difficult to isolate because they are usually present
only in minute quantities in tissues that are often not easily
accessible, and their numbers appear to decrease with age. Second,
adult stem cells appear to be a less desirable source of cultured
tissue than ES cells because they have a shorter life span and less
capacity for self-renewal. Third, adult stem cells are believed to
be tissue specific and not pluripotent, generally capable of giving
rise only to new cells of a few types closely related to their
tissue of origin.
[0011] One particularly notable difference between ES cells and
adult stem cells is that ES cells in suspension culture are capable
of forming aggregates of cells known as embryoid bodies. These
embryoid bodies usually contain germ cells of all three lineages
that differentiate into various lineage-committed tissues.
Therefore, embryoid bodies can be useful in the preparation of
different types of differentiated cells in culture. To date, no
other isolated adult stem cell lines have been reported that are
capable of forming structures similar to embryoid-like bodies in
culture.
[0012] Recently, however, it has been suggested that some adult
stem cells have the capacity to be pluripotent. The most fully
characterized are the hematopoetic stem cells known as bone marrow
stromal cells or mesenchymal stem cells (Jiang et al., (2002)
Nature 418:41-48). These were the first adult stem cells found to
have pluripotent properties. Pluripotent adult stem cells have also
been isolated from liver (U.S. Publ. No. 2003/0186439), mouse inner
ear (Li and Heller, (2003) Nat. Med. 9:1293-1299), and amniotic
fluid (Prusa et al., (2003) Hum. Reprod. 18:1489-1493). Pluripotent
adult stem cells have also been recently described in many tissues
such as skeletal muscle, brain, and intestinal epithelium (Howell
et al., (2003) Ann. N.Y. Acad. Sci. 996:158-173.). Still, while
these papers report isolated or identified adult stem cells that
are pluripotent, these "pluripotent" adult stem cells, unlike ES
cells, differentiate into only a few lineages. In addition, none of
the isolated adult stem cells reported to date appear to be capable
of forming embryoid-like bodies in culture in a manner similar to
ES cells.
[0013] II. Corneoscleral Limbus
[0014] Similar to the other sources of adult stem cells referenced
above, it is known that adult stem cells are present in the
corneoscleral limbus of the eye. These cells participate in the
dynamic equilibrium of the corneal surface and replace superficial
epithelial cells that are shed and sloughed off during
eye-blinking. Severe damage to the limbal stem cells from chemical
or thermal burns, contact lenses, severe microbial infection,
multiple surgical procedures, cryotherapy, or diseases such as
Steven-Johnson syndrome or ocular cicatrical pemphigoid can lead to
destruction of limbal stem cells and limbal stem cell deficiency
which can lead to an abnormal corneal surface, photophobia, and
reduced vision (Anderson et al., (2001) Br. J. Opthalmol.
85:567-575). This damage cannot be repaired without the
re-introduction of a source of limbal stem cells (Tseng et al.,
(1998) Arch. Ophthalmol. 116:431-41; Tsai et al., (2000) N. Engl.
J. Med. 343:86-93; Henderson et al., (2001) Br. J. Ophthalmol.
85:604-609). Thus limbal stem cells, with their high proliferative
capacity, are clearly crucial for the maintenance of a viable
ocular surface because they provide an unbroken supply of corneal
epithelial cells necessary to maintain the equilibrium of the
corneal surface (Tseng, (1996) Mol. Biol. Rep. 23:47-58).
[0015] Experiments conducted in the 1980s first indicated the
existence of limbal cells in the corneal epithelium (Schermer et
al., (1986) J. Cell Biol. 103:49-62; Cotsarelis et al., (1989) Cell
57:201-209). Although it was later suggested that the transcription
factor P-63 was a specific marker for human corneal stem cells,
this marker is also expressed in other epithelial cells such as
skin, and therefore is not specific to corneal stem cells. In
addition, although P-63 expression has been shown to be principally
limited to the basal limbal region in human corneas (Moore et al.,
(2002) DNA Cell Biol. 21:443-51), in mice expression of this
transcription factor was maximal in paracentral cornea tissue
rather than limbus (Moore et al., (2002) DNA Cell Biol.
21:443-451). Therefore, currently there is no known definitive stem
cell marker for limbal epithelial stem cells.
[0016] It would be desirable to identify a source of adult stem
cells that are capable of self-renewal in culture and that are
pluripotent and ES cell-like in their ability to differentiate into
cells of all three major lineages: ectoderm, mesoderm and endoderm.
Further, it would be desirable to isolate and culture these adult
stem cells, and to induce them to differentiate into various cell
types.
BRIEF SUMMARY OF THE INVENTION
[0017] The present disclosure describes the isolation of mammalian
pluripotent embryonic-like stem cells (ELSCs) derived from
non-embryonic tissue, preferably corneal limbal tissue. In
particular, the present disclosure provides isolated mammalian
pluripotent ELSCs which: [0018] (i) are capable of proliferating in
an in vitro culture, [0019] (ii) maintain the potential to
differentiate into cells of endoderm, mesoderm, and ectoderm
lineage in culture, and [0020] (iii) are capable of forming
embryoid-like bodies when placed in suspension culture.
[0021] In preferred embodiments the isolated ELSCs are human ELSCs.
In related preferred embodiments, the ELSCs are derived from
corneoscleral limbus tissue, preferably human tissue. In other
preferred embodiments, the isolated ELSCs remain substantially
undifferentiated in an in vitro culture for at least about 20
passages, more preferably at least about 50 passages, and most
preferably at least about 100 passages in culture. Preferably,
after multiple passages in culture the substantially
undifferentiated ELSCs maintain normal karyotype and high
telomerase activity. In further embodiments, the isolated ELSCs
have the potential to terminally differentiate into cells or
tissues of endoderm, mesoderm, or ectoderm lineage.
[0022] The present disclosure also provides isolated mammalian
pluripotent ELSCs which: [0023] (i) are isolated from corneoscleral
limbus, [0024] (ii) are capable of proliferating in an in vitro
culture, and [0025] (iii) maintain the potential to differentiate
into lineage-committed endodermal, ectodermal, or mesodermal
cells.
[0026] In preferred embodiments the isolated ELSCs are human ELSCs,
which are more preferably SSEA-4 positive. In related embodiments,
the corneoscleral limbus is isolated from a human subject.
Preferably, the isolated ELSCs are capable of forming embryoid-like
bodies when placed in suspension culture. In other preferred
embodiments, the isolated ELSCs remain substantially
undifferentiated in an in vitro culture for at least about 20
passages, more preferably at least about 50 passages, and most
preferably at least about 100 passages in culture. Preferably,
after multiple passages in culture the substantially
undifferentiated ELSCs maintain normal karyotype and high
telomerase activity. In further embodiments, the isolated ELSCs
have the potential to terminally differentiate into cells or
tissues of endoderm, mesoderm, or ectoderm lineage.
[0027] The present disclosure also provides methods of isolating a
population of mammalian pluripotent embryonic-like stem cells
(ELSCs), comprising the steps of: [0028] (a) isolating corneal
limbal tissue from a donor; [0029] (b) culturing the corneal limbal
tissue to expand corneal limbal cells in culture; and [0030] (c)
isolating a population of pluripotent ELSCs from the cultured
corneal limbal cells by sorting the corneal limbal cells to select
for one or more undifferentiated cell-specific surface markers.
[0031] In preferred embodiments the isolated population of
pluripotent ELSCs are human ELSCs, which are more preferably SSEA-4
positive. In other embodiments, the donor of the corneal limbal
tissue is human. In certain embodiments, the corneal limbal tissue
is cultured in culture media such as DMEM or F12, further
supplemented with a nutrient serum and one or more soluble factors
selected from the group consisting of dimethyl sulphoxide (DMSO),
recombinant human epidermal growth factor (rhEGF), insulin, sodium
selenite, transferrin, basic fibroblast growth factor (bFGF), and
leukemia inhibitory factor (LIF). Preferably, the corneal limbal
tissue is cultured until the corneal limbal cells in the culture
become confluent. In certain embodiments, the corneal limbal tissue
is cultured on an extracellular matrix, for example Matrigel.TM.,
laminin, collagen-IV, poly-L-lysine, gelatin, poly-L-ornithin,
fibronectin, and combinations thereof, or mammalian amniotic
membrane. When the corneal limbal tissue is cultured on an
extracellular matrix, the above methods preferably further comprise
the step of dissociating the cultured corneal limbal cells from the
extracellular matrix prior to isolating the pluripotent ELSCs.
[0032] In preferred embodiments, the corneal limbal cells are
sorted using methods well known to those of skill in the art, for
example magnetic-affinity cell sorting (MACS) or
fluorescence-activated cell sorting (FACS) to isolate a population
of pluripotent ELSCs. In other embodiments, the one or more
undifferentiated cell-specific markers selected for to isolate
pluripotent ELSCs include but are not limited to SSEA-4, SSEA-3,
CD73, CD105, CD31, CD54, and CD117. In preferred embodiments,
corneal limbal cells are sorted to select for SSEA-4 positive
ELSCs. In certain embodiments, the sorted ELSCs comprise at least
about 80%, 90%, 95%, 98%, or 99% pluripotent ELSCs that are SSEA-4
positive. In preferred embodiments, the isolated population of
pluripotent ELSCs comprise at least about 70%, 80%, 90%, 95%, 98%,
or 99% pluripotent ELSCs. Preferably the isolated population of
pluripotent ELSCs are further cultured to produce an embryonic-like
stem cell line. In certain embodiments, the pluripotent ELSCs are
cultured in culture media such as DMEM or F12, further supplemented
with a nutrient serum and one or more soluble factors selected from
the group consisting of DMSO, rhEGF, insulin, sodium selenite,
transferrin, bFGF, and LIF.
[0033] In alternate embodiments, pluripotent ELSCs isolated by the
methods disclosed herein are capable of proliferating and
maintaining the potential to differentiate in vitro or in vivo into
cells or tissues of endoderm, mesoderm or endoderm lineage.
Preferably, the isolated pluripotent ELSCs are also capable of
forming embryoid-like bodies, for example when placed in suspension
culture. In other preferred embodiments, the isolated ELSCs remain
substantially undifferentiated in an in vitro culture for at least
about 20 passages, more preferably at least about 50 passages, and
most preferably at least about 100 passages in culture. Preferably,
after multiple passages in culture the substantially
undifferentiated ELSCs maintain normal karyotype and high
telomerase activity. In further embodiments, the isolated ELSCs
have the potential to terminally differentiate into cells or
tissues of endoderm, mesoderm, or ectoderm lineage.
[0034] In further embodiments the isolated pluripotent ELSCs,
preferably human ELSCs, are further differentiated in culture into
endodermal lineage-committed cells or tissues, mesodermal
lineage-committed cells or tissues, or ectodermal lineage-committed
cells or tissues. Alternatively, the isolated pluripotent ELSCs are
further differentiated into endodermal lineage-committed cells or
tissues, mesodermal lineage-committed cells or tissues, or
ectodermal lineage-committed cells or tissues in vivo. In other
embodiments, these ELSCs are further differentiated by exposing the
ELSCs to one or more agents known to induce differentiation of
pluripotent embryonic stem (ES) cells, including but not limited to
acidic fibroblast growth factor, bFGF, platelet-derived growth
factor (PDGF), insulin, retinoic acid, transferrin,
insulin-transferrin-selenious acid (ITS), dexamethasone, sodium
butyrate, DMSO, nerve growth factor (NGF), Cytosine beta-d-Arabino
Furanoside (Ara C), glial cell line-derived neurotrophic factor
gene (GDNF), transforming growth factor .beta.3 (TGF-.beta.3),
ascorbic acid, N-acetyl Cysteine, dibutaryl cyclic AMP, Neurturin,
transforming growth factor .beta.1 (TGF-.beta.1), insulin-like
growth factor I or II (IGF-I or IGF-II), epidermal growth factor
(EGF), bone morphogenic proteins 2 (BMP-2), .beta.
glycerophosphate, ascorbic acid 2 phosphate, 5-Aza-deoxy-cytidine,
oncostatin, hepatocyte growth factor (HGF), progesterone,
nicotinamide, or any combination thereof.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0035] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present disclosure. The present disclosure may be
better understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0036] FIG. 1. Limbal composite grafts (LCG): (a) H & E stained
LCG (whole mount); (b) LCG probed using immunofluorescence for
SSEA-4 antigen; (c) LCG analysis by RT-PCR for expression of the
pluripotent stem cell markers Oct-4, Nanog, and Rex 1, as well as
GAPDH as a positive control; and (d) SSEA-4 positive cells isolated
from LCG by flow cytometry.
[0037] FIG. 2. Immunophenotyping of ELSCs: ELSCs cultured for 20
passages were labeled with FITC-coupled antibodies against SSEA-4,
CD105, CD73, CD54, CD45, CD34, CD123, CD133, CD 123, and HLA-DR.
The ELSCs demonstrated positive labeling with SSEA-4, CD105, CD73,
and CD54 antibodies, indicating that the cells maintain
pluripotency after 20 passages, and that ELSCs are not
hematopoietic in origin.
[0038] FIG. 3. Gene expression profiling by RT-PCR of the
undifferentiated stem cell markers Oct-4, Nanog, Rex1, and TDGF1
after 5, 10, 15, and 20 passages of the isolated ELSCs. GAPDH
expression was also analyzed as a positive control. hEF cells were
used as a negative control for expression of the undifferentiated
stem cell markers, while NTERA (NT) cells were used as a positive
control for expression of these markers.
[0039] FIG. 4. Karyotyping of ELSCs. ELSCs isolated after 13
passages maintained a normal karyotype (analysis by CYTOVISION
software).
[0040] FIG. 5. Phase-contrast micrographs of ELSCs and ELBs
(10.times.): (a) micrograph of passage 15 ELSCs; (b) micrograph of
ELBs formed from ELSCs after 4 days of suspension culture; and (c)
micrograph demonstrating initiation of differentiation from ELBs
formed from ELSCs.
[0041] FIG. 6. Gene expression profiling by RT-PCR of the
undifferentiated stem cell markers Oct-4, Nanog, Rex1, and TDGF1 in
ELSCs (UD) and in ELBs collected on day 2 (2 d), day 4 (4 d), day 8
(8 d), day 12 (12 d), and day 14 (14 d) of ELB formation. GAPDH
expression was also analyzed as a positive control. hEF cells were
used as a negative control for expression of the undifferentiated
stem cell markers.
[0042] FIG. 7. Gene expression profiling by RT-PCR of lineage
markers NFH and Keratin (neuroectoderm lineage markers); c-Actin
(mesoderm lineage marker); and AFP and Albumin (endoderm lineage
markers) in ELSCs (UD) and in ELBs collected on day 2 (2 d), day 4
(4 d), day 8 (8 d), day 12 (12 d), and day 14 (14 d) of ELB
formation. Positive controls (PC) for each marker were as follows:
fetal-brain tissue extract for NFH and Keratin; fetal-heart tissue
extract for c-Actin; and fetal-liver tissue extract for AFP and
Albumin. The negative controls (NC) were (-)RT products.
[0043] FIG. 8. Gene expression profiling by RT-PCR of the following
markers in ELSCs (UD) and in ELBs collected on day 2 (2 d), day 4
(4 d), day 8 (8 d), day 12 (12 d), and day 14 (14 d) of ELB
formation: PECAM, which is an endothelial lineage marker; KGF and
Collagen I, which are stromal cell markers; and p63, which is a
corneal epithelial stem cell marker. Positive controls (PC) for
each marker were as follows: fetal-heart tissue extract for PECAM;
hEF cells for KGF and Collagen I; and limbal tissue extract for
p63. The negative controls (NC) were (-)RT products.
[0044] FIG. 9. Immunofluorescence assays of neuronal cells
differentiated from ELSCs through the formation of ELBs
(10.times.). Immunological characterization of neuronal cells
differentiated from ELSCs showed positive immunofluorescence for
the neuronal markers .beta.-tubulin III, Neurofilament, O4,
Glutamate, GABA, Tyrosine hydroxylase, Serotonin, Nestin.
[0045] FIG. 10. Cellular and functional characterization of various
differentiated cell-types from ELSCs through the formation of ELBs:
(a) initiation of differentiation from ELBs; (b) Von Kossa staining
of osteoblasts; (c) Alcian Blue staining of chondrocytes; (d) Oil
Red-O staining of adipocytes; (e) immunofluorescence of myocytes
with anti-myogenin antibody; (f) phase contrast micrograph of
beating cardiomyocytes (10.times.); (g) immunofluorescence of
cardiomyocytes with anti-cTnT antibody; (h) phase contrast
micrograph of mature hepatocytes derived from ELSCs; (i)
immunofluorescence of hepatocytes with anti-albumin antibody; (j)
PAS staining of mature hepatocytes showing insoluble glycogen
deposits; (k) immunofluorescence of pancreatic beta-islet cells
with anti-PDX-1 antibody.
[0046] FIG. 11. Gene expression profiling of differentiated cells
of various lineages derived from ELSCs by RT-PCR. Positive
expression of the following markers was found: c-Actin (cardiac
cell marker); NCS (functional cardiomyocyte marker); Myogenin
(myocyte marker); Alpha-Fetoprotein (AFP) (early mesendoderm
marker); Albumin (hepatocyte marker); PECAM (endothelial cell
marker); Insulin (pancreatic islet cell marker); Somatostatin
(pancreatic islet cell marker); .beta.-tubulin (neuronal cell
marker); and Tyrosine hydroxylase (TH) (dopaminergic neuronal
marker). GAPDH expression was also analyzed as a positive
control.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present disclosure relates to the isolation of mammalian
pluripotent embryonic-like stem cells (ELSCs) from non-embryonic
cells or tissues. In preferred embodiments, the present disclosure
utilizes mammalian eye structures as a source of ELSCs, preferably
corneal limbus tissue. In preferred embodiments, the ELSCs are
derived from corneoscleral or corneal limbus tissue of a human
donor. ELSCs of the present disclosure are particularly
advantageous due to several unique properties of these cells: (1)
ELSCs are capable of differentiating into cells of a variety of
different lineage-committed or differentiated cell and tissue
types, including cells and tissues derived from all three germ
layers, endoderm, ectoderm, and mesoderm; (2) ELSCs are
self-renewing and capable of propagating in culture for at least
about 20 to about 100 population doublings or more while
maintaining pluripotency, high telomerase activity, and normal
karyotype; and (3) ELSCs are capable of forming embryoid-like
bodies (ELBs).
[0048] The pluripotent ELSCs of the present disclosure are
undifferentiated or substantially undifferentiated cells that have
the potential to differentiate into almost any cell type.
Morphological characteristics of undifferentiated cells are well
known to those of skill in the art. For example, human ES cells may
be morphologically identified by high nucleus to cytoplasm ratios,
prominent nucleoli, and compact colony formation, with often
distinct cell borders and colonies that are often flatter than
mouse ES cells. Human ES cells are also preferably immunoreactive
with markers for human pluripotent ES cells, for example SSEA-3,
SSEA-4, GCTM-2 antigen, and TRA 1-60, as described by Thomson et
al., (Science 282:1145-1147, 1998) and Reubinoff et al. (Nature
Biotech. 18:399-403, 2000). As used herein, "non-embryonic cells or
tissues" refer to any cells or tissues that are derived from cells
or tissues other than embryonic cells, embryonic tissue, fetal
primordial germ cells, or fetal gonadal ridge tissue. In
particular, non-embryonic cells or tissues include cells or tissues
from adult mammals, and can also include cells or tissues from
juvenile mammals. ELSCs can differentiate into cells that are
committed to a particular germ lineage but still able to give rise
to various progeny cells of different cell types within that
lineage, as well as cells that are terminally-differentiated.
[0049] ELSCs of the present disclosure can be propagated in an in
vitro culture, where they are capable of differentiating cells that
are derivitives of each of the three germ layers (ectoderm,
mesoderm, and endoderm). As used herein, the term "differentiation"
refers to a process whereby undifferentiated pluripotent stem cells
or precursors cells acquire a more specialized fate. For example,
endodermal cells include but are not limited to epithelial cells
(e.g., corneal epithelial cells), hepatocytes, beta islet cells,
pancreatic cells (e.g., islet, acinar and ductal cells),
parenchymal cells of the trachea, bronchi, lungs, gastrointestinal
tract, bladder, pharynx, thyroid, thymus, parathyroid glands,
tympanic cavity, pharyngotympanic tube, tonsils, and the like;
mesodermal cells include but are not limited to myocytes (e.g.,
smooth muscle, skeletal muscle, cardiac myocytes, cardiomyocytes),
adipocytes (e.g., white fat and brown fat adipocytes),
chondrocytes, hematopoietic cells (e.g., erythrocytes),
lymphocytes, monocytes, macrophages, plasma cells, B cells, natural
killer cells and mast cells, endothelial cells, microglia,
dendritic cells, megakaryocytes, osteoblasts, osteoclasts,
chondroclasts, lymphoid cells, and cells of the tonsils, spleen,
kidney, ureter, bladder, testes, ovaries, uterus, and the like; and
ectodermal cells include but are not limited to neurons (e.g.,
dopaminergic, GABAergic, serotonergic, glutamatergic, and motor
neurons), glial cells (e.g., oligodendrocytes, astrocytes),
epithelial cells, ependymal cells, retinal cells, pineal body
cells, posterior pituitary cells, ganglia, peripheral nerve cells,
Schwann cells, sensory nerve endings, adrenal medulla, melanocytes,
mesenchymal cells, parafollicular "C" (calcitonin secreting) cells,
enterochromaffin cells, and cells of the heart valves, heart
outflow tract, epidermis, hair, nails, sweat glands, salivary
glands, sebaceous glands, mammary glands, anterior pituitary, inner
ear, lens of the eye, and the like.
[0050] ELSCs are also capable of forming embryoid-like bodies
(ELBs) in culture, for example in suspension culture. As used
herein, the term "embryoid-like bodies" or "ELBs" refer to an
aggregation of differentiated cells generated when pluripotent
ELSCs are grown in suspension culture, or overgrow in monolayer
cultures. ELBs may also have undifferentiated cells in the
aggregation of cells. ELBs typically contain cells derived from all
three germ layers, ectoderm, mesoderm and endoderm. Functionally,
ELBs may be similar or identical to embryoid bodies generated in
culture from ES cells, for example human ES cells. Embryoid bodies
and ELBs are distinguished from each other in the present
disclosure primarily by source, i.e., embryoid bodies are derived
from ES cells, while ELBs are derived from ELSCs.
[0051] The present disclosure further describes the isolation of
pluripotent ELSCs and uses of those cell lines. The use of ELSCs as
a source of pluripotent stem cells has numerous advantages. First,
the use of ELSCs does not raise many of the ethical concerns that
are associated with research using cells derived from embryonic or
fetal cells and tissue. Second, the use of ELSCs may make
autologous pluripotent stem cells available for medical therapies
as the source of differentiated cells and tissues without the
intermediate step of cloning. It is generally desirable that
transplanted cells or tissues be genetically identical to the
recipient of the transplant in order to avoid problems with tissue
rejection. However, it is not generally possible to obtain ES cells
that are genetically identical to a patient in need of treatment.
The use of ELSCs can surmount this problem if the donor of the
ELSCs is also the recipient of transplanted cells or tissue derived
from the ELSCs.
[0052] While adult stem cells have been previously isolated, none
of these adult stem cell lines have had the characteristics of the
ELSCs of the present disclosure. For example, previously isolated
adult stem cell lines have generally only been able to
differentiate into a few cell types, unlike the ELSCs of the
present disclosure. In a preferred embodiment of the present
disclosure, ELSCs are derived from corneal limbus tissue, which is
a safe, simple, and efficient source of pluripotent ELSCs.
Therefore, the present disclosure obviates the problems associated
with conventional sources of pluripotent stem cells.
[0053] A significant advantage of the use of corneal limbal tissue
as a source of ELSCs is the relative ease in obtaining corneal
limbal tissue from a donor. The process requires only minor
surgery, unlike the more invasive procedures that may be used to
obtain other types of adult stem cells. The corneal limbal tissue
is found in the cornea, which is a transparent, avascular tissue
that is located at the outer surface of the anterior eye. It
provides protection from environmental insult, and allows for the
efficient transmission of light into the eye. The cornea is
comprised of two main compartments: (1) the anterior non-cornified
stratified squamous epithelial layer and (2) the underlying
substantia propria. The human cornea harbors three known cell
types: corneal epithelial cells; stromal keratocytes (corneal
fibroblast); and an underlying layer of stromal associated corneal
endothelial cells. Corneal epithelium is a cellular multiplayer
that is five to seven cells thick and covers the anterior surface
of the cornea. Ordinarily, a natural turnover of corneal epithelial
cells takes place in which superficial epithelial cells are shed
from the epithelial surface and replaced by those from below. Basal
epithelial cells, migrating inward from the periphery, replenish
the population of deeper corneal epithelial cells.
[0054] Corneal limbus (also known as corneoscleral limbus) is an
annular transitional zone approximately 1 mm wide between the
cornea and the bulbar conjunctiva and sclera. It appears on the
outer surface of the eyeball as a slight furrow marking the line
between the clear cornea and the sclera. It is highly vascular and
is involved in the metabolism of the cornea. Limbal and conjuctival
epithelial cells, together with a stable pre-ocular tear film
maintain the integrity of the cornea. While it is known that the
source of the replenished corneal epithelial cells are adult stem
cells, the exact location and properties of these cells were
unknown. The adult stem cells previously isolated from the eye are
P-63 positive, and are responsible for maintaining corneal
integrity (Pellegrini et al., (2001) Proc. Acad. Natl. Sci. USA,
98:3156-61). The plasticity of these corneal stem cells was
recently reported by Seigel et al. (Mol. Vis. 9:159-63, 2003). The
existence of a second population of stem cells that are pluripotent
and have similar properties to ES cells was unknown. The present
disclosure describes the localization of ELSCs of the eye to the
corneoscleral limbus. A typical procedure for isolating corneal
limbal tissue is to surgically remove a small biopsy consisting of
2-3 mm of limbal tissue from the superior or temporal quadrant of
the corneal surface of the donor's eye. Procedures for obtaining
such biopsies from the corneal limbus are known to those of skill
in the art.
[0055] After limbal tissue is biopsied from a donor, it is placed
in culture, preferably on an extracellular matrix or bio-coated
surface, for example extracellular matrix or bio-coated petri
dishes. Examples of extracellular matrices useful for culturing
limbal tissue include but are not limited to Matrigel.TM. and its
equivalents, mammalian amniotic membrane, laminin, collagen-IV,
poly-L-lysine, gelatin, poly-L-ornithin, fibronectin, or platelet
derived growth factor (PDGF), either alone or in combination with
other extracellular matrix materials. Matrigel.TM. and human
amniotic membrane are particularly preferred for culturing biopsied
limbal tissue. Preferred methods of using extracellular matrix
materials are described in the examples below. With bio-coated
surfaces, a preferred method of culturing the limbal tissue is to
subject the explants to dry incubation for several minutes on a
bio-coated tissue culture plate. The explants are then affixed to
the tissue culture dish with a small amount of culture medium so
that they stick to the bio-coated tissue culture surface. After
several hours to a day, media is gently added and cells are
incubated for approximately 4-5 days at 37.degree. C. in a CO.sub.2
incubator, changing the media every alternate day.
[0056] The preferred media used for culturing the cells of the
limbal tissue is Dulbecco's Modified Eagles Medium (DMEM) or
DMEM:F-12 (1:1), preferably supplemented with a nutrient serum, for
example a serum or serum-based solution that supplies nutrients
effective for maintaining the growth and viability of the cells
(e.g., knock-out serum or heat-inactivated human serum), as well as
growth factors. As used herein, the term "growth factor" refers to
proteins that bind to receptors on the cell surface with the
primary result of activating cellular proliferation and
differentiation. The growth factors used for culturing limbal
tissue are preferably selected from epidermal growth factor (EGF),
basic fibroblast growth factor (bFGF), leukemia inhibitory factor
(LIF), insulin, sodium selenite, human transferrin, or human
leukemia inhibitory factor (hLIF), as well as combinations thereof.
However, any suitable culture media known to those of skill in the
art may be used. In certain embodiments, the limbal cells are
treated with cytokines or other growth factors which cause the
ELSCs to preferably proliferate in the culture.
[0057] After the limbal cells are cultured for several days,
preferably 7 to 21 days or until the cells become confluent, ELSCs
can isolated from the culture. In preferred embodiments, the limbal
cells are first dissociated from the extracellular matrix,
preferably through enzymatic digestion, for example using
trypsin-EDTA or dispase solutions. The pluripotent ELSCs can be
isolated from the other limbal cells in the culture using a variety
of the methods known to those of skill in the art such as
immunolabeling and fluorescence sorting, for example solid phase
adsorption, FACS, MACS, and the like. In preferred embodiments, the
ELSCs are isolated through sorting, for example immunofluorescence
sorting of certain cell-surface markers. Two methods of sorting
well known to those of skill in the art are are magnetic-affinity
cell sorting (MACS) and fluorescence-activated cell sorting
(FACS).
[0058] Sorting techniques such as immunofluorescence-staining
techniques involve the use of appropriate stem cell markers to
separate ELSCs from other cells in the culture. Appropriate stem
cell markers that may be used to isolate ELSCs from cultured limbal
cells include but are not limited to SSEA-4, SSEA-3, CD73, CD105,
CD31, CD54, and CD117. In preferred embodiments, pluripotent ELSCs
are isolated by MACS through the use of a cell surface marker such
as SSEA-4. By this means, enriched populations of cell-surface
marker positive ELSCs are obtained from the mixed population of
limbal cells. Alternatively, the cells can be sorted to remove
undesirable cells by selecting for cell-surface markers not found
on the pluripotent ELSCs. In the case of ELSCs isolated from limbal
tissue, the ELSCs were found to be negative for the following
cell-surface markers: CD34, CD45, CD14, CD133, CD106, CD11c, CD123,
and HLA-DR.
[0059] The enriched ELSCs cultures obtained by sorting have at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,
or 99% pluripotent ELSCs. In preferred embodiments the isolated
cells will be at least about 50%, 70%, 80%, 90%, 95%, 98%, or 99%
SSEA-4 positive ELSCs cells. In alternative embodiments, mixed cell
cultures containing pluripotent ELSCs are screened for the presence
of ELSCs that express certain gene markers. In the case of mixed
limbal cell cultures, populations of pluripotent ELSCs can be
identified by the expression of gene markers such as OCT-4, Nanog,
TDGF, UTX-1, FGF-4, Sox 2, Rex 1, as well as other gene marker of
undifferentiated cells.
[0060] The ELSCs isolated from the limbal cell culture are cultured
or passaged in an appropriate medium to allow the ELSCs to remain
in a substantially undifferentiated state. Although colonies of
undifferentiated ELSCs within the population may be adjacent to
neighboring cells that are differentiated, the culture of ELSCs
will nevertheless remain substantially undifferentiated when the
population is cultured or passaged under appropriate conditions,
and individual undifferentiated ELSCs constitute a substantial
proportion of the cell population. ELSCs cultures that are
substantially undifferentiated contain at least about 20%
undifferentiated ELSCs, and may contain at least about 40%, 60%,
80%, or 90% ELSCs. For example, ELSCs must be kept at an
appropriate cell density and repeatedly dissociated and subcultured
while frequently exchanging the culture medium to prevent them from
differentiating. When ELSCs are passaged they may be dispersed into
small clusters or into single-cell suspensions. Typically, a single
cell suspension of cells is achieved and then seeded onto another
tissue culture grade plastic dish.
[0061] For general techniques relating to cell culture and
culturing ES cells, which can be applied to culturing ELSCs, the
practitioner can refer to standard textbooks and reviews, for
example: E. J. Robertson, "Teratocarcinomas and embryonic stem
cells: A practical approach" ed., IRL Press Ltd. 1987; Hu and
Aunins (1997), Curr. Opin. Biotechnol. 8:148-153; Kitano (1991),
Biotechnology 17:73-106; Spier (1991), Curr. Opin. Biotechnol.
2:375-79; Birch and Arathoon (1990), Bioprocess Technol. 10:251-70;
Xu et al. (2001), Nat. Biotechnol. 19(10):971-4; and Lebkowski et
al. (2001) Cancer J. 7 Suppl. 2:S83-93, each incorporated herein by
reference.
[0062] The isolated ELSCs are cultured in an appropriate cell
culture medium such as DMEM or DMEM:F-12 medium, preferably
supplemented with a nutrient serum, for example a serum or
serum-based solution that supplies nutrients effective for
maintaining the growth and viability of the cells (e.g., knock-out
serum or heat-inactivated human serum), as well as growth factors.
In preferred embodiments, the medium is supplemented with growth
factors such as EGF, basic FGF, LIF, insulin, transferrin, sodium
selenite, and fibronectin. In some embodiments, the ELSCs are
cultured on a feeder layer. Methods for culturing pluripotent stem
cells on feeder layers are well known to those of skill in the art
(U.S. Pat. No. 5,843,780, WO 99/20741, incorporated herein by
reference). In other embodiments, the ELSCs are cultured on an
extracellular matrix. An extracellular matrix provides conditions
for supporting cell growth, for example similar to the conditions
provided by feeder cells. ELSCs may also be grown in the presence
of conditioned medium that can support growth of ELSCs cells, for
example in a feeder-free culture. Conditioned medium is prepared by
culturing a first population of cells in a medium for a sufficient
period of time to produce "conditioned" medium which will support
the culturing of ELSCs without substantial differentiation.
[0063] The isolated ELSCs can be serially passaged for at least 20,
40, 60, 80, 100 or more passages, without substantially
differentiating. ELSCs also can be frozen for further use at
various time points without loss of differential potential, i.e.,
the cells will retain the ability to differentiate into derivative
of endodermal, ectodermal, or mesodermal lineage under appropriate
conditions. In preferred embodiments, the isolated ELSCs retain
high telomerase activity and normal karyotypes for at least 20, 40,
60, 80, 100 or more passages.
[0064] In another embodiment, isolated pluripotent ELSCs are
identified and characterized by the ability to form ELBs in
culture, for example in suspension culture. Preferably the ELBs can
be further cultured to differentiate into cells of ectodermal,
mesodermal, and endodermal lineages. Methods for culturing
pluripotent stem cells to generate embryoid bodies are disclosed in
U.S. Pat. No. 6,602,711, incorporated herein by reference. These
same methods can also be used to generate ELBs from the ELSCs
disclosed herein. For example, ELSCs are dissociated using trypsin,
and cultured on bacteriological plates that have a non-adhesive
surface, thereby preventing attachment of the ELSCs to the surface
of the plate. The ELSCs are preferably cultured in an appropriate
cell culture medium such as knockout DMEM or DMEM:F-12 medium,
preferably supplemented with a nutrient serum, for example a serum
or serum-based solution that supplies nutrients effective for
maintaining the growth and viability of the cells (e.g., fetal calf
serum or fetal bovine serum), as well as growth factors. In
preferred embodiments, the medium is supplemented with growth
factors such as insulin, transferrin, or sodium selenite. The cells
are cultured until they form ELBs. Preferably the ELBs are cultured
until they reach sufficient size or desired differentiation, for
example after 3-10 days of culture, preferably 4-14 days. When ELBs
are subsequently cultured to differentiate into particular cell
types, the ELBs are allowed to grow to a sufficient size to
facilitate differentiation into the selected cell type. The ELBs
may be plated onto a substrate, for example a substrate coated with
extracellular matrix components, including but not limited to
poly-L-lysine, poly-L-ornithine, laminin, collagen, fibronectin,
Matrigel.RTM., or combinations thereof. The ELBs may be plated
directly onto a substrate with or without dispersing the cells.
[0065] ELSCs disclosed herein can be utilized for various
applications, such as therapeutic and diagnostic applications, as
well as for in vitro and in vivo assessment and screening of
various compounds such as small molecule drugs for their effects on
these cells, as well as differentiated cells derived from ELSCs.
The differentiated cells may be either lineage-committed progenitor
cells, or terminally-differentiated cells. Examples of
differentiated cell types that may be derived from pluripotent
ELSCs include but are not limited to neuronal cells, corneal cells,
osteoblasts, chondrocytes, adipocytes, beta-islets, cardiomyocytes,
hepatocytes, and the like. The ELSCs and cells and tissues
differentiated therefrom of the present disclosure can be used to
treat any subject in need of treatment, including but not limited
to humans, primates, and domestic, farm, pet, or sports animals,
such as dogs, horses, cats, sheep, pigs, cattle, rats, mice, and
the like. These cells can also be used to prepare cDNA expression
libraries to analyze the expression patterns of ELSCs as well as
cells derived therefrom, and to prepare monoclonal or polyclonal
antibodies that are specific to markers for the particular cells
used, using techniques that are well known to those of skill in the
art.
[0066] These cells can also be use therapeutically to the benefit
of individuals suffering from debilitating diseases, conditions,
injuries, and disorders, for example in tissue reconstitution or
regeneration in subjects such as human patients. As used herein,
the terms "therapeutically", "to treat", "treatment", or "therapy"
refer to both therapeutic treatment and prophylactic or
preventative measures. Therapeutic treatment includes but is not
limited to reducing or eliminating the symptoms of a particular
disease, condition, injury or disorder, or slowing or attenuating
the progression of, or curing an existing disease or disorder.
Subjects in need of such therapy will be treated by a
therapeutically effective amount of such cells to tissues to
restore or regenerate function. As used herein, a "therapeutically
effective amount" of cells or tissues is an amount sufficient to
arrest or ameliorate the physiological effects in a subject caused
by the loss, damage, malfunction, or degeneration of particular
cell-types or tissue-types. The therapeutically effective amount of
cells or tissues used will depend on the needs of the subject, the
subject's age, physiological condition and health, the desired
therapeutic effect, the size of the area of tissue that is to be
targeted for therapy, the site of implantation, the extent of
pathology, the chosen route of delivery, and the treatment
strategy. These cells or tissues may be administered to the patient
in a manner that permits the cells or tissue to graft to the
intended tissue site and reconstitute or regenerate the
functionally deficient area.
[0067] The following is a brief but by no means exhaustive list of
human diseases and conditions potentially treatable through the
administration of ELSCs or differentiated cells or tissues derived
therefrom: neurodegenerative disorders and neuronal diseases such
as Parkinson's disease, Alzheimer's disease, Huntington's disease,
Lewy body dementia, pancreatic diseases such as diabetes and
juvenile onset diabetes mellitus, cardiovascular and heart diseases
such as cardiac infarcts, Acquired Immunodeficiency Disease
Syndrome (AIDS), hematopoietic diseases such as lymphoma and
leukemia, cerebellar ataxia, progressive supranuclear palsy,
amyotrophic lateral sclerosis (ALS), epilepsy, multiple sclerosis,
burns, stroke, ischemia, trauma to the nervous system, neurotoxic
injury, and spinal cord injuries.
[0068] ELSCs of the present disclosure may be induced to
differentiate by any appropriate method known to those of skill in
the art. Many such methods are well known to those of skill in the
art for differentiating ES cells or adult stem cells into specific
cell types, for example neuronal precursor cells, neuronal cells,
or glial cells (U.S. Ser. No. 09/970,382, WO 01/88104, WO
03/000868, WO 01/68815, WO 01/83715, and U.S. Ser. Nos. 10/157,288
and 10/127,740), hematopoietic cells (U.S. Pat. No. 6,280,718, WO
01/34776), cardiomyocytes (WO 03/006950), hepatocytes (U.S. Pat.
Nos. 6,458,589 and 6,506,574), endothelial cells (WO 03/40319),
insulin-producing cells (WO 02/92756), and endocrine cells (WO
02/59278), all of which are specifically incorporated herein by
reference. Although these methods were originally adapted for
differentiating ES cells or adult stem cells into specific cell
types, they may also be adapted to differentiate the ELSCs
described herein. These methods can include differentiation through
the formation of colonies, ELBs, or other aggregates (WO 01/62899,
specifically incorporated herein by reference), as well as methods
promoting differentiation into certain cell lineages by withdrawing
serum or factors that inhibit differentiation and/or adding factors
that promote differentiation. Differentiation of cells may also be
facilitated by the use of particular extracellular matrices, for
example poly-o-orinthine, laminin, or Matrigel.TM.. ELSCs can also
be differentiated directly into committed precursor cells or fully
differentiated cells, for example without forming ELBs as an
intermediate step.
[0069] Preferred methods of inducing differentiation of ELSCs
include the use of differentiation agents, including but not
limited to progesterone, putrescine, laminin, insulin, sodium
selenite, transferrin, neurturin, sonic hedgehog (SHH), noggin,
follistatin, retinoic acid, epidermal growth factor (EGF), any type
of fibroblast growth factor, cytosine .beta.-d-Arabino furanoside
(Ara-C), growth and differentiation factor 5 (GDF-5), members of
the neurotrophin family (nerve growth factor (NGF), neurotrophin 3
(NT-3), neurotrophin 4 (NT-4), brain derived neurotropic factor
(BDNF)), transforming growth factor .alpha. (TGF-.alpha.),
transforming growth factor beta-1 (TGF .beta.1), transforming
growth factor beta-3 (TGF .beta.3), platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF-1), bone morphogenic
proteins (BMP-2, BMP-4), glial cell derived neurotrophic factor
(GDNF), midkine, ascorbic acid, ascorbic acid 2 phosphate,
dibutyryl cAMP, dopamine, ligands to receptors that complex with
gp130 (e.g., LIF, CNTF, SCF, IL-11, and IL-6),
insulin-transferrin-selenious acid (ITS), dexamethasone, sodium
butyrate, dimethyl sulfoxide (DMSO), N-acetyl Cysteine,
insulin-like growth factor I or II (IGF-I or IGF-II), .beta.
glycerophosphate, 5-Aza-deoxy-cytidine, oncostatin, hepatocyte
growth factor (HGF), nicotinamide, or combinations thereof. As used
herein, the term "fibroblast growth factor" or "FGF" refers to any
suitable fibroblast growth factor, derived from any organism that
expresses such factors, and functional fragments thereof. A variety
of FGFs are known to those of skill in the art, and include but are
not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2
(basic fibroblast growth factor), FGF-3 (int-2), FGF-4 (hst/K-FGF),
FGF-5, FGF-6, FGF-7, FGF-8, and FGF-9. Differentiation nutrient
mediums may also contain additives that help sustain cultures of
neural cells, for example N2 and B27 additives (Gibco). Pluripotent
ELSCs can be induced to differentiate in various available culture
media, including but not limited to DMEM, DMEM-F-12, MCDB,
Neurobasal medium, neurturin, N2, B27, and the like, or
combinations thereof.
[0070] The presence of differentiated cells in a cell culture can
be determined by any one of many methods known to those of skill in
the art. For example, determination of differentiated cells can be
accomplished by methods such as flow cytometry, immunochemistry,
immunofluorescence staining, or other staining techniques, for
example Von Kossa staining of osteoblasts, Alcian Blue staining of
chondrocytes, or Oil Red-O staining of adipocytes, to detect the
presence of cell surface markers, proteins, or other types of
genetic markers. Alternately, identifying differentiated cells may
be accomplished by detecting expression of certain genes or gene
products such as RNA or proteins using RT-PCR, HPLC, and the
like.
[0071] The following is an exemplary list of methods for
differentiating pluripotent ELSCs into particular cell types. This
list is by no means exhaustive, and is intended for illustrative
purposes only. In one specific embodiment, pluripotent ELSCs can be
induced to differentiate into neurons by culturing the cells in a
neurobasal medium supplemented with B-27, N2, insulin-transferrin,
and selenite in the presence of retinoic acid, basic FGF, and Ara C
for approximately 4 to 14 days. In another specific embodiment,
pluripotent ELSCs can be induced to differentiate into hepatocytes
directly or by exposing ELBs to acidic FGF, basic FGF, HGF,
oncostatin, dexamethasone, insulin, transferrin-selenious acid
(ITS), DMSO, 5-azacytidine, sodium butyrate, or combinations
thereof. In another specific embodiment, pluripotent ELSCs can be
differentiated into cardiomyocytes directly or by exposing ELBs to
TGF-.beta.1, IGF-I, IGF-II, BMP-4, basic FGF, FGF-4, PDGF-BB,
5-aza-deoxycytidine, insulin, EGF, or combinations thereof. In
still another specific embodiment, pluripotent ELSCs can be
differentiated into beta-islet cells either directly or by exposing
ELBs to N.sub.2, B.sub.27, nicotinamide, basic FGF, TGF-.beta.1, or
combinations thereof.
[0072] In a further specific embodiment, pluripotent ELSCs can be
differentiated into chondrocytes either directly or by exposing
ELBs to TGF.beta.3, ascorbic acid 2 phosphate, or combinations
thereof. In another specific embodiment, pluripotent ELSCs can be
differentiated into osteoblasts directly or by exposing ELBs to
dexamethasone, .beta.-glycerophosphate, ascorbic acid 2 phosphate,
hydrocortisone, or combinations thereof. In yet another specific
embodiment, pluripotent ELSCs can be differentiated into adipocytes
directly or by exposing ELBs to dexamethasone,
isobutylmethylxanthine (IBMX), indomethacin, insulin, or
combinations thereof. In a further specific embodiment, pluripotent
ELSCs can be differentiated into myocytes directly or by exposing
ELBs to 5-Azacytidine, PDGF-BB, or combination thereof. The present
disclosure also provides a method of cryopreservation of
pluripotent ELSCs, for example, wherein the cells are cryopreserved
in 10% dimethyl sulfoxide (DMSO) or another appropriate medium and
stored in liquid nitrogen.
[0073] The present disclosure also contemplates the use of
pluripotent ELSCs for cell-based therapies. As reported in the
literature, the ability to regenerate human tissues that are
substantially damaged due to disease or injury is reduced
significantly in adults. Pluripotent ELSCs disclosed herein may be
induced to terminally differentiate into appropriate cell or tissue
types, or to differentiate into appropriate lineage-committed
progenitor cells, which can then be administered or transplanted
into a mammalian subject for cell replacement therapy or tissue
regeneration. Alternatively, ELSCs may be directly administered to
a subject. Therefore, the methods of the present disclosure may be
useful in the treatment of many diseases, injuries, or other
detrimental condition. Pluripotent ELSCs of the present disclosure
can be induced to differentiate either in vitro or in vivo.
[0074] ELSCs generated according to the present disclosure can also
be used to study the cellular and molecular biology of development,
functional genomics, as well as the generation of differentiated
cells for use in therapeutic or prophylactic transplantation,
treatment, drug screening, or in vitro drug discovery. For example,
the ELSCs can be used for genomic analysis, to produce mRNA, cDNA,
or genomic libraries, to produce specific polyclonal or monoclonal
antibodies, including but not limited to humanized monoclonal
antibodies (WO 01/51616, specifically incorporated herein by
reference), or to screen for the effects of different test
compounds or biologically active molecules on ELSCs and cells or
tissues derived therefrom, such as pharmaceutical compounds in drug
research. The test compounds or biologically active molecules
screened may be derived for example from plants, plant-based
extracts, or synthetic sources. ELSCs can also be used to screen
for factors (such as small molecule drugs, peptides,
polynucleotides, and the like) or conditions (such as cell culture
conditions or manipulations) that affect the characteristics of
ELSCs in culture, and the differentiation of ELSCs into various
specific cell and tissue types.
[0075] Differentiated cells derived from ELSCs, for example
neuronal cells, beta-islets, cardiomyocyte, hepatocyte, corneal
cells, osteoblasts, chondrocytes, and adipocytes, can be used to
generate human body organs by 3-D reconstruction, for example
tissues in the human brain may be reconstructed by 3-D culturing of
the neurons derive from human ELSCs. Similarly, other human body
organs or parts such as liver, heart, kidney, skin, eye, ear, and
the like may be derived and reconstructed from pluripotent ELSCs.
ELSCs of the present disclosure may also be used as carrier
vehicles for various therapetically active molecules or genes to be
delivered at various sites of the human body, for example by
genetically manipulating and differentiating the ELSCs as required,
and delivering the cells or tissue to a target site in a donor for
gene therapy. The present disclosure therefore provides methods of
using pluripotent ELSCs with their unique capability to
differentiate into cells of all three germ layer lineages for
pharmaceutical interventions and for human-based cell assays for
drug discovery, analysis, and testing.
[0076] The following examples are included to demonstrate preferred
embodiments of the disclosure. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
[0077] 1) Collection of Limbal Tissue Biopsies
[0078] Prior to initiating the collection of limbal tissue biopsies
from human patients, Institutional Review Board approval was
obtained. Informed consent was obtained from each patient and
donor, and all human subjects were treated according to the
Helsinki Accord. A 2-3 mm limbal biopsy of the donor eye was
collected surgically from superior or temporal quadrants of the
corneal surface by lamellar keratectomy. After excision, biopsies
were immediately placed in a 2 ml transport vial filled with
transport medium. The transport medium consisted of Dulbecco's
Modified Eagles Medium (DMEM) and Ham's F-12 Medium (DMEM:F-12;
1:1) supplemented with 5% fetal bovine serum (FBS) or 5% human
serum collected from cord blood, 0.5% dimethyl sulphoxide (DMSO), 2
ng/ml recombinant human epidermal growth factor (rhEGF), 5 .mu.g/ml
insulin, 5 .mu.g/ml transferrin, 5 .mu.g/ml sodium selenite, 0.5
.mu.g/ml hydrocortisone, 0.1 nmol/l cholera toxin A, 50 .mu.g/ml
gentamycin, and 1.25 .mu.g/ml amphotericin B. Blood samples were
also collected from each donor and transported along with each
limbal tissue biopsy to a centrally located cGMP facility. Blood
samples were immediately tested for infectious diseases, including
Hepatitis B virus (HBV), Hepatitis C virus (HCV), Syphillis, and
CMV.
[0079] 2) Preparation of Extracellular Matrices for Limbal
Biopsies
[0080] Throughout the studies described herein, suitable
extracellular matrix carriers such as Matrigel.TM., fibrinogen,
PDGF, laminin, EGF, collagen V, or human amniotic membrane were
used to culture limbal tissue biopsies. In certain studies, the
extracellular matrix carrier was treated with attachment factors
such as laminin, collagen V, PDGF, EGF, or fibrinogen, either
singly or in combination, along with growth factors such as EGF,
insulin-like growth factor-1 (IGF-1), insulin, either singly or in
combination. In the present disclosure, Matrigel.TM. (BD
Biosciences) is the preferred extracellular matrix carrier.
[0081] To prepare Matrigel.TM. coated tissue culture plates,
Matrigel.TM. was slowly thawed overnight at 4.degree. C. to avoid
the formation of a gel. After complete thawing, 10 ml of cold
knockout DMEM was added to the bottle containing 10 ml Matrigel.TM.
and mixed. The mixture was kept on ice, mixed well, and aliquots of
1 ml were prepared and stored at -20.degree. C. until needed for
coating plates. As needed, each Matrigel.TM. aliquot was slowly
thawed at 4.degree. C. for at least 2 hours to avoid the formation
of a gel. The aliquot was then diluted 1:15 in cold knockout DMEM,
and 1 ml of Matrigel.TM. solution was added to coat 35 mm or 60 mm
plates. After pouring the solution into the plates, the plates were
allowed to set for 1-2 hours at room temperature or overnight at
4.degree. C. After setting, any remaining Matrigel.TM. solution was
removed from the plate prior to use, and the plates were washed
with knockout DMEM prior to use.
[0082] 3) Preparation of Amniotic Membrane Cultures for Limbal
Biopsies
[0083] Amniotic membrane cultures were used to culture limbal
tissue biopsies isolated from human subjects. The preparation of
these amniotic membrane culture began with the collection of human
placental membranes. Placental membranes were collected from
elective Cesarean section operations and transported to laboratory
facilities in a transport medium consisting of Dulbecco's phosphate
buffered saline (DPBS) supplemented with 50 unit/ml penicillin, 50
.mu.g/ml streptomycin, 50 .mu.g/ml neomycin, and 2.5 .mu.g/ml
amphotericin B. Placental membrane was transported to the
laboratory within 3 hours of surgery. Blood samples were also
collected from each donor and sent for infectious disease
diagnostic tests as described above.
[0084] Once received, the placenta was washed with washing medium
to remove mucus and blood clots. The washing medium consisted of
Dulbecco's phosphate buffered saline (DPBS) supplemented with 50
unit/ml penicillin, 50 .mu.g/ml streptomycin, 100 .mu.g/ml
neomycin, and 2.5 .mu.g/ml amphotericin B. Placental tissue was
removed from the amniotic membrane using sterile scissors, and the
amniotic membrane was washed thoroughly at least 7 times to remove
substantially all blood clots. Next, the chorion was peeled off of
the amniotic membrane with blunt forceps, and the epithelial side
of the amniotic membrane was washed 5 times with the washing
medium. The amniotic membrane was then placed on a sterile
nitrocellulose membrane with the epithelial side of the membrane
facing up. The membrane was cut into 5 cm.times.5 cm area pieces
and each piece was placed in a cryo-vial filled with freezing
medium consisting of 50% glycerol in DMEM. Each batch of processed
amniotic membrane was checked for sterility, as well as the absence
of mycoplasma or endotoxin contamination before being used for
limbal culture. The pieces of amniotic membrane were each stored at
-80.degree. C.
[0085] Amniotic membrane cultures for culturing limbal tissue
biopsies were prepared from these pieces of amniotic membrane by
first thawing the pieces at room temperature for 20 minutes. Each
amniotic membrane was then carefully removed from the
nitrocellulose membrane using blunt forceps, preferably without
tearing the surface, and placed on a sterile glass slide in a 100
mm petri plate. Next, a small volume of trypsin (1.0-1.5 ml of
0.05% Trypsin-EDTA) was added to cover the amniotic membrane, and
the membrane was incubated at 37.degree. C. for 30 minutes. After
incubation, the epithelial layer of the amniotic membrane was
scraped off with a cell scraper under sterile aseptic conditions.
The amniotic membrane was then washed 3 times with washing
solution. The processed and treated amniotic membrane, which
functions as an extracellular carrier matrix in culture, was placed
on a culture plate with a 0.4 .mu.M track-etched polyethylene
terephthalate (PET) membrane insert (Falcon, USA, 3090). The
amniotic membrane was fastened to the PET insert, for example by
using number 10 Ethilon non-absorbent suture or by using a medical
grade silicon O-ring.
[0086] Regardless of the means, the amniotic membrane should be
spread on the membrane insert in such a way that the denuded
epithelial side of the membrane faces the inner side of the insert
and the stromal side of the membrane faces out of the insert. The
amniotic membrane was stretched uniformly before being secured to
the insert, for example by inserting the silicon O-ring into the
bottom of the amniotic membrane, or suturing the amniotic membrane
to the basement membrane of the insert. The entire set-up was
incubated in a 6-well dish filled with culture medium for at least
2 hours in DMEM/F12 (Gibco-BRL) media supplemented with 10% FBS (ES
tested) (Hyclone), 5 .mu.g/ml transferrin (Gibco), 0.1 .mu.g/ml
cholera toxin (Sigma), 50 U/ml penicillin-streptomycin (Sigma), 5
.mu.g/ml gentamicin (Sigma), 5 ng/ml Na-selenite (Sigma), 10 ng/ml
EGF (Sigma), and 0.5% DMSO (Sigma). The amniotic membrane was
washed two times with culture medium and again incubated in culture
medium for 30 minutes, after which the amniotic membrane was ready
for culturing limbal tissue biopsies.
[0087] 4) Culturing of Limbal Biopsies to Produce Limbal Composite
Grafts
[0088] Limbal tissue from the limbal biopsies was initially washed
several times with culture medium DMEM/F12 (Gibco-BRL) media
supplemented with 10% FBS (ES tested) (Hyclone), 5 .mu.g/ml
transferrin (Gibco), 0.1 .mu.g/ml cholera toxin (Sigma), 50 U/ml
penicillin-streptomycin (Sigma), 5 .mu.g/ml gentamicin (Sigma), 5
ng/ml Na-selenite (Sigma), 10 ng/ml EGF (Sigma), and 0.5% DMSO
(Sigma). The biopsies were then trimmed of any sclera and
conjunctiva tissues and cut into 6 to 7 pieces. These limbal tissue
pieces were subsequently cultured on Matrigel.TM. coated plates or
on amniotic membrane cultures, which were prepared as described
above. Some of the limbal tissue pieces were enzymatically treated
with 0.25% trypsin-EDTA (Gibco-BRL, USA) for 30 minutes, while
others were treated with 0.25% trypsin-EDTA overnight at 4.degree.
C. The epithelial layer of each biopsy piece was removed using
blunt forceps, and the stromal were cultured cells on
Matrigel.TM.-coated 35 mm plates in culture medium consisting of
Dulbecco's Modified Eagles Medium (DMEM) and F-12 (DMEM:F-12; 1:1)
supplemented with 10% knock-out serum or 10% heat-inactivated human
serum collected from cord blood, 0.5% dimethyl sulphoxide (DMSO), 2
ng/ml recombinant human epidermal growth factor (rhEGF), 5 .mu.g/ml
insulin, 5 .mu.g/ml transferrin, 5 .mu.g/ml sodium selenite, 0.5
.mu.g/ml hydrocortisone, 4 ng/ml bFGF, 10 ng/ml hLIF, 50 .mu.g/ml
gentamycin, and 1.25 .mu.g/ml amphotericin B. The entire culture
process to generate limbal composite grafts was carried out at
37.degree. C. in air (5% CO.sub.2) for 7 to 21 days or until the
cells became confluent, with the culture medium being changed every
alternate day.
[0089] To determine whether the limbal composite grafts (LCG)
contained pluripotent ELSCs, the LCGs were analyzed by
immunofluorescence and flow cytometry to detect the presence of the
cell surface marker SSEA-4, which is a marker for human pluripotent
ES cells. FIG. 1(a) shows Hematoxylin and Eosin (H & E)
staining of a LCG (whole mount). Hematoxylin stains negatively
charged nucleic acids such as nuclei and ribosomes blue, while
Eosin stains proteins pink. As shown in FIG. 1(b), when a LCG is
exposed to an SSEA-4 antibody (1:100 dilution), the LCG is clearly
positive for SSEA-4, as indicated by green immunofluoresence.
Molecular characterization of the LCG was also performed using
RT-PCR analysis to detect OCT-4, Nanog, and Rex-1 expression, each
of which are pluripotency markers that are down-regulated upon
differentiation. FIG. 1(c) shows expression of each of these
pluripotent stem cell markers, with GAPDH acting as a positive
control. Finally, FIG. 1(d) shows the isolation of SSEA-4 positive
cells (63%) from a LCG by flow cytometry after the cells were
subjected to magnetic affinity cell sorting (MACS) (see below).
[0090] 5) Isolation of Pluripotent Embryonic-Like Stem Cells from
Limbal Composite Grafts
[0091] After 7 to 21 days of limbal cell culture, the cultured
cells were subjected to magnetic affinity cell sorting (MACS) to
isolate pluripotent ELSCs. The cultured cells were first dispersed
using 0.05% trypsin-EDTA. The trypsin was neutralized by adding an
equal amount of culture medium that contained a trypsin inhibitor
or fetal calf serum. The cells were subsequently pipeted into a
single cell suspension, and counted using a hemocytometer. Next,
the cells were spun down and resuspended to a concentration of
10.sup.7 cells per 200 .mu.l of PBS. The cells were incubated for
30 minutes at 4.degree. C. with 1 .mu.l of primary antibody SSEA-4
(DSHB, USA; 1:40). After incubation with SSEA-4 primary antibody,
the cells were washed twice with PBS to remove any unbound
antibody. A 20 .mu.l suspension of secondary antibody beads
(Miltenyi Biotech, Germany; 1:4) that bind to the SSEA-4 primary
antibody was added to 200 .mu.l of the cell suspension, mixed well,
and incubated at 4.degree. C. for 20 minutes. The cells were washed
three times with PBS to remove any unbound secondary antibody.
[0092] The cell suspension were then passed through a MACS magnetic
column according to the manufacturer's instructions (Miltenyi
Biotech, Germany) to isolate SSEA-4 positive cells. The negative
fraction was collected first, and the column was washed twice with
PBS. Next, the column was removed from the magnet and the positive
fraction with SSEA-4 positive cells was collected. The SSEA-4
positive cells, are also pluripotent ELSCS, were washed twice and
seeded on an extracellular matrix carrier in culture medium.
Preferably, the extracellular matrix carrier was
Matrigel.TM.-coated plates and the culture medium was DMEM and F-12
(DMEM:F-12; 1:1), supplemented with 10% knock-out serum or 10%
heat-inactivated human serum collected from cord blood, DMSO
(0.5%), rhEGF (2 ng/ml), insulin (5 .mu.g/ml), transferrin (5
.mu.g/ml), sodium selenite (5 .mu.g/ml), gentamycin (50 .mu.g/ml)
and amphotericin B (1.25 .mu.g/ml), hLIF (10 .eta.g/ml), and bFGF
(4 .eta.g/ml). The ELSCs were cultured for an additional week at
37.degree. C. in a CO.sub.2 incubator or until the cultures became
confluent.
[0093] After confluence, the ELSCs in culture were dissociated and
re-plated on fresh bio-coated tissue culture dishes at a plating
dilution of 1:3. The ELSCs were then expanded and serially passaged
for at least 100 population doublings. ELSCs that were serially
passaged could also be frozen for further use without any loss of
differential potential. Telomerase activity was still detected in
the cultured cells after 50 passages.
EXAMPLE 2
[0094] Analysis and Characterization of Pluripotent Embryonic-Like
Stem Cells:
[0095] As outlined in Example 1, pluripotent ELSCs were derived
from limbal tissue biopsies. Although not wishing to be limited to
any particular theory, it appears that corneal limbus has
essentially two stem-cell types that are segregated into two zones.
The top layer of the limbus is composed mainly of corneal
epithelial stem cells that are P-63 positive, while the basal layer
is composed mainly of stromal cells. It appears that the
pluripotent ELSCs disclosed herein, predominantly reside in the
stromal layer, and may migrate towards the epithelial zone as
needed.
[0096] To better understand the nature of the pluripotent ELSCs
derived from limbal tissue, and the undifferentiated status of
these cells, ELSCs were analyzed using flow cytometry,
immunofluorescence, and molecular analysis for the presence or
absence of various cellular markers for undifferentiated and
differentiated cells. Karyotype and telomerase activity were also
analyzed at various passages to determine whether these cells
maintain an undifferentiated state after serial passages.
[0097] 1) Flow Cytometry Analysis
[0098] Only a few cell surface markers that are immunoreactive with
pluripotent embryonic stem cells are known. To determine whether
the ELSCs isolated herein are also immunoreactive with these cell
surface markers, the ELSCs were analyzed for the presence of
various cell surface cluster differentiation (CD) markers and stage
specific embryonic antigen (SSEA) markers that are usually
expressed on pluripotent ES cells. Analysis was carried out after
every passage. The presence of the following markers was also
analyzed using flow cytometry: SSEA-1, SSEA-3, SSEA-4, CD11c, CD14,
CD34, CD45, CD54, CD73, CD105, CD106, CD123, CD133, stem cell
factor (SCF), and HLA-DR markers. Antibodies to SSEA-1, SSEA-3, and
SSEA-4, and CD markers have previously been used for flow cytometry
analysis.
[0099] Pluripotent ELSCs isolated in Example 1 were trypsinized
after expansion using 0.25% trypsin-EDTA for 2-3 minutes. After
inactivation of the trypsin, the cells were passed through a 40
micron filter mesh to remove any remaining cellular clumps that
could interfere with staining. The cells were then centrifuged and
resuspended in wash buffer at a concentration of 1.times.10.sup.6
cells/ml. The wash buffer consisted of phosphate buffer
supplemented with 1% fetal bovine serum. Aliquots of
1.times.10.sup.5 cells were added to control and test tubes and
incubated with the following antibodies, each of which was
conjugated with either fluorescein isothiocyanate (FITC) or
phycoerythrin (PE): SSEA-1, SSEA-3, SSEA-4, CD11c, CD14, CD31,
CD34, CD45, CD54, CD73, CD105, CD106, CD117, CD123, CD133, or
HLA-DR antibody. The tubes were vortexed briefly and incubated in
the dark for 1 hour at 4.degree. C. The cells were washed 3-4 times
with wash buffer and resuspended in 500 .mu.l of wash buffer. Flow
cytometry was performed on a FACS Calibur flow cytometer
(Becton-Dickinson), and cells were identified by light scatter.
Logarithmic fluorescence was evaluated on 10,000 gated events, and
control samples were used to adjust the background fluorescence.
Analysis was performed using CELL QUEST software (Becton
Dinkinson). The percent of positive cells was determined with
respect to the control tube events. Results are summarized in Table
1 below: TABLE-US-00001 TABLE 1 Results of the various stem cell
markers analyzed for pluripotent embryonic-like stem cells by flow
cytometry SI. No Markers Results % Cells positive SSEA-1 Negative
0% SSEA-3 Positive 19% 1 SSEA-4 Positive 98% 2 CD11c Negative 0% 3
CD14 Negative 0% 4 CD34 Negative 0% 5 CD45 Negative 0% 6 CD54
Positive 51% 7 CD73 Positive 98% 8 CD105 Positive 98% 9 CD106
Negative 0% 10 CD117 Positive 44% 11 CD123 Negative 0% 12 CD133
Negative 0% 13 HLA-DR Negative 0% 14 CD-31 Positive 98%
[0100] Results of immunophenotyping pluripotent ELSCs cultured for
20 passaged are shown in FIG. 2. As outlined above, the ELSCs were
labeled with FITC-coupled antibodies against SSEA-4, CD105, CD73,
CD54, CD45, CD34, CD123, CD133, CD 123 and HLA-DR. The ELSCs were
analyzed using FACS-calibur. The results in FIG. 2 are consistent
with the results shown in Table 1. As shown, ELSCs are positive for
SSEA-4, CD54, CD73, and CD105 markers, and negative for CD34, CD45,
CD106, CD123, CD133, and HLA-DR markers.
[0101] Pluripotent ELSCs were found to have similar characteristics
to previously isolated primate ES cells derived from the inner cell
mass of a blastocyst (U.S. Pat. No. 6,200,806), namely the ELSCs
were positive for the stage-specific embryonic antigen markers
SSEA-3 and SSEA-4, and negative for the SSEA-1 marker. The
expression of SSEA-3 and SSEA-4, and the lack of expression of
SSEA-1, in limbal-tissue-derived pluripotent ELSCs is similar to
that of human ES cells, indicating that these pluripotent cells may
have similar properties.
[0102] Pluripotent ELSCs were also analyzed for cluster
differentiation markers, and were found to be negative for CD11c,
CD14, CD34, CD 45, CD106, CD123, CD133, and HLA-DR markers. This
data demonstrates that ELSCs isolated from limbus tissue are not
hematopoietic in origin since they are negative for the CD34 and
CD45 markers. The remaining CD markers analyzed, CD11c, CD14,
CD106, CD123, CD133 and HLA-DR markers, are only known to be
expressed in differentiated cells. Therefore, the absence of these
markers on the ELSCs demonstrates that these cells are
undifferentiated and are not generating cells of differentiated
lineages while passaged in culture under the indicated conditions.
The expression pattern shown in FIG. 2 also suggests that ELSCs can
be maintained in an undifferentiated state, (i.e., maintain
"stemness") for at least 20 passages, and that the ELSCs are not
hematopoetic in origin.
[0103] Interestingly, expression of CD73 and CD105 by ELSCs
suggests that these cells are mesenchymal in origin. Therefore, it
is hypothesized that ELSCs are derived from the lower stromal cell
layer of limbus tissue that contains mesenchymal and fibroblastic
cells rather than the upper epithelial cell layer. ELSCs, however,
also expressed the CD54 marker, which is known to be an endothelial
cell marker. To further understand this finding, the cells were
analyzed by RT-PCR for expression of another endothelial marker,
the PECAM gene (see FIG. 11).
[0104] Pluripotent ELSCs were further assayed for their
pluripotency and undifferentiated status through the use of the
cellular markers OCT-4, TRA-1-60, TRA-1-80, and Alkaline
phosphatase. In order to determine whether these genes were
expressed by ELSCs, cultured ELSCs were collected at day 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 and
subjected to gene expression analysis. Strong expression of each of
these undifferentiated markers was observed in pluripotent ELSCs.
The results of this gene profiling again demonstrate that the
pluripotent ELSCs of the present disclosure are undifferentiated
stem cells.
[0105] 2) Molecular Analysis
[0106] As found from the experiments and results set forth above,
an array of unique markers expressed in ELSCs isolated from limbal
tissue have been found. To further characterize the ELSCs, the
cells were analyzed by RT-PCR for expression of the following
pluripotent stem cell marker genes: Oct-4, Nanog, Rex1, and TDGF1.
Expression of Oct-4, Nanog, Rex1, and TDGF1 are down regulated upon
differentiation. Expression of the "housekeeping" gene GAPDH, which
is ubiquitously expressed in all cells, was also analyzed as a
positive control. The identity of the RT-PCR products was confirmed
by sequencing.
[0107] Briefly, total RNA of pluripotent ELSCs was isolated at
every passage, for example passage 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, using the TRIzol method
(Gibco-BRL). Next, 1 .mu.g of total RNA treated with RNase-OUT
ribonuclease inhibitor (Invitrogen Inc, USA) was used for cDNA
synthesis by reverse-transcription using reverse transcriptase
(Invitrogen Inc, USA) and oligo dT (Invitrogen Inc, USA) to prime
the reaction. For each polymerase chain reaction (PCR) reaction, 2
ul of cDNA was amplified by PCR using Abgene 2.times.PCR master mix
and the appropriate primers. PCR primers were selected to
distinguish between cDNA and genomic DNA by using individual
primers specific for different exons. The primers used to amply
Oct-4, Nanog, Rex1, and TDGF1 cDNAs are set forth below in Table 2.
The PCR amplification conditions used in the thermal cycler (ABI
Biosystems 9700) to amplify the PCR products were as follows: (1)
94.degree. C., 1 minute; (2) 94.degree. C., 30 seconds; annealing
Tm .degree. C., 45 seconds; 72.degree. C., 1 minute, for 30 cycles
of amplification; (3) 72.degree. C., 5 minutes; and (4) final hold
at 4.degree. C. until the samples were analyzed. TABLE-US-00002
TABLE 2 Annealing PCR Product Temp size Gene Primer Sequence
(.degree. C.) (bp) GAPDH 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' SEQ ID
NO:1 60 890 5'-CATGTGGGCCATGAGGTCCACCAC-3' SEQ ID NO:2 Oct-4
5'-CGRGAAGCTGGAGAAGGAGAAGCTG-3' SEQ ID NO:3 58 247
5'-CAAGGGCCGCAGCTTACACATGTTC-3' SEQ ID NO:4 Nanog
5'-CCTCCTCCATGGATCTGCTTATTCA-3' SEQ ID NO:5 52 262
5'-CAGGTCTTCACCTGTTTGTAGCTGAG-3' SEQ ID NO:6 Rex1
5'-GCGTACGCAAATTAAAGTCCAGA-3' SEQ ID NO:7 56 306
5'-CAGCATCCTAAACAGCTCGCAGAAT-3' SEQ ID NO:8 TDGF1
5'-GCCCGCTTCTCTTACAGTGTGATT-3' SEQ ID NO:9 55 499
5'-TAGTACGTGCAGACGGTGGTAGTTCT-3' SEQ ID NO:10
[0108] The results of this experiment at the fifth (P5), tenth
(P10), fifteenth (P15) and twentieth (P20) passages of the isolated
ELSCs are shown in FIG. 3. Expression of Oct-4, Nanog, Rex1 and
TDGF1 markers for "stemness" was also analyzed in hEF and NTERA
cell lines as negative and positive controls, respectively. The
NTERA cell line is an established terato-carcinoma cell line which
expresses pluripotent stem cell markers. GAPDH expression appeared
to be approximately the same in each sample, indicating that the
amount of RNA used for each RT-PCR reaction was quantitatively
similar. The same data is reproduced in a tabulated manner in Table
3. TABLE-US-00003 TABLE 3 Gene P5 P10 P15 P20 hEF NTERA GAPDH ++ ++
++ ++ ++ ++ Oct-4 + + ++ + + +++ Nanog ++ ++ ++ + - ++ Rex-1 ++ ++
++ ++ ++ ++ TDGF1 ++ ++ ++ + - ++
[0109] Conspicuous expression of all four markers of pluripotent
undifferentiated cells was observed in the pluripotent ELSCs at
each of the various passages. It appears that expression of at
least two of the pluripotency markers, Nanog and TDGF1, gradually
decreased, over time. It is possible that this decrease is due to
an increased percentage of differentiated cells with the ELSCs as
they are passaged in culture, because it is known that expression
of these genes is down regulated in differentiated cells.
Nevertheless, these results clearly indicate that ELSCs isolated
from limbal tissues are pluripotent and have embryonic stem
cell-like properties. Although the hEF cell line was a negative
control, expression of both Oct-4 and Rex-1 was found in the cell
line. Interestingly, there have been reports of undifferentiated
stem cell markers being expressed in certain committed cell lines
(Abeyta et al., (2004) Hum. Mol. Genet. 13:601-608).
[0110] 3) Telomerase Activity Analysis
[0111] Maintenance of telomerase activity in a pluripotent stem
cell line is important for the long-term pluripotency of the cell
line. Therefore, telomerase activity of the presently disclosed
ELSCs was evaluated over time. ELSCs extracts from passage 5 (P5),
10 (P10), 15 (P15), and 20 (p20) were prepared, and protein
concentration in each extract was estimated. The extracts were next
evaluated using the telomeric repeat amplification protocol (TRAP),
which is an assay designed for highly sensitive qualitative
detection of telomerase activity. Telomerase activity was then
detected by photometric enzyme immunoassay. Briefly, after
estimation of protein concentration, samples were placed in a
thermocycler and PCR was performed per the protocol. After PCR
amplification, the amplified products were denatures, and detected
by ELISA.
[0112] Telomerase PCR ELISA was done according to manufacturer's
protocol (Roche Molecular Biochemicals), and all proper positive
and negative controls provided in the kit were used. The telomerase
PCR ELISA allows highly specific amplification of
telomerase-mediated elongation products combined with
non-radioactive detection following the ELISA protocol. Care was
taken during the protocol to remove inhibitors of Taq polymerase,
which could result in a false negative result. In the first step of
the protocol, telomerase in the extract adds telomeric repeats
(TTAGGG SEQ ID NO:11) to the 3' end of biotin-labeled synthetic
primer. Next, these elongation products are amplified by PCR using
primers that generate PCR products that contain the
telomerase-specific six nucleotide increments. An aliquot of the
PCR products are denatured and hybridized to a
digoxigenin-(DIG)-labeled, telomeric repeat-specific detection
probe. The resulting product is immobilized via the biotin labeled
primer to a strepatavidin-coated microtiter plate. After detection
with an antibody, which is conjugated to peroxidase, telomerase
activity is detected by formation of a colored product. High
expression of telomerase activity was seen in the extracts of all
passages tested, indicating the high proliferative capacity of
pluripotent ELSCs.
[0113] 4) Karyotype Analysis
[0114] To determine whether the pluripotent ELSCs of the present
disclosure maintain a normal karyotype in culture, ELSCs were
karyotyped using a standard G-banding technique (Genetics Lab.,
Reliance Life Sciences Pvt. Ltd., Mumbai) and compared to published
human karyotypes. The karyotyping was performed using the
CYTOVISION software. ELSCs from passage 13 (P13) were karyotyped
and found to have `normal karyotype`, that is, the cells at P13
were found to be euploid, all human chromosomes were present, and
the chromosomes were not noticeably altered (FIG. 4). ELSCs were
also found to have normal karyotypes at passage 20.
EXAMPLE 3
[0115] Differentiation and Analysis of Pluripotent Embryonic-Like
Stem Cells:
[0116] 1) Generation of Embryoid-Like Bodies from Pluripotent
Embryonic-Like Stem Cells
[0117] To determine whether the undifferentiated human pluripotent
ELSCs could form embryoid-like bodies (ELBs) in culture, the cells
were first allowed to proliferate and the cell cultures expanded.
Next, the cells were cultured on bacteriological plates having a
non-adhesive surface that prevented attachment of the ELSCs, and
stimulated differentiation of these cells. Briefly, ELSCs were
dissociated by briefly exposing them to a 0.05% trypsin-EDTA
solution, and subsequently cultured as a suspension culture in ES
cell medium containing DMEM:F-12 or knockout DMEM, supplemented
with 10-20% fetal calf serum, cord blood serum, or knockout serum
replacement. The media was also supplemented with
.beta.-mercaptoethanol, L-glutamine, insulin, human transferrin,
sodium selenite, but did not contain bFGF or hLIF. The cells were
incubated in suspension culture for about 4 days and the media was
changed every other day. The medium was changed by transferring the
suspension of aggregates to a centrifuge tube, allowing the
aggregates to settle down, aspirating the old medium, replacing it
with fresh medium, and returning the aggregates and fresh medium to
the culture dish.
[0118] At the end of 4 days, ELBs were collected by spinning down
the aggregates at low speed (1000 rpm, 5 minutes) and resuspending
the ELBs in the same ES cell medium described above. FIG. 5 shows
this differentiation process using phase contrast micrographic
pictures (10.times.). First, FIG. 5(a) shows ELSCs grown in culture
after 15 passages. FIG. 5(b) shows ELBS, which formed after
culturing ELSCs for 4 days in suspension culture. Finally, FIG.
5(c) shows initiation of differentiation from the ELBs.
[0119] To further characterize the process of differentiation from
ELSCs to ELBs, the molecular analysis protocol described above in
Example 2 was repeated on undifferentiated ELSCs (UD), as well as
ELBs after 2 days (EB2), 4 days (EB4), 8 days (EB8), 12 days
(EB12), and 14 days (EB14) of differentiation in suspension
culture. The cells were analyzed by RT-PCR for expression of the
following pluripotent stem cell marker genes: Oct-4, Nanog, Rex1,
and TDGF1, the expression of which are down regulated upon
differentiation. The primers used to amply Oct-4, Nanog, Rex1, and
TDGF1 cDNAs are set forth above in Table 2. In addition, expression
of the "housekeeping" gene GAPDH was also analyzed as a positive
control. Expression of each of these markers was also analyzed in
hEF, which functioned as a negative control. The identity of the
RT-PCR products was confirmed by sequencing.
[0120] The results of this experiment are shown in FIG. 6. GAPDH
expression appeared to be approximately the same in each sample,
indicating that the amount of RNA used for each RT-PCR reaction was
quantitatively similar. The same data is reproduced in tabulated
form in Table 4. TABLE-US-00004 TABLE 4 Gene UD EB2 EB4 EB8 EB12
EB14 hEF GAPDH ++ ++ ++ ++ ++ ++ ++ Oct-4 ++ ++ - - - - + Nanog ++
++ ++ + + + + Rex-1 ++ + - - - + TDGF1 ++ + + - - - -
[0121] Expression of all four markers was again found in the
undifferentiated ELSCs, and expression of each of the genes also
appeared to gradually decreased over time as cells in the ELBs
differentiated. Although it is surprising that Oct-4, Nanog
expression was found in the hEF cell line, there have been reports
of undifferentiated stem cell markers being expressed in certain
committed cell lines. Although the hEF cell line was a negative
control, expression of Oct-4, Nanog, and Rex-1 was found in the
cell line. Interestingly, there have been reports of
undifferentiated stem cell markers being expressed in certain
committed cell lines (Abeyta et al., (2004) Hum. Mol. Genet.
13:601-608).
[0122] In addition to examining expression of undifferentiated
markers, the same set of cells were analyzed for expression of
various genes for ectoderm, mesoderm and endodermal lineages, as
well as expression of genes indicating endothelial lineage, stromal
cells, and corneal epidermal stem cells. Expression of the
following gene markers was analyzed, again by RT-PCR using the
protocol outlined in Example 2: Neurofilament Heavy Chain (NFH) and
Keratin (ectodermal lineage markers); cardiac-Actin (c-Actin)
(mesodermal lineage marker); Alpha-Fetoprotein (AFP) and Albumin
(endodermal lineage markers); PECAM (endothelial lineage marker),
Keratinocyte Growth Factor (KGF) and Collagen I (stromal cell
markers); and p63 (corneal epithelial stem cell marker). The
primers used to amply each of these markers are set forth below in
Table 5. TABLE-US-00005 TABLE 5 Annealing PCR Temp Product Gene
Primer Sequence (.degree. C.) size (bp) NFH
5'-TGAACACAGACGCTATGCGCTCAG-3' SEQ ID NO:12 58 400
5'-CACCTTTATGTGAGTGGACACAGAG-3' SEQ ID NO:13 Keratin
5'-AGGAAATCATCTCAGGAGGAAGGGC-3' SEQ ID NO:14 56 780
5'-AAAGCACAGATCTTCGGGAGCTACC-3' SEQ ID NO:15 c-Actin
5'-TCTATGAGGGCTACGCTTTG-3' SEQ ID NO:16 50 630
5'-CCTGACTGGAAGGTAGATGG-3' SEQ ID NO:17 AFP
5'-AGAACCTGTCACAAGCTGTG-3' SEQ ID NO:18 50 680
5'-GACAGCAAGCTGAGGATGTC-3' SEQ ID NO:19 Albumin
5'-CCTTTGGCACAATGAAGTGGGTAACC-3' SEQ ID NO:20 58 450
5'-CAGCAGTCAGCCATTTCACCATAGG-3' SEQ ID NO:21 PECAM
5'-GTCATGGCCGTCGAGTA-3' SEQ ID NO:22 50 260
5'-CTCCTCGGCATCTTGCTGAA-3' SEQ ID NO:23 Collagen I
5'-CCATCCAAACCACTGAAACC-3' SEQ ID NO:24 55 600
5'-TGACGAGACCAAGAACTG-3' SEQ ID NO:25 KGF
5'-GATACTGACATGGATCCTGCC-3' SEQ ID NO:26 55 300
5'-CACAATTCCAACTGCCACTG-3' SEQ ID NO:27 p63
5'-CAGACTCAATTTAGTGAG-3' SEQ ID NO:28 48 550
5'-AGCTCATGGTTGGGGCAC-3' SEQ ID NO:29
[0123] FIGS. 7 and 8 shows the results of the gene expression
pattern of different gene markers in the ELSCs (UD) and in ELBs
collected after 2, 4, 8, 12, and 14 days of culture. The positive
controls used for each RT-PCR were fetal-brain tissue extract as a
neuroectodermal lineage control (for NFH and Keratin), fetal-heart
tissue extract as a mesodermal and endothelial lineage control (for
c-Actin and PECAM), fetal-liver tissue extract as a endodermal
lineage control (for AFP and Albumin), hEF cells as a stromal cell
control (for Collagen I and KGF), and limbal tissue extract as a
corneal epidermal stem cell control (for p63), while the negative
control was (-)RT product. The same data as shown in FIGS. 7 and 8
are reproduced in a tabulated manner in Table 6. TABLE-US-00006
TABLE 6 Gene UD EB2 EB4 EB8 EB12 EB14 PC NC NFH - - + ++ - - ++ -
Keratin - ++ - - ++ - ++ - c-Actin - - - + ++ - +++ - AFP - ++ - +
- - ++ - Albumin - - - + ++ - ++ - PECAM ++ ++ ++ ++ ++ ++ ++ - Col
I + + + ++ ++ - ++ - KGF ++ ++ + ++ ++ - + - p63 ++ + + + - - -
-
[0124] Expression of Keratin, KGF, Collagen-1, and P-63 in the
ELSCs and ELBs indicates that the cells are derived from limbal
tissue. Keratin is an early ectodermal lineage marker, while
expression of KGF and Collagen-I at nearly all stages is expected
because they are both markers for fibroblastic cells, and ELSCs are
isolated from the stromal layer. It is unclear why expression of
Collagen-I appears to be upregulated at day 8 and 12 of ELB
formation, but is not present at day 14 of ELB formation. P-63
expression was found in the population of undifferentiated ELSCs,
indicating that these cells also contain a population of corneal
limbal stem cells. P-63 expression decreased as the ELSCs
differentiate during ELB formation.
[0125] The lineage markers NFH, c-Actin, AFP, and Albumin do not
appear to be expressed in the ELSCs, but are expressed at various
stages of differentiation in the ELBs. NFH, which is an early
neuro-ectodermal marker, is not expressed in undifferentiated
ELSCs, but is expressed at day 4 and 8 of ELB formation. NFH was
not expressed in later stages of ELB formation, however, because
expression of this marker is downregulated upon maturation of
neuronal cells. c-Actin, which is a mesodermal lineage marker, is
highly expressed by day 12 of ELB formation. AFP, which is an early
mesendodermal marker, is highly expressed at day 2 of ELB
formation, but is gradually downregulated as the ELBs
differentiate. Expression of Albumin, which is a mature hepatic
cell marker, is gradually upregulated as ELBs differentiate. PECAM
is expected to be expressed at all stages of embryonic development,
and is closely correlated with the pluripotency of reported cells
(Furusawa et al., (2004) Biol. Reprod. 70:1452-57). The
differential expression of appropriate lineage markers in ELBs at
various stages of differentiation indicates the ability of ELSCs to
differentiate into cells of all three lineages.
[0126] 2) Differentiation of Pluripotent Embryonic-Like Stem Cells
Into Neurons
[0127] To determine whether ELSCs of the present disclosure can
differentiate into neurons, ELBs derived from ELSCs were cultured
for approximately 4-10 days in an appropriate medium and plated
directly onto a suitable substrate with an extracellular matrix
component such as polyornithine, laminin, or fibronectin. The ELBs
were cultured in a suitable nutrient medium adapted to promote
differentiation of the cells into neuroprogenitor cells. Then the
cells were further cultured under conditions that encouraged
differentiation and maturation into specific neuronal phenotypes,
including GABAergic and dopaminergic neurons.
[0128] Derivation of GABAergic Neurons
[0129] ELBs were cultured in serum-free neuronal induction medium
composed of basal medium DMEM:F12 along with additives that help
sustain cultures of neural cells, for example N2 (1-15%) and B27
(1-20%). The medium was also supplemented with one or more growth
factors selected from Retinoic acid (20-80 ng/ml), GDNF (1-10
.mu.g/ml), Ara-C (10-50 ng/ml), and Neurotrophin-3 (5-20 .mu.g/ml).
The cells were grown for 6 days, which resulted in the
differentiation of neuroprogenitor cells.
[0130] Next, the neuroprogenitor cells were grown in a neural
differentiation medium containing Neurobasal medium supplemented
with N2 (1-15%), B27 (1-20%), and growth factors including insulin
(5-20 .mu.g/ml), Transferrin (4-10 .mu.g/ml), FGF-8 (50-200 ng/ml),
and Ara-C (10-50 ng/ml) for 12 days with the media being change
every second day. To generate cells having a mature neuronal-like
morphology, the cells were grown in neural differentiation medium
that was supplemented with other neuronal growth factors such as
neutrotrophin-3 and GDNF. In particular, Neurotrophin-3 (5-20
.mu.g/ml) and GDNF (1-10 .mu.g/ml) were added to the
differentiation medium on day 6 to day 12 of differentiation.
[0131] Derivation of Dopaminergic Neurons
[0132] The ELBs were cultured in serum-free defined medium composed
of DMEM:F12 supplemented with N2 (1-15%) and B27 (1-20%), along
with one or more antioxidants, such as DMSO (1-10%), Butylated
hydroxyanisole (50-400 .mu.M), and forskolin (5-20 .mu.M), for
initiation of neuronal induction. After 4-7 days of culture, one or
a combination of growth factors such as Retinoic acid (20-80
ng/ml), GDNF (1-10 .mu.g/ml), Shh (50-200 ng/ml), FGF-8 (50-200
ng/ml), or bFGF (10-50 ng/ml) were added to the medium to
facilitate neuronal differentiation. The cells were grown in this
medium for an additional 7-10 days. Changes in cell morphology of
the cultured cells were observed within 48 hours. The percentage of
responsive cells increased progressively with incubation under
antioxidant and serum-free conditions. The neuroprogenitor cells
were next grown in neuronal maturation media containing Neurobasal
medium supplemented with N2 (1-15%), B27 (1-20%), GDNF (1-10
.mu.g/ml), Retinoic acid (20-80 ng/ml), db-cAMP (10-200 .mu.M), and
IL-1b (1-5 .mu.g/ml). Under these culture conditions, about 30-40%
of the cells extended neurite processes and stained positive for
.beta.-tubulin, which evidenced their ability to form neurons. The
growth factors present in the neuronal induction medium contribute
to the overall increase in percentage of neuronal cells, and
further induce these precursor cells to adopt the dopaminergic
phenotype.
[0133] Characterization of Differentiated Neurons
[0134] The differentiated neuronal cell types generated according
to the above protocols were evaluated both by the overall
morphology of the cells, as well as the phenotypes identified by
immunofluorescence. Immunofluorescence analysis was carried out at
day 12 and day 25 of differentiation of GABAergic and dopaminergic
neurons, respectively. First, the isolated cells were grown on
2-well chamber slides precoated with extracellular matrices, rinsed
with PBS, and fixed for 10 minutes with 4% paraformaledyde at room
temperature. Next, the cells were permeabilized with 0.2% Triton
X-100 in PBS for 5 minutes, blocked with 1% bovine serum albumin
(BSA)/PBS for 2 hours, and incubated with a primary antibody
(antibody dilutions were made in 1% BSA/Tris-buffered saline)
overnight at 4.degree. C.
[0135] The cells were stained with the following primary
antibodies: early neuronal marker .beta.-tubulin III (1:500); late
neuronal marker Microtubule associated protein 2 (MAP-2) (1:200);
gamma aminobutyric acid (GABA) (1:200); Glutamate (1:500); Nestin
(1:50); Neurofilament (1:500); Tyrosine hydroxylase (TH) (1:800);
Serotonin (1:500) and Oligodendrocyte (1:500). All primary
antibodies were obtained from Chemicon Inc., USA. Next, the cells
were incubated with the appropriate FITC-labeled secondary
antibody. After each step, the cells were washed three times with
PBS. The chamber slides were observed under a fluorescence
microscope to evaluate the immunopositive areas. This
immunofluorescence analysis, as shown in FIG. 9, demonstrated that
many of the differentiated cells were immunoreactive to the neuron
specific markers MAP-2, .beta.-tubulin III, and Neurofilament, as
well as the phenotype specific markers TH (marker for dopaminergic
neurons), GABA (marker for GABAergic neurons), Glutamate (marker
for glutamatergic neurons), and Serotonin (marker for serotonergic
neurons). Only a few cells expressed the non-neuronal marker O4,
which is present in Oligodendrocytes (glial cells).
[0136] ELSCs and differentiated neuronal cell types generated above
were also analyzed for expression of .beta.-tubulin and Tyrosine
hydroxylase (TH) by RT-PCR as previously described above using the
following primers: TABLE-US-00007 TABLE 7 Annealing PCR Temp
Product Gene Primer sequence (.degree. C.) size (bp) .beta.-tubulin
5'-GGAACATAGCCGTAAACTGC-3' SEQ ID NO:30 60 317
5'-AGTTCACTGYGCCTGAACTTACC-3' SEQ ID NO:31 TH
5'-TGTCAGAGCAGCCCGAGGTC-3' SEQ ID NO:32 63 417
5'-CCAAGAGCAGCCCATCAAAG-3' SEQ ID NO:33
[0137] Expression of c-Actin and NCX is indicative of adult
cardiomyocyte formation. The PCR primers used to amplify the cDNAs
were specific for exon sequence of each gene, thereby allowing
amplification of cDNA only, and not genomic DNA encoding the genes.
FIG. 11 shows the results of RT-PCR analysis for the expression of
cardiomyocyte specific markers in ELSCs and differentiated
cardiomyocytes derived therefrom. As shown, both c-Actin and NCX
are expressed in the differentiated cardiomyoctyes, and not in the
undifferentiated ELSCs. The housekeeping gene GAPDH was used as a
positive control as previously described.
[0138] Functional Characterization of Dopaminergic Neurons by
RP-HPLC
[0139] The functional capacity of ELSC-derived dopaminergic neurons
to produce dopamine was evaluated by directly measuring the
extracellular dopamine levels using Reverse Phase HPLC (RP-HPLC).
The concentration of dopamine detected in culture supernatant was
determined by comparison with a standard solution of dopamine
injected into the column immediately before and after each
analysis. Approximately 5.times.10.sup.6 cells were trypsinized and
pelleted by centrifugation. The cells were then sonicated in cold
1N perchloric acid with antioxidants (0.2 g/l sodium
metabisulphite), and centrifuged at 15,000 rpm/min for 20 minutes
at 4.degree. C. Next, the culture supernatant was immediately
stabilized with 7.5% orthophosphoric acid and sodium
metabisulphite, and stored at -70.degree. C. for subsequent
determination of the extracellular dopamine concentration by
RP-HPLC. Dopamine levels in the culture supernatant (48 hours after
the last medium change) at day 25 of differentiation was
approximately 70 .mu.g/ml.
[0140] Differentiated neuronal cells derived from ELSCs (e.g.,
glutamatergic, GABAergic, serotonergic, and dopaminergic neurons,
as well as oligodendrocytes) may be utilized for various
applications, such as therapeutic application, as well as in vitro
and in vivo assessment and screening of various compounds such as
small molecule drugs for their effects on neuronal cells. The
neuronal cells may be used, for example, to treat or prevent
various neurological or neurodegenerative disorders or diseases
including but limited to Parkinson's disease, Alzheimer's disease,
Huntington's disease, Lewy body dementia, multiple sclerosis,
cerebellar ataxia, progressive supranuclear palsy, spinal cord
injury, amyotrophic lateral sclerosis (ALS), epilepsy, stroke,
ischemia, injury or trauma to the nervous system, neurotoxic
injury, and the like, in which neuronal cells, neurons, or glial
cells are injured or die in the central nervous system or spinal
cord. Additionally, the neuronal cells derived from pluripotent
ELSCs can also used to treat neurological disorders associated with
cognition and psychology including but not limited to anxiety
disorders, mood disorders, obsessive-compulsive disorders (OCD),
personality disorders, attention deficit disorder (ADD), attention
deficit hyperactivity disorder (ADHD), and schizophrenia.
[0141] 3) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Osteoblasts
[0142] To determine whether ELSCs of the present disclosure could
differentiate into osteoblasts, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into osteoblasts. For example,
the ELBs were cultured in DMEM supplemented with 10-15% fetal
bovine serum in the presence dexamethasone (10-100 nM),
glycerophosphate, (1-10 mM), ascorbic acid 2 phosphate (0.1-0.5
mM), bone morphogenic protein 2 (BMP2) (1-10 ng/ml), and
hydrocortisone (0.05-0.1 .mu.M). The cells were cultured for
approximately 28 days.
[0143] The cells isolated from the above differentiation protocol
were analyzed to confirm the presence of osteoblasts in the
culture. First, the differentiated cells were analyzed for calcium
deposits, which are indicative of osteoblasts, by Von Kossa
staining (Pittenger et al., (1999) Science 284:143-147,
incorporated herein by reference). FIG. 10(b) shows Von Kossa
staining of abundant calcium deposits (deep brown bodies) in
osteoblasts derived from ELSCs after 17 days of differentiation
culture.
[0144] 4) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Chondrocytes
[0145] To determine whether ELSCs of the present disclosure could
differentiate into chondrocytes, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into chondrocytes. For
example, the ELBs were cultured in DMEM supplemented with 10-15%
knockout serum and in the presence of TGF beta-3 (10-100 ng/ml),
ascorbic acid (0.01-0.05 mM), 1.times.ITS, and sodium pyruvate (1-5
mM). The cells were cultured for approximately 21 days.
[0146] The cells isolated from the above differentiation protocol
were analyzed to confirm the presence of chondrocytes in the
culture. The differentiated cells were analyzed for the presence of
glycogen deposits, which are indicative of chondrocytes, by
staining with Alcian Blue (Pittenger et al., (1999) Science
284:143-147). FIG. 10(c) shows Alcian Blue staining of sulfated
proteoglycan deposits in chondrocytes derived from ELSCs after 17
days of differentiation culture.
[0147] 5) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Adipocytes
[0148] To determine whether ELSCs of the present disclosure could
differentiate into adipocytes, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into adipocytes. For example,
the ELBs were cultured in DMEM supplemented with 10-15% knockout
serum and in the presence of dexamethasone (1 .mu.M-100 mM),
isobutylmethylxanthine (IBMX) (10-50 ng/ml), insulin (10-20 ng/ml),
indomethac (2-20 mM), and insulin-like growth factor (IGF) (10-100
ng/ml). The cells were cultured for approximately 14 days.
[0149] The cells isolated from the above differentiation protocol
were analyzed to confirm the presence of adipocytes in the culture.
The differentiated cells were analyzed for the presence of
cytoplasmic lipid droplets, which are indicative of adipocytes, by
staining with Oil Red-O (Pittenger et al., (1999) Science
284:143-147). FIG. 10(d) shows Oil Red-O staining of abundant
deposits of lipid droplets in adipocytes derived from ELSCs after
12 days of differentiation culture.
[0150] 6) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Hepatocytes
[0151] To determine whether ELSCs of the present disclosure could
differentiate into hepatocytes, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into hepatocytes. For example,
the ELBs were cultured in DMEM supplemented with 10-15% knockout
serum, EGF (10-100 .eta.g/ml), hepatocyte growth factor (HGF) (5-50
.eta.g/ml), bFGF (5-20 .eta.g/ml), FGF-4 (5-50 .eta.g/ml), IL-6
(10-100 .eta.g/ml), acidic FGF (50-100 .eta.g/ml), human oncostatin
(10-50 .eta.g/ml), insulin-transferrin-selenious acid (ITS)
(1.times.), dexamethasone (10-100 .eta.M), sodium butyrate (1-5
mM), DMSO (0.5-1%), and 5-azacytidine (1-10 .mu.M). The growth
factors were added together or at different time points to the
cultured cells as early growth factors, mid-stage growth factors,
or late stage growth factors. The cells were cultured for 20
days.
[0152] The morphology of the differentiated hepatocytes generated
above was examined by phase contrast microscopy (FIG. 10(h)) and by
hematoxylin-eosin staining. The hepatocytes were also analyzed for
gene expression using RT-PCR, immunological characterization by
immunofluorescence using anti-albumin antibody, and functional
characterization by evidence of stored glycogen in the cells as
detected by periodic-acid-Schiff's staining (PAS). These analyses
were carried out at the end of differentiated stage, preferably 20
days after hepatocyte differentiation as disclosed in the above
protocol. FIG. 10(d) shows insoluble glycogen deposits in mature
hepatocytes by PAS staining. Moreover, as a continuation of
extensive functional characterization of ELSC-derived hepatocytes,
the potential of these hepatocytes as a prospective drug screening
tool is being confirmed by evaluating the hepatocytes for
glucose-6-phosphatase activity, uptake of LDL, and albumin
production, as well as analyzing cytochrome p450 and Urea assays of
the differentiated cells.
[0153] To prepare the differentiated hepatocyte cells for
immunofluorescence analysis, 21-day old hepatocytes (oval-shaped)
were first fixed with paraformaldehyde (Sigma-Aldrich) for 20
minutes. Next, the hepatocytes were rinsed once with PBS at room
temperature (RT), and either stored at 4.degree. C. or directly
permeabilized with 0.2% Triton X-100 for 5 minutes at RT. After
aspirating the fixative, the hepatocytes were washed three times (5
minutes each) with PBS, and blocked with PBS containing 1% BSA for
1 hour at RT. After 2 more washes with 1.times.PBS, the hepatocytes
were incubated with a primary antibody solution diluted in
1.times.PBS-1% BSA overnight at RT. The primary antibody used was
to cytokeratin 18 (CK18), 1:200 (Chemicon, Inc, USA). CK18 is
expressed on hepatocyte plasma membrane surface (Wells et al.,
(1997) J. Biol. Chem. 272:28574-581). The next day, the hepatocytes
were washed with 1.times.PBS three times (10 minutes each) on a
rocker, and incubated with a secondary antibody dilution containing
a fluorescent label FITC in 1.times.PBS-1% BSA at RT for 1 hour on
a rocker. After three washes with PBS (5 minutes each), the
hepatocytes were exposed to 1 mg/ml DAPI solution. The hepatocytes
were washed twice with 1.times.PBS (5 minutes each), and mounted on
slides with DPX mountant. FIG. 10(e) shows that hepatocytes
differentiated from ELSCs stain positive for the anti-CK18
antibody.
[0154] Hepatocytes differentiated from ELSCs were also analyzed for
expression of AFP and Albumin by RT-PCR as described above. FIG. 11
shows expression of AFP and Albumin that is indicative of both
early and mature hepatocyte formation.
[0155] 7) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Pancreatic Beta-Islet Cells
[0156] To determine whether ELSCs of the present disclosure could
differentiate into pancreatic beta-islet cells, ELBs derived from
ELSCs were cultured for approximately 4-10 days in an appropriate
medium and plated directly onto a suitable substrate with an
extracellular matrix component such as polyornithine, laminin, or
fibronectin. The ELBs were cultured in a suitable nutrient medium
adapted to promote differentiation of the cells into pancreatic
beta-islet cells. For example, the ELBs were cultured in DMEM
supplemented with 10-15% knockout serum and in the presence of N2
supplement (1%), B27 supplement (2%), forskolin (10 .mu.M), and
cyclopamine (10 .mu.M). The cells were cultured for approximately
12 days.
[0157] The cells isolated from the above differentiation protocol
were analyzed to confirm the presence of pancreatic beta-islet
cells in the culture using immunofluorescence analysis, as
described above for differentiated hepatocytes. The differentiated
cells were analyzed using immunofluorescence for staining with an
anti-PDX-1 antibody, which is indicative of beta-islet cells.
Insulin-promoting factor-1 (PDX-1) is a transcription factor
expressed by beta-islet cells of the pancreas. FIG. 10(k) shows
positive immunofluorescence in beta-islet cells derived from ELSCs
after staining with anti-PDX-1 antibody. The beta-islet cells were
also analyzed for gene expression of insulin and somatostatin using
RT-PCR as previously described using the following primers:
TABLE-US-00008 TABLE 8 Annealing PCR Temp Product Gene Primer
sequence (.degree..degree.C.) size (bp) Insulin
5'-CCCTGCTGGCCCTGCTCTT-3' SEQ ID NO:34 58 212
5'-AGGTCTGAAGGTCACCTGCT-3' SEQ ID NO:35 Somatostatin
5'-GTTTCTGCAGAAGTCTCTGG-3' SEQ ID NO:36 56 222
5'-AGTTCTTGCAGCCAGCTTTG-3' SEQ ID NO:37
[0158] FIG. 11 shows expression of insulin and somatostatin by
differentiated cells, which are indicative of beta-islet cells.
[0159] 8) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Cardiomyocytes
[0160] To determine whether ELSCs of the present disclosure could
differentiate into cardiomyocytes, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into cardiomyocytes. For
example, the ELBs were cultured in DMEM/F-12 (1:1) supplemented
with 15% knockout serum and 100 mM L-glutamine in the presence of
EGF (50 .eta.M), TGF beta-3 (10 .eta.g/ml), bFGF (50 .eta.g/ml),
PDGF-BB (50 .eta.g/ml), and ITS (1.times.). A well known
cardiomyocyte inducing factor for human ES cells,
5-Azadeoxycitidine (5-10 .eta.M), was not found to be useful for
differentiating ELSCs into cardiomyocytes. The cardiotropic factors
were added together or at different time points to the
differentiation media as early growth factors, mid-stage growth
factors, or late stage growth factors. The cells were cultured for
approximately 21 days, and were carefully monitored for contracting
embryoid-like bodies (i.e., beating cardiac cells), for example
through a phase contrast microscope.
[0161] The cells isolated from the above differentiation protocol
were evaluated morphologically to confirm the presence of
ELSC-derived cardiomyoctyes, as shown in FIG. 10(f). In addition,
the ELSC-derived cardiomyocytes were analyzed for expression of
cardiac troponin T (cTnT), which is a marker characteristic of
cardiomyocytes, using the anti-cTnT antibody (Santacruz, USA). FIG.
10(g) shows that these cells are recognized by anti-cTnT antibody,
which is indicative of cardiomyocytes. Both ELSCs and
cardiomyocytes differentiated from ELSCs were analyzed for
expression of c-Actin and Na--Ca exchanger (NCX) by RT-PCR as
previously described above using the following primers:
TABLE-US-00009 TABLE 9 Annealing PCR Temp Product Gene Primer
sequence (.degree. C.) size (bp) c-Actin 5'-TCTATGAGGGCTACGCTTTG-3'
SEQ ID NO:38 50 630 5'-CCTGACTGGAAGGTAGATGG-3' SEQ ID NO:39 NCX
5'-ATGCTTCGATTAAGTCTCCCAC-3' SEQ ID NO:40 50 630
5'-TAAAGCCAGGTATAGGCAAAGA-3' SEQ ID NO:41
[0162] Expression of c-Actin and NCX is indicative of adult
cardiomyocyte formation. The PCR primers used to amplify the cDNAs
were specific for exon sequence of each gene, thereby allowing
amplification of cDNA only, and not genomic DNA encoding the genes.
FIG. 11 shows the results of RT-PCR analysis for the expression of
cardiomyocyte specific markers in ELSCs and differentiated
cardiomyocytes derived therefrom. As shown, both c-Actin and NCX
are expressed in the differentiated cardiomyoctyes, and not in the
undifferentiated ELSCs. Additionally, the potential of these
ELSC-derived cardiomyocytes in cell therapy for cardiac diseases is
being assessed by extensive functional characterization of the
cardiomyocytes by electrophysiology, as well as testing using in
vivo animal models.
[0163] 9) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Myocytes
[0164] To determine whether ELSCs of the present disclosure could
differentiate into myocytes, ELBs derived from ELSCs were cultured
for approximately 4-10 days in an appropriate medium and plated
directly onto a suitable substrate with an extracellular matrix
component such as polyornithine, laminin, or fibronectin. The ELBs
were cultured in a suitable nutrient medium adapted to promote
differentiation of the cells into myocytes. For example, the ELBs
were cultured in DMEM supplemented with 10-15% knockout serum and
in the presence of 5-Azacytidine (5-10 .mu.M) and PDGF-BB (10-50
ng/ml). The cells were cultured for approximately 12 days.
[0165] The cells isolated from the above differentiation protocol
were analyzed for expression of Myogenin, which is a member of the
gene family encoding muscle-specific basic-helix-loop-helix
transcription factors that is activated in myoblasts at the onset
of differentiation. Antibodies to Myogenin (Santacruz, USA) were
used to confirm the presence of myocytes in the culture. FIG. 10(e)
shows that these cells are recognized by anti-Myogenin antibody.
Both ELSCs and cells differentiated from ELSCs were also analyzed
for expression of Myogenin by RT-PCR as previously described above.
FIG. 11 shows expression of Myogenin in differentiated cells, which
is indicative of myocytes.
[0166] 10) Differentiation of Pluripotent Embryonic-Like Stem Cells
into Endothelial Cells
[0167] To determine whether ELSCs of the present disclosure could
differentiate into endothelial cells, ELBs derived from ELSCs were
cultured for approximately 4-10 days in an appropriate medium and
plated directly onto a suitable substrate with an extracellular
matrix component such as polyornithine, laminin, or fibronectin.
The ELBs were cultured in a suitable nutrient medium adapted to
promote differentiation of the cells into endothelial cells. For
example, the ELBs were cultured in DMEM supplemented with 10-15%
knockout serum and in the presence of VEGF (20 ng/ml), bFGF (50
ng/ml), and BMP-4 (1-10 ng/ml). The cells were cultured for
approximately 21 days.
[0168] Both ELSCs and endothelial cells differentiated from ELSCs
were analyzed for expression of PECAM by RT-PCR as previously
described above. FIG. 11 shows expression of PECAM in both ELSCs
and differentiated cells, which is indicative of endothelial
cells.
[0169] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents that are chemically or physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
12 1 26 DNA Homo sapiens 1 tgaaggtcgg agtcaacgga tttggt 26 2 24 DNA
Homo sapiens 2 catgtgggcc atgaggtcca ccac 24 3 25 DNA Homo sapiens
3 cgrgaagctg gagaaggaga agctg 25 4 25 DNA Homo sapiens 4 caagggccgc
agcttacaca tgttc 25 5 25 DNA Homo sapiens 5 cctcctccat ggatctgctt
attca 25 6 26 DNA Homo sapiens 6 caggtcttca cctgtttgta gctgag 26 7
23 DNA Homo sapiens 7 gcgtacgcaa attaaagtcc aga 23 8 25 DNA Homo
sapiens 8 cagcatccta aacagctcgc agaat 25 9 21 DNA Homo sapiens 9
ggaagaacta ccagaaacgc g 21 10 21 DNA Homo sapiens 10 agatgatcag
ccagaggaaa a 21 11 26 DNA Homo sapiens 11 aagaggacaa gaaggactaa
aaatat 26 12 25 DNA Homo sapiens 12 gtagagatcc agcataaaga gaggt
25
* * * * *