U.S. patent application number 10/917853 was filed with the patent office on 2005-02-24 for banking of multipotent amniotic fetal stem cells.
Invention is credited to Haas, Martin.
Application Number | 20050042595 10/917853 |
Document ID | / |
Family ID | 34198044 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050042595 |
Kind Code |
A1 |
Haas, Martin |
February 24, 2005 |
Banking of multipotent amniotic fetal stem cells
Abstract
Stem cells, including those designated as multipotent amniotic
fluid stem cells (MAFSC) cells are found in the amniotic fluid of
mammals, including humans. MAFSCs are fetal, multipotent stem cells
that can be used for any desired stem cell utility, including
treatment of individuals in need of tissue replacement or gene
therapy. Methods of banking MAFSCs derived from the amniotic fluid
cells of pregnant individuals are disclosed. Amniotic fluid-derived
cells are banked for the purpose of access to transplantation
antigen-compatible or syngeneic multipotent stem cells.
Inventors: |
Haas, Martin; (San Diego,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
34198044 |
Appl. No.: |
10/917853 |
Filed: |
August 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60495513 |
Aug 14, 2003 |
|
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60495437 |
Aug 14, 2003 |
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Current U.S.
Class: |
435/2 ; 435/366;
702/19 |
Current CPC
Class: |
C12N 2501/115 20130101;
C12N 2501/39 20130101; C12N 2501/11 20130101; C12N 5/0607 20130101;
C12N 2500/25 20130101; C12N 2501/135 20130101; C12N 5/0605
20130101 |
Class at
Publication: |
435/002 ;
702/019; 435/366 |
International
Class: |
A01N 001/02; G01N
033/48; G06F 019/00; G01N 033/50; C12N 005/08 |
Claims
What is claimed is:
1. A cell bank system, comprising: a plurality of preserved, viable
samples, containing amniotic fluid-derived cells, wherein said
samples are from a plurality of individuals; and a database
containing one or more data fields that allow for specific
identification and retrieval of individual samples.
2. The cell bank system of claim 1, wherein said database includes
data fields allowing association of an individual sample with a
person from whom the sample was obtained.
3. The cell bank system of claim 1, wherein the samples are
cryopreserved.
4. The cell bank system of claim 1, wherein individual samples are
retrievable by the person from whom the sample was obtained.
5. The cell bank system of claim 1, wherein said samples contain
MAFCS.
6. The cell bank system of claim 1, wherein said samples contain
amniotic fluid.
7. The cell bank system of claim 1, wherein said samples contain
multipotent or pluripotent stem cells.
8. A method for banking stem cells, comprising: obtaining a
plurality of viable samples containing amniotic-fluid-derived stem
cells, wherein said samples are from the amniotic fluid of a
plurality of individual fetuses; preserving and storing the samples
in a manner that preserves viability of at least some of said stem
cells; storing data relating to the identity of individual samples;
and providing for retrieval of said individual samples by or on
behalf of the individual from whom the sample was obtained.
9. The method of claim 8, wherein the samples are
cryopreserved.
10. The method of claim 8, wherein said samples contain MAFCS.
11. The method of claim 8, wherein said samples contain amniotic
fluid.
12. The method of claim 11, wherein said amniotic fluid comprises
about 15% to 35% (V/V) of said medium.
13. The method of claim 8, wherein said samples contain multipotent
or pluripotent stem cells.
14. A collection comprising a plurality of viable stem cells
derived from amniotic fluid of different fetuses.
15. The collection of claim 14, wherein said stem cells are
cryopreserved.
16. The collection of claim 14, wherein said cells are fresh cells
isolated from amniotic fluid.
17. The collection of claim 14, wherein said cells are cultured
MAFSC cells.
18. A method of preparing amniotic fluid derived stem cells (MAFSC)
for a potential future use, comprising: obtaining amniotic fluid
containing live cells; culturing MAFSC cells isolated from said
amniotic fluid; and cryopreserving said MAFSC cells.
19. A storage bank of multiple samples of amniotic fluid-derived
cells or MAFSCs taken from multiple individuals, wherein the
samples are preserved in such a way as to be viable upon
recovery.
20. The storage bank of claim 19, wherein the cells are
cryopreserved in a medium comprising DMSO.
21. The storage bank of claim 19, wherein at least some of the
samples are differentiable human stem cells.
22. The storage bank of claim 21, wherein said human stem cells are
multipotent.
23. The storage bank of claim 21, wherein said human stem cells are
pluripotent.
24. The cell bank system of claim 1, wherein said amniotic
fluid-derived cells comprise pluripotent stem cells.
25. The cell bank system of claim 1, wherein said amniotic
fluid-derived cells comprise multipotent stem cells characterized
by a) the ability to grow in continuous culture for at least 60
generations, and b) the presence of at least one marker selected
from the group consisting of: SSEA3, SSEA4, Tra1-60, Tra1-81,
Tra2-54, and Oct-4.
26. The cell bank system of claim 1, wherein said amniotic
fluid-derived cells comprise multipotent stem cells characterized
by a) the ability to grow in continuous culture for at least 60
generations, and b) the presence of all of the markers SSEA3,
SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4.
27. The cell bank system of claim 25 wherein said stem cells are
further characterized by expressing at least one marker selected
from the group consisting of: HLA Class I, CD13, CD44, CD49b, and
CD105.
28. The cell bank system of claim 1, wherein said amniotic
fluid-derived cells comprise pluripotent stem cells characterized
by a) the ability to grow in continuous culture for at least 60
generations, and b) the presence of at least one of the markers
selected from the group consisting of: SSEA3, SSEA4, Tra1-60,
Tra1-81, Tra2-54, and Oct-4.
29. The cell bank system of claim 1, wherein said amniotic
fluid-derived cells comprise pluripotent stem cells characterized
by a) the ability to grow in continuous culture for at least 60
generations, and b) the presence of the following markers: SSEA3,
SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4.
30. The cell bank system of claim 29 wherein said stem cells are
further characterized by expressing at least one marker selected
from the group consisting of: HLA Class I, CD13, CD44, CD49b, and
CD105.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Application No. 60/495,513, filed Aug. 14,
2003, and U.S. Provisional Application No. 60/495,437, filed Aug.
14, 2003, the disclosures of which are incorporated by reference
herein in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the field of stem cell research.
Specifically, the invention relates to the preservation and banking
of amniotic fluid-derived cells, or multipotent amniotic
fluid-derived stem cells of individuals. The cryopreserved amniotic
fluid-derived cells can be stored indefinitely. Thawed cells can be
used to grow stem cell lines which can create differentiated cells,
such as specific cell types or tissue types. These differentiated
cells are capable of being transplanted into the individual or into
unrelated matching individuals if needed.
[0004] 2. Description of the Related Art
[0005] Stem cells can give rise to many types of differentiated
cells, and thus may be useful to treat many types of diseases. Stem
cells have the ability to divide for indefinite periods in culture
and to give rise to specialized cells. There are several types of
stem cells, such as embryonic stem cells, which are
undifferentiated cells from the embryo, and adult stem cells, which
are undifferentiated cells derived from various mature tissues.
[0006] Embryonic stem cells have the potential to become a wide
variety of specialized cell types. This ability of an embryonic
stem cell to become a variety of cell types is termed
"pluripotent." Embryonic stem cells can be differentiated into a
host of cell types and tissue types which can be used for basic
research, drug discovery, treatment and prevention of diseases. For
example, U.S. Pat. No. 6,506,574 to Rambhatla, which is
incorporated by reference herein in its entirety, discloses methods
of differentiating embryonic stem cell cultures into hepatocyte
lineage cells. Other methods for the preparation of embryonic stem
cells are disclosed, for example, in U.S. Pat. No. 6,200,806 to
Thomson; U.S. Pat. No. 5,670,372 to Hogan, and U.S. Pat. No.
6,432,711 to Dinsmore, each of which is incorporated by reference
herein in its entirety.
[0007] Human Embryonic Stem cells (hES) are derived from the inner
cell mass of the blastocyst, the earliest stage of embryonic
development of the fertilized egg. The blastocyst is a
preimplantation stage of the embryo, a stage before the embryo
would implant in the uterine wall. When cultured on an inactivated
feeder layer of cells according to conditions described by Thompson
and colleagues (Thomson, et al., (1995) Proc. Natl. Acad. Sci.
U.S.A. 92:7844-7848; Thomson, et al. (1998) Science 282:1145-1147;
Marshall, et al., (2001) Methods Mol. Biol. 158:11-18, each of
which is incorporated by reference herein in its entirety the inner
layer cells of the blastocyst can be grown in vitro indefinitely in
an undifferentiated state. Properly propagated hES cells have
unlimited potential to double while maintaining their capacity of
differentiating into the three layers of the embryo, Ectoderm (Ec),
Mesoderm (me) and Endoderm (En); they are pluripotent. When grown
as pluripotent hES, the cells maintain a euploid karyotype and are
not prone to senescence. hES cells have been differentiated in
vitro into skin and brain (Ec), heart, muscle, kidney and blood
(Me), and into pancreatic, thyroid and lung cells (En) (Fraichard,
et al., (1995) J Cell Sci. 108:3181-3188; Itskovitz-Eldor, et al.,
(2000). Mol. Med. 6:88-95; Lee, et al., (2000) Nat. Biotechnol.
18:675-679; Liu, et al., (2000) Proc. Natl. Acad. Sci. U.S.A.
97:6126-6131; Lumelsky, et al., (2001) Science 292:1389-1394;
Maltsev, et al., (1993). Mech. Dev. 44:41-50; Odorico, et al.,
(2001) Stem Cells 19:193-204. Potocnik, et al., EMBO. J.
13:5274-5283; Reubinoff, et al., (2000) Nat. Biotechnol.
18:399-404; Schuldiner, et al., (2001) Proc. Natl. Acad. Sci. USA
97:1997:11307-11312; Kim, et al., (2002) Nature 418:50-56;
Wichterle, et al., (2002) Cell 110:385-397), each of which is
incorporated by reference herein in its entirety.
[0008] Human embyronic stem cells display a distinct group of cell
surface antigens, SSEA-3, SSEA-4, TRA-2-54 (alkaline phosphatase),
TRA-1-60 and TRA-1-81, in addition to expressing specific
transcription factors OCT-4, NANOG, SOX-2, FGF-4 and REX-1
(Henderson, et al., (2002) Stem Cells 20:329-337; Draper, et al.,
(2002). J. Anat. 200:249-258; Mitsui et al., (2003) Cell
113:631-642; Chambers et al., (2003) Cell 113:643-655), each of
which is incorporated by reference herein in its entirety.
[0009] Additionally, hES cells (i) are capable of symmetrical
division in vitro without differentiating; (ii) can integrate into
all fetal tissues during in vivo development; (iii) are capable of
colonizing the germ line and give rise to egg or sperm cells; (iv)
develop into teratocarcinomas in immunologically impaired adult
mice--another measure of pluripotency, and lack the G1 checkpoint
in the cell cycle like somatic cells but spend most of their time
in S phase.
[0010] Stem cells can also be derived from nonembryonic sources.
For example, an additional class of human stem cells are the
mesenchymal or adult stem cells (MSC). Adult stem cells are
undifferentiated, like embryonic stem cells, but are present in
differentiated tissues. Adult stem cells are capable of
differentiation into the cell types from the tissue that the adult
stem cell originated. Adult stem cells (MSC) have been derived from
the nervous system (McKay, R. (1997) Science 276:66-71.
Shihabuddin, et al., (1999) Mol. Med. Today 5:474-480), bone marrow
(Pittenger, et al., (1999) Science 284:143-147; Pittenger, M. F.
and Marshak, D. R. (2001). In: Mesenchymal stem cells of human
adult bone marrow. Marshak, D. R., Gardner, D. K., and Gottlieb, D.
eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press) 349-374); adipose tissue (Gronthos, et al., (2001) J. Cell.
Physiol. 189:54-63), dermis (Toma, et al., (2001) Nature cell Biol.
3:778-784) and pancreas and liver (Deutsch, et al., (2001)
Development 128:871-881), each of which is incorporated by
reference herein in its entirety.
[0011] The political, moral and ethical issues around hES cells, as
well as the perceived difficulties of expanding undifferentiated
adult stem cells in culture, while maintaining a genetically normal
genome, are major barriers in the development of human cell
replacement therapy. Further, what is needed in the art is a method
of isolating, storing, banking, and retrieving novel sources of
multipotent or pluripotent human stem cells so that they can be
revived and utilized at a later date.
SUMMARY OF THE INVENTION
[0012] Embodiments of the invention include a cell bank system
having preserved, viable samples from many individuals, where the
samples contain amniotic fluid-derived cells, and a database
allowing for specific identification and retrieval of individual
samples. The database may include information allowing association
of an individual sample with the sample donor, and the sample may
be retrieved by the donor. The samples may contain amniotic fluid,
and may contain multipotent or pluripotent stem cells. The amniotic
fluid-derived cells comprise pluripotent stem cells, or can be
multipotent stem cells. For example, the amniotic fluid-derived
cells can be multipotent stem cells characterized by a) the ability
to grow in continuous culture for at least 60 generations, and b)
the presence of at least one, or two, or three, or four, or five,
or all of the markers selected from the group consisting of: SSEA3,
SSEA4, Tra1-60, Tra1-81, Tra2-54, and Oct-4. The stem cells can
further express at least one marker selected from the group
consisting of: HLA Class I, CD13, CD44, CD49b, and CD105. The
amniotic fluid-derived cells can be pluripotent stem cells
characterized by a) the ability to grow in continuous culture for
at least 60 generations, and b) the presence of at least one, or
two, or three, or four, or five, or all of the markers selected
from the group consisting of: SSEA3, SSEA4, Tra1-60, Tra1-81,
Tra2-54, and Oct-4. The stem cells can further express at least one
marker selected from the group consisting of: HLA Class I, CD13,
CD44, CD49b, and CD105.
[0013] Additional embodiments of the invention include methods for
banking stem cells by obtaining viable samples containing
amniotic-fluid-derived stem cells, which were derived from the
amniotic fluid of individual fetuses, then preserving and storing
the samples in a way that allows viability of at least some of the
stem cells, storing data relating to the identity of the individual
samples; and allowing the retrieval of individual samples by the
individual from whom the sample was obtained. The samples may be
cryopreserved, and may contain MAFCS, amniotic fluid, multopotent
and/or pluripotent stem cells.
[0014] Further embodiments of the invention include methods for
banking stem cells by obtaining viable samples containing
amniotic-fluid-derived stem cells derived from the amniotic fluid
of individual fetuses, preserving and storing the samples in a way
that preserves viability of at least some of the stem cells,
storing data relating to tissue compatibility characteristics of
individual samples; and providing for retrieval of an individual
sample by an individual sharing tissue compatibility
characteristics of the sample. Such samples may be cryopreserved,
and may contain MAFCS, amniotic fluid, multopotent and/or
pluripotent stem cells.
[0015] Yet further embodiments of the invention include collections
having numerous viable stem cell samples derived from amniotic
fluid of different fetuses. Such samples may be cryopreserved, and
may contain MAFCS, amniotic fluid, multopotent and/or pluripotent
stem cells.
[0016] Additional embodiments of the invention include methods of
preparing amniotic fluid-derived cells for a potential future use,
by obtaining and cryopreserving amniotic fluid containing live
amniotic stem cells. The cryopreserving step may be performed in a
medium which contains DMSO, preferably at 1% to 80%, and more
preferably 10% to 40% (V/V) DMSO. Amniotic fluid may also be
present, preferably at about 15% to 35% (V/V).
[0017] Further embodiments of the invention include methods for
preparing amniotic fluid derived stem cells or multipotent amniotic
fluid derived stem cells (MAFSC) for a potential future use by
obtaining amniotic fluid containing live cells, then culturing
MAFSC cells isolated from said amniotic fluid, then cryopreserving
the MAFSC cells. The cryopreserving step may be performed in a
medium which contains DMSO, preferably at 1% to 80%, and more
preferably 10% to 40% (V/V) DMSO. Amniotic fluid may also be
present, preferably at about 15% to 35% (V/V).
[0018] Additional embodiments include storage banks of multiple
samples of amniotic fluid-derived cells or MAFSCs taken from
multiple individuals, where the samples are preserved in such a way
as to be viable upon recovery. The cells may be cryopreserved in a
medium having DMSO. At least some of the samples can be
differentiable human stem cells, which may be multipotent or
pluripotent.
[0019] Additional embodiments include data storage media containing
a database having a first data item associated with each of many
preserved amniotic-fluid-derived stem cell samples, containing
location information for the samples; and a second data item
associated with each sample containing identifying information for
the sample. The second data item can include, for example, the
identity of a fetus from whom the sample was obtained, tissue
compatibility information, and/or histocompatibility information
for the stem cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0020] A newly discovered source of human stem cells is described
herein and in co-pending application Ser. No. 60/495,437, which is
incorporated by reference herein in its entirety.
[0021] The cells, coined Multipotent Amniotic Fetal Stem Cells
(MAFSC), are immortal in culture, maintain euploidy for >1 year
in culture, share markers with human ES cells, and are capable of
differentiating into all three germ layers of the developing
embryo, Endoderm, Mesoderm and Ectoderm. These human stem cells are
found in the amnion harvested during the first trimester of human
pregnancies.
[0022] Both fresh amniotic fluid derived cells, and the cultured
MAFSC cells derived from them, may be stored indefinitely. When a
need arises, an aliquot of the cells can be thawed, cultured, and
used as needed. The long term storage of amniotic fluid derived
cells allows an individual to have a supply of stem cells taken
while the individual is still in the womb, to be stored in such a
way as to provide a supply of cells for emergency or other uses
throughout the individual's lifetime. The long term storage or
"banking" of amniotic fluid-derived cells or cultured MAFSCs is
disclosed herein.
[0023] While amniotic fluid contains multiple
morphologically-distinguisha- ble cell types, the majority of the
cells are prone to senescence and are lost from cultures grown
under MAFSC culture conditions. More than 80% of amniotic fluid
harvests from normal 16-18 week pregnancies give rise to continuous
MAFSC lines. The MAFSCs may be harvested from anmniotic fluid from
pregnant females at any stage in the gestation period.
[0024] MAFSC are of fetal origin, and have a normal diploid
karyotype. Additionally, MAFSC cells are devoid of tumorgenic
properties: unlike hEC cells, human MAFSC cells do not grow into
teratocarcinomas when injected into SCID mice. This property may be
especially useful in using MAFSC cells or their derivatives for
human gene therapy purposes.
[0025] The term "stem cell" refers to any cells that have the
ability to divide for indefinite periods of time and to give rise
to specialized cells. The term "long term stem cells" refers more
specifically to those stem cells that are capable of self-renewal
over indefinite periods of time.
[0026] The MAFSC cells of the invention have been shown to be
multipotent, as several main cell types have been derived from
them. As used herein, the term "multipotent" refers to the ability
of MAFSC to differentiate into several main cell types. The MAFSC
cells may also be propagated under specific conditions to become
"pluripotent." The term "pluripotent stem cells" describes stem
cells that are capable of differentiating into any type of body
cell, when cultured under conditions that give rise to the
particular cell type.
[0027] The MAFSCs may be isolated as described, for example, in
Example 1. Briefly, the sample of amniotic fluid (AF) can be was
removed from a pregnant female at any time during thee gestation
period. Cells to be cultured are then removed from the amniotic
fluid, preferably by centrifugation or filtration. The cells can
then be plated on medium as disclosed in Example 1, or other
suitable growth medium.
[0028] Typically, the cells are grown in a nutrient medium such as
the medium shown in Example 1. Expansion of the undifferentiated
amniotic fluid-derived cells can be achieved by culturing the cells
in defined media containing low amounts of serum or no serum at
all, using, for example, recombinant growth promoting factors. The
term "undifferentiated" refers to cells that have not become
specialized cell types. A "nutrient medium" is a medium for
culturing cells containing nutrients that promote proliferation.
The nutrient medium may contain any of the following in an
appropriate combination: isotonic saline, buffer, amino acids,
antibiotics, serum or serum replacement, and exogenously added
factors.
[0029] The MAFSCs are preferably isolated from humans. However, the
MAFSCs may be isolated in a similar manner from other species.
Examples of species that may be used to derive the MAFSCs include
but are not limited to mammals, humans, primates, dogs, cats,
goats, elephants, endangered species, cattle, horses, pigs, mice,
rabbits, and the like.
[0030] The amniotic fluid-derived cells and MAFSC can be recognized
by their specific cell surface proteins or by the presence of
specific cellular proteins. Typically, specific cell types have
specific cell surface proteins. These surface proteins can be used
as "markers" to determine or confirm specific cell types.
Typically, these surface markers can be visualized using
antibody-based technology or other detection methods. The surface
markers of the isolated MAFSC cells derived from
independently-harvested amniotic fluid samples were tested for a
range of cell surface and other markers, using monoclonal
antibodies and FACS analysis (see Examples 2 and 3, and Table 1).
These cells are characterized by the following cell surface
markers: SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54 but are
distinguished from mouse ES cells in that these cells do not
express the cell surface marker SSEA1. Additionally, MAFSC express
the stem cell transcription factor Oct-4.
Storage Bank of Amniotic Fluid-Derived Cells
[0031] The anmiotic fluid-derived cells can be stored or "banked"
in a manner that allows the cells to be revived as needed in the
future. An aliquot of the undifferentiated cells can be removed at
any time, to be grown into cultures of many undifferentiated cells
and then differentiated into a particular cell type or tissue type,
and may then be used to treat a disease or to replace
malfunctioning tissues in a patient. Since the cells are harvested
from the amniotic fluid, the cells can be stored so that an
individual can have access to his or her own undifferentiated cells
for an entire lifetime. Alternatively, the cells can be used by
individuals other than the original donor.
[0032] In addition, although the principal exemplary disclosure of
the present application relates to amniotic stem cells, the cell
banking and retrieval disclosure herein is similarly applicable to
any stem cell types, including those derived from embryos,
placenta, umbilicus, infants, children, and adults.
[0033] In one embodiment of the present invention, a stem cell bank
is provided for storing amniotic fluid-derived cell samples. In
additional embodiments of the present invention, methods for
administering such a stem cell bank are provided. U.S. Published
Patent Application No. 20030215942, which is incorporated by
reference herein in its entirety, provides an example of a stem
cell bank system.
[0034] Using methods such as those described above and in Examples
5 and 6, below, the isolation and in vitro propagation of stem cell
samples from amniocentesis harvests and their cryopreservation
facilitates the establishment of a "bank" of transplantable human
stem cells. The method described herein allows viable stem cells of
any individual to be obtained from the amniotic fluid (for example,
from an amniocentesis procedure) and be available for use at any
time in the future. Any number of individuals may have cells stored
in this manner. Because it is possible to store smaller aliquots of
AF or MASFCs, the banking procedure could take up a relatively
small space. Therefore, the cells of many individuals could be
stored or "banked" on a short term or long term basis, with
relatively little expense.
[0035] In some embodiments of the present invention, a portion of
the sample is made available for testing, either before or after
processing and storage.
Cryopreservation Methods
[0036] The fresh amniotic fluid-derived cells, or the cultured
MAFSC cells may be preserved so that the cells can be revived on
demand. A preferred method is cryopreservation. One example of a
suitable cryopreservation method is shown in Example 4. Typically,
the surrounding fluid for preservation contains amniotic fluid. The
use of amniotic fluid as part of the cryopreservation medium,
rather than the use of other types of media or serum, allows for
the cells to remain in the preferred undifferentiated form.
Exposure of primary amniotic fluid (AF) cells or MAFSC cells to
serum may cause the cells to become more susceptible to controlled
differentiation, which may make them less suitable for future
multipotent or pluripotent uses, once they are removed from
storage. Preferably, the cryopreservation medium contains between
about 10% and 50% amniotic fluid. More preferably, the
cryopreservation medium contains between about 20% and 30% amniotic
fluid. Most preferably, the cryopreservation medium contains about
24% to about 27% amniotic fluid. Preferably, the amniotic fluid is
filtered. Most preferably, the filtration occurs through a 0.1
.mu.m filter.
[0037] Another component of the cryopreservation medium is DMSO
(dimethylsulfoxide). Preferably, DMSO is present at approximately
1% to 80% (V/V). More preferably, DMSO is present at approximately
5% to 30% (V/V). Most preferably, DMSO is present at approximately
8% to 12% DMSO.
[0038] The AF cells or MAFSCs can be stored indefinitely under
liquid nitrogen. The cells can be kept for >100 years without
the incidence of any damage: they can then be thawed, grown and
differentiated as required. The cells are preferably frozen in the
above described medium at a controlled rate. Preferably, this rate
is from about 0.1.degree. C./min to about 10.degree. C./min. More
preferably, the freezing rate is from about 0.2, 0.3, 0.4.degree.
C./min to about 4, 6, 8, or 9.degree. C./min. Most preferably, the
freezing rate is from about 0.5.degree. C./min to about 2.degree.
C./min.
[0039] Frozen cells are then stored under liquid nitrogen until
needed. The cells may be stored indefinitely, once frozen. Care
should be taken to prevent the possibility of accidental thawing or
warming of the frozen cells at any time during their storage
period. In some embodiments of the invention, the cells may be
preserved by methods other than cryopreservation.
[0040] Typing of Amniotic Fluid-Derived Cell Samples
[0041] In some embodiments of this invention, the amniotic
fluid-derived cells can be further classified according to certain
identifying features, such as by HLA typing, either before or after
processing and storage. The term "type or "typing" refers to any
characteristics of an amniotic fluid-derived cell sample that may
be relevant for any possible use of the sample. Determination of
which tests are relevant and how to perform them is entirely
conventional and will change with technological developments.
Typing also includes any method that identifies a stem cell product
in such a way that the stem cell sample may be matched to a certain
individual. For the purposes of this invention, matching indicates
that the stem cell sample is suitable for transplantation into a
specific individual.
[0042] The type information may include, for example, genotype or
phenotype information. Genotype information may refer to a specific
genetic composition of a specific individual organism, for example,
whether an individual organism has one or more specific genetic
variants up to all the variations in that individual's genome, for
example, whether the individual is a carrier of genetic variations
that influence disease or the HLA type of that individual.
Phenotype information may include any observable or measurable
parameter.
[0043] In some embodiments of the invention, the amniotic
fluid-derived cells may be typed using HLA typing methods. For
example, stem cells can be typed using the high-throughput HLA
typing-methods described in U.S. Pat. No. 6,670,124, which is
incorporated by reference herein in its entirety. A high throughput
HLA typing method may include obtaining a biological sample
containing template nucleic acid from a subject, amplifying the
template nucleic acid with labeled HLA allele-specific primers,
hybridizing the amplification products with immobilized HLA
locus-specific capture oligonucleotides and using detection methods
to determine the HLA genotype of the subject.
[0044] Other typing methods may be used. One typing method for HLA
identification purposes is restriction fragment length polymorphism
analysis. Restriction fragment length polymorphism analysis relies
upon the strong linkage between allele-specific nucleotide
sequences within the exons that encode functionally significant HLA
class II epitopes. Another method, PCR-SSO, relies upon the
hybridization of PCR amplified products with sequence-specific
oligonucleotide probes to distinguish between HLA alleles (Tiercy
et al., 1990, Blood Review 4: 9-15, Saiki et al., 1989, Proc. Natl.
Acad. Sci., U.S.A. 86: 6230-6234; Erlich et al. (1991) Eur. J.
Immunogenet. 18(1-2): 3355; Kawasaki et al. (1993) Methods Enzymol.
218:369-381), each of which is incorporated by reference herein in
its entirety.
[0045] Another molecular typing method that can be used in the
present invention, PCR-SSP, uses sequence specific primer
amplification (Olerup and Zetterquist (1992) Tissue Antigens 39:
225-235, which is incorporated by reference herein in its
entirety). The SSCP-Single-Stranded Conformational Polymorphism
method may also be used. These and other standard techniques for
HLA typing are known in the art, e.g., DNA typing or serological
and cellular typing (Terasaki et al., 1964, Nature, 204:998, which
is incorporated by reference herein in its entirety).
[0046] In some embodiments of the invention, in order to allow for
the availability of cells which can be used for any individual,
even if those who do not have stored amniotic fluid-derived cell
samples, many amniotic fluid-derived cell samples can be banked
that possess a range of genetic characteristics and that display a
range of antigens to allow for sufficient matching of HLA
specificities for the use by any potential recipient.
[0047] Organizing the Amniotic Fluid-Derived Cell Samples
[0048] This invention also provides methods of recording the
amniotic fluid-derived cell samples so that when a stem cell sample
needs to be located, it can be easily retrieved. Any indexing and
retrieval system can be used to fulfill this purpose. Any suitable
type of storage system can be used so that the stem cells can be
stored. The amniotic fluid-derived cell samples can be designed to
store individual samples, or can be designed to store hundreds,
thousands and even millions of different amniotic fluid-derived
cell samples.
[0049] The stored amniotic fluid-derived cell samples can be
indexed for reliable and accurate retreival. For example, each
sample can be marked with alphanumeric codes, bar codes, or any
other method or combinations thereof. There may also be an
accessible and readable listing of information enabling
identification of each stem cell sample and its location in the
bank and enabling identification of the source and/or type of stem
cell sample, which is outside of the bank. This indexing system can
be managed in any way known in the art, e.g., manually or
non-manually, e.g. a computer and conventional software can be
used.
[0050] In some embodiments of the invention, the amniotic
fluid-derived cell samples are organized using an indexing system
so that the sample will be available for the donor's use whenever
needed. In other embodiments of the invention, the amniotic
fluid-derived cell samples can be utilized by individuals other
than the original donor. Once recorded into the indexing system,
the amniotic fluid-derived cell sample can be made available for
matching purposes, e.g., a matching program will identify an
individual with matching type information and the individual will
have the option of being provided the matching stem cell
sample.
[0051] The storage banking system can comprise a system for storing
a plurality of records associated with a plurality of individuals
and a plurality of amniotic fluid-derived cell samples. Each record
may contain type information, genotypic information or phenotypic
information associated with the stem cell samples or specific
individuals. In a specific embodiment, the system will include a
cross-match table that matches types of the stem cell samples with
types of individuals who with to receive a stem cell sample.
[0052] In a particular embodiment, the database system stores
information for each stem cell sample in the bank. Certain
information is stored in association with each sample. The
information may be associated with a particular donor, for example,
an identification of the donor and the donor's medical history.
Alternatively, a stem cell sample may be anonymous and not
associated with a specific donor. Alternatively, or additionally,
the information may be sample type information. For example, the
information might include the volume of the stem cell sample or the
total nucleated cells count in the product. The stored information
may also include match and typing information. For example, each
stem cell sample may be HLA typed and the HLA type information may
be stored in association with each sample. The information stored
may also be availability information. The information stored with
each sample is searchable and identifies the sample in such a way
that it can be located and supplied to the client immediately.
[0053] Accordingly, Embodiments of the invention utilize
computer-based systems that contain information such as the donor,
date of submission, type of cells submitted, types of cell surface
markers present, genetic information relating to the donor, or
other pertinent information, and storage details such as
maintenance records and the location of the stored samples, and
other useful information.
[0054] The term "a computer-based system" refers to the hardware,
software, and any database used to store, search, and retrieve
information about the stored cells. The computer-based system
preferably includes the storage media described above, and a
processor for accessing and manipulating the data. The hardware of
the computer-based systems of this embodiment comprise a central
processing unit (CPU) and a database. A skilled artisan can readily
appreciate that any one of the currently available computer-based
systems are suitable.
[0055] In one particular embodiment, the computer system includes a
processor connected to a bus that is connected to a main memory
(preferably implemented as RAM) and a variety of secondary storage
devices, such as a hard drive and removable medium storage device.
The removable medium storage device can represent, for example, a
floppy disk drive, a DVD drive, an optical disk drive, a compact
disk drive, a magnetic tape drive, etc. A removable storage medium,
such as a floppy disk, a compact disk, a magnetic tape, etc.
containing control logic and/or data recorded therein can be
inserted into the removable storage device. The computer system
includes appropriate software for reading the control logic and/or
the data from the removable medium storage device once inserted in
the removable medium storage device. Information relating to the
amniotic fluid-derived cells can be stored in a well known manner
in the main memory, any of the secondary storage devices, and/or a
removable storage medium. Software for accessing and processing
these sequences (such as search tools, compare tools, etc.) reside
in main memory during execution.
[0056] As used herein, "a database" refers to memory that can store
any useful information relating to the amniotic fluid-derived cell
collections and the donors. Additionally, a "database" refers to a
memory access component that can access manufactures having
recorded thereon information relating to the amniotic fluid-derived
cell collections.
[0057] The data relating to the stored amniotic fluid-derived cells
can be stored and manipulated in a variety of data processor
programs in a variety of formats. For example, the data can be
stored as text in a word processing file, such as Microsoft WORD or
WORDPERFECT, an ASCII file, a html file, or a pdf file in a variety
of database programs familiar to those of skill in the art, such as
DB2, SYBASE, or ORACLE.
[0058] A "search program" refers to one or more programs that are
implemented on the computer-based system to search for details or
compare information relating to the cryopreserved samples within a
database. A "retrieval program" refers to one or more programs that
can be implemented on the computer-based system to identify
parameters of interest in the database. For example, a retrieval
program can be used to find samples that fit a particular profile,
samples having specific markers or DNA sequences, or to find the
location of samples corresponding to particular individuals.
[0059] Storage Facility
[0060] The amniotic fluid-derived cell samples of the invention may
be transported to and from a cell storage facility, interim
facility or processing area by methods known in the art. Storage of
the amniotic fluid-derived cell samples may be short term or long
term. In some embodiments, the stored cells may be cryogenically
preserved but any storage method suitable for long term storage may
be used, such as, for example, the addition of amino acids,
inosine, adenine, or other compounds to the cells. Any storage
method may be used in this invention providing that the stored
product retain viability for the therapeutic or other purposes.
[0061] There is no upper limit on the number of amniotic
fluid-derived cell samples that can be stored in one cell bank. In
one embodiment, hundreds of stem cell products from different
individuals will be stored at one bank or storage facility. In
another embodiment, up to millions of products may be stored in one
storage facility. A single storage facility may be used to store
amniotic fluid-derived cell samples, or multiple storage facilities
may be used.
[0062] In some embodiments of the present invention, the storage
facility may have a means for any method of organizing and indexing
the stored cell samples, such as, for example, automated robotic
retrieval mechanisms and cell sample manipulation mechanisms. The
facility may include micromanipulation devices for processing such
amniotic fluid-derived cell samples. Known conventional
technologies can be used for efficient storage and retrieval of the
amniotic fluid-derived cell samples. Exemplary technologies include
but are not limited to Machine Vision, Robotics, Automated Guided
Vehicle System, Automated Storage and Retrieval Systems, Computer
Integrated Manufacturing, Computer Aided Process Planning,
Statistical Process Control, and the like. Less sophisticated
storage facilities may be used as well, such as, for example, large
areas maintained at appropriate temperatures having numerous racks
on which are indexed and stored the amniotic fluid-derived cell
samples of the invention.
[0063] Potential Recipients of the Amniotic Fluid-Derived Cells
[0064] The type information or other information associated with
the individual in need of a amniotic fluid-derived cell sample may
be recorded into a system that can be used to identify an
appropriate matching stem cell product, such as, for example, a
database system, an indexing system, and the like. Once recorded in
the system, a match can be made between the type of the individual
and a donor amniotic fluid-derived cell sample. In preferred
embodiments, the donor sample is from the same individual as the
individual in need of the sample. However, similar but not
identical donor/recipient matches can also be used. The matching
amniotic fluid-derived cell sample is available for the individual
possessing the matching type identifier. In one embodiment of this
invention, the individual's identification information is stored in
connection with the cell sample. In some embodiments, the matching
process occurs around the time of harvesting the sample, or can
occur at any time during processing, storage, or when a need
arises. Accordingly, in some embodiments of the invention, the
matching process occurs before the individual is in actual need of
the amniotic fluid-derived cell sample.
[0065] When the amniotic fluid-derived cell sample is needed by an
individual, it may be retrieved and made available for research,
transplantation or other purposes within minutes, if desired. The
stem cell sample may also be further processed to prepare it for
transplantation or other needs.
[0066] Thawing the Banked Cells and Use of the Thawed Cells
[0067] When the cells are to be used, they can be thawed under
controlled conditions. An example of one suitable method is shown
in Example 8. Preferably, the thawing is performed at about
37.degree. C. A water bath set at 37.degree. C. may be used for
this purpose.
[0068] The thawed samples can then be tested for viability and
growth characteristics. Typically, over 99% viability is attained.
The cells can be grown in any suitable medium, once the DMSO is
diluted to less than about 1% of the cell culture volume. The
growth properties, viability, karyotype and differentiation ability
of frozen and thawed cells were found to be identical to fresh AF
cells and to MAFSC cells upon freezing, respectively.
[0069] It was found that the previously stored, thawed cells could
be grown and differentiated as if they had not been frozen.
Therefore, once thawed, the cells can be used for creating cultures
of undifferentiated cells, or for creating differentiated cell
types or tissue types as disclosed herein and in co-pending U.S.
patent provisional application Ser. No. 60/495,437, which is
incorporated by reference herein in its entirety.
[0070] The thawed MAFSCs may be grown in an undifferentiated state
for as long as desired, and can then be cultured under certain
conditions to allow progression to a differentiated state. By
"differentiation" is meant the process whereby an unspecialized
cell acquires the features of a specialized cell such as a heart,
liver, muscle, pancreas or other organ or tissue cell. The MAFSCs,
when cultured under certain conditions, have the ability to
differentiate in a regulated manner into three or more
subphenotypes. Once sufficient cellular mass is achieved, cells can
be differentiated into endodermal, mesodermal and ectodermal
derived tissues in vitro and in vivo. This planned, specialized
differentiation from undifferentiated cells towards a specific cell
type or tissue type is termed "directed differentiation." Examples
of such cell types that may be prepared from MAFSCs using directed
differentiation include but are not limited to fat cells, cardiac
muscle cells, epithelial cells, liver cells, brain cells, blood
cells, neurons, or glial cells.
[0071] General methods relating to stem cell differentiation
techniques that may be useful for differentiating the MAFSCs of
this invention can be found in general texts such as:
Teratocarcinomas and embryonic stem cells: A practical approach (E.
J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in
Mouse Development (P. M. Wasserman et al. eds., Academic Press
1993); Embryonic Stem Cell Differentiation in vitro (M. V. Wiles,
Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic
Stem Cells: Prospects for Application to Human Biology and Gene
Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998);
and in Stem cell biology (L. M. Reid, Curr. Opinion Cell Biol.
2:121, 1990), each of which is incorporated by reference herein in
its entirety.
[0072] Differentiation agents, maturation agents, or maturation
factors may be useful to allow progression to certain cell types.
Examples of differentiation agents, that may be used include but
are not limited to agents, such as N-butyrate, which are useful for
differentiating embryonic stem cells to liver cells are described
in U.S. Pat. No. 6,506,574, to Rambhatla et al. Optionally,
maturation agents, or maturation factors, such as, for example,
growth factors, peptide hormones, cytokines, ligand receptor
complexes, corticosteroids, and even organic solvents like DMSO
have been found to effect differentiation of embryonic stem cells
(U.S. Pat. No. 6,506,574, which is incorporated by reference herein
in its entirety.
[0073] Treatment of Individuals Using Amniotic Fluid-Derived Cells
that have been Thawed from a Cell Bank
[0074] The isolated amniotic fluid-derived cells or their
derivatives may be used to treat diseases in humans or animals. As
used herein the term "treat" or "treatment" refer to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent, slow down (lessen), or reverse an
undesired physiological change or disorder. The term "treat" also
refers to the characterization of the type or severity of disease
which may have ramifications for future prognosis, or need for
specific treatments. For purposes of this invention, beneficial or
desired clinical results include, but are not limited to,
alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0075] To treat a human or animal in need of treatment, the
amniotic fluid-derived cells can be either regenerated into
segments of a desired tissue, then transplanted into the patient,
or can be regenerated into a whole tissue that will be used to
replace the failing tissue, or can be injected into a tissue of
interest as whole cells, where they will regenerate at the injected
location.
[0076] The banked amniotic fluid-derived cells may be used in the
individual's postnatal life for purposes such as regenerative
medical attention (as shown in Examples 8 and 9) or for cosmetic
purposes. The banked cells will be readily available to prepare
replacement tissue or cells as needed. Additionally, the amniotic
fluid-derived cells and tissues derived from amniotic fluid-derived
cells can also be shared with individuals of similar, but not
necessarily identical genetic make-up. A directed search of the
database information can be used to find samples of similar but not
necessarily identical genetic make-up in situations where
individuals of similar genetic background are not known or are not
available.
[0077] It may be possible to replace any type of failing tissue
with amniotic fluid-derived cells. Accordingly, amniotic
fluid-derived cells that have been retrieved from a cell bank
system may be differentiated into tissues such as liver, endocrine
tissues, lung, blood cells, neuronal or astroglial cells, or
others, which may then be used for transplantation to cure or treat
diseases. Examples of tissues which may be (at least partially)
replaced include, but are not limited to pancreatic tissue or
cells, lung tissue, heart tissue, ocular tissue, nerve tissue,
brain tissue, muscle tissue, skin, or others.
[0078] Examples of diseases that may be treated with amniotic
fluid-derived cell-derived cells or tissues include but are not
limited to cirrhosis of the liver, pancreatitis, diabetes,
Parkinson's disease, spinal cord injury, stroke, burns, heart
disease, certain types of cancer, osteoarthritis, rheumatoid
arthritis, leukemia, lymphoma, genetic blood disorders, Examples of
diseases that can be treated with amniotic fluid-derived
cell-derived stem cells include but are not limited to Acute
Lymphoblastic Leukemia, Acute Myelogenous Leukemia, Acute
Biphenotypic Leukemia, and Acute Undifferentiated Leukemia; Chronic
Myelogenous Leukemia, Chronic Lymphocytic Leukemia, Juvenile
Chronic Myelogenous Leukemia, Juvenile Myelomonocytic Leukemia,
Refractory Anemia, Refractory Anemia with Ringed Sideroblasts,
Refractory Anemia with Excess Blasts, Refractory Anemia with Excess
Blasts in Transformation, Chronic Myelomonocytic Leukemia, Aplastic
Anemia, Fanconi Anemia, Paroxysmal Nocturnal Hemoglobinuria, Pure
Red Cell Aplasia, Acute Myelofibrosis, Agnogenic Myeloid
Metaplasia, myelofibrosis, Polycythemia Vera, Essential
Thrombocythemia, Non-Hodgkin's Lymphoma, Hodgkin's Disease,
Chediak-Higashi Syndrome, Chronic Granulomatous Disease, Neutrophil
Actin Deficiency, Reticular Dysgenesis, Mucopolysaccharidoses,
Hurler's Syndrome, Scheie Syndrome, Hunter's Syndrome, Sanfilippo
Syndrome, Morquio Syndrome, Maroteaux-Lamy Syndrome, Sly Syndrome,
Beta-Glucuronidase Deficiency, Adrenoleukodystrophy, Mucolipidosis
II, Krabbe Disease, Gaucher's Disease, Niemann-Pick Disease, Wolman
Disease, Metachromatic Leukodystrophy, Familial Erythrophagocytic
Lymphohistiocytosis, Histiocytosis-X, Hemophagocytosis, Inherited
Erythrocyte Abnormalities, Beta Thalassemia Major, Sickle Cell
Disease, Inherited Immune System Disorders, Ataxia-Telangiectasia,
Kostmann Syndrome, Leukocyte Adhesion Deficiency, DiGeorge
Syndrome, Bare Lymphocyte Syndrome, Omenn's Syndrome, Severe
Combined Immunodeficiency, Common Variable Immunodeficiency,
Wiskott-Aldrich Syndrome, X-Linked Lymphoproliferative Disorder,
Other Inherited Disorders, Lesch-Nyhan Syndrome, Cartilage-Hair
Hypoplasia, Glanzmann Thrombasthenia, Osteopetrosis, Inherited
Platelet Abnormalities, Amegakaryocytosis, Congenital
Thrombocytopenia, Plasma Cell Disorders, Multiple Myeloma, Plasma
Cell Leukemia, Waldenstrom's Macroglobulinemia, Breast Cancer,
Ewing Sarcoma, Neuroblastoma, Renal Cell Carcinoma, brain disorders
such as Alzheimer's disease, and the like (see, for example,
hypertext transfer protocol (http) on the world wide web at the
following link: marrow.org/index.html, which is incorporated by
reference herein in its entirety).
[0079] Genetic Modification of MAFSCs before or after Banking
[0080] MAFSCs or MAFSC-derived cells may also be genetically
modified by transduction with any suitable gene of interest, as
shown in Example 10. General techniques useful to genetically
modify the MAFSC cells (or their derivatives) can be found, for
example, in standard textbooks and reviews in cell biology, tissue
culture, and embryology. Methods in molecular genetics and genetic
engineering are described, for example, in Molecular Cloning: A
Laboratory Manual, 2nd Ed. (Sambrook et al., 1989); Oligonucleotide
Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I.
Freshney, ed., 1987); the series Methods in Enzymology (Academic
Press, Inc.); Gene Transfer Vectors for Mammalian Cells (I. M.
Miller & M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology and Short Protocols in Molecular Biology, 3rd
Edition (F. M. Ausubel et al., eds., 1987 & 1995); and
Recombinant DNA Methodology II (R. Wu ed., Academic Press
1995).
[0081] Transduction of MAFSC cells can be accomplished by each of
many techniques, DNA or RNA gene/sequence insertion of a suitably
promoted gene construct, electroporation of said genes, infection
by retroviral, lentiviral or other viral vector constructs encoding
a gene of interest, mechanical gene introduction or the transfer by
any means of specific protein, glycoprotein or phosphoprotein
entities by any of a number of general techniques used for such
purpose. The nucleic acid molecule of interest can be stably
integrated into the genome of the host MAFSC cell, or the nucleic
acid molecule and can also be present as an extrachromosomal
molecule, such as a vector or plasmid. Such an extrachromosomal
molecule can be auto-replicating. The term "transfection," as used
herein, refers to a process for introducing heterologous nucleic
acid into the host MAFSC or MAFSC-derived cell or tissue. A
transfected MAFSC cell refers to a MAFSC cell into which a
heterologous nucleic acid molecule has been introduced. One example
of a useful genetic modification of a MAFSC cell was the insertion
of the "TERT" gene (telomerase reverse transcriptase). MAFSC Cells
that have had the Tert gene transduced by retroviral gene
transduction expressed this gene to high levels and had acquired an
immortal phenotype, i.e. they did not senesce after >200
population doublings. Further, genetic modification of MAFSC cells
can be used for purposes of propagation of stable
non-differentiated cells, for the purpose of accomplishing stable
or transient MAFSC cell differentiation or for gene therapy
purposes, such as the administration of a gene encoding a
functional protein product to an individual that lacks a functional
copy of a gene of interest. If desired, MAFSC cells or their
derivatives can also be genetically modified to inhibit the
expression of certain genes, using gene manipulation methods known
in the art.
[0082] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. A more complete
understanding can be obtained by reference to the following
specific examples which are provided herein for purposes of
illustration only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Isolation and Expansion of Undifferentiated Cells Derived from
Amniotic Fluid
[0083] Approximately 2 to 5 ml of fresh amniotic fluid was
harvested from women undergoing routine amniocentesis at 16 to 21
weeks of pregnancy (2.sup.nd trimester). Second trimester amniotic
fluid contained approximately 1-2.times.10.sup.4 live cells per ml.
The cells were pelleted in a clinical centrifuge and resuspended in
15 ml "MAFSC" medium. MAFSC medium was composed of low glucose
Dulbecco Modified Eagle's Medium (GIBCO, Carlsbad, Calif.) and MCDB
201 medium (SIGMA, Saint Louis, Mo.) at a one to one ratio and
contained 2% Defined Fetal Calf Serum (HYCLONE, Logan, Utah),
1.times. insulin-transferrin-selenium,
linoleic-acid-bovine-serum-albumin (ITS+1, SIGMA), 1 nanomolar
dexamethasone (Sigma), 100 .mu.m ascorbic acid 2-phosphate (Sigma),
4 .mu.m/ml gentamycin, 10 ng/ml of rhEGF (R&D Systems,
Minneapolis, Minn.), 10 ng/ml rrPDGF-BB (R&D) and 10 ng/ml
rhFGF-basic (R&D). The wells of 6-well culture dishes were
prepared for cell plating by coating for one hour at room
temperature with 2.5 ml of fibronectin (stock of 10 .mu.g
fibronectin/ml of sterile water) immediately prior to cell plating.
The fibronectin solution was removed prior to cell plating and the
wells were not washed after removal of the fibronectin solution.
The cells were then seeded in 2.5 ml of medium in each well.
[0084] The cells in MAFSC culture appeared under the inverted phase
microscope as large suspension cells that divided on average once
every 4 days, but ceased dividing 8-12 days after seeding. The
growth medium of MAFSC cultures was changed with complete MAFSC
medium every two days making sure to not lose the suspended cells.
After 8-10 days, small numbers of adherent cells emerged which grew
into large colonies of >10.sup.5 cells in 14-15 days. On
average, 0-1 adherent colonies grew out per 2.times.10.sup.4 live
cells seeded. Hence, a sample of 5 ml of fresh amniotic fluid gave
rise to 3-5 adherent cell colonies, resulting in a single
colony/clone in the majority of the wells of 6-well cell culture
clusters.
[0085] Cells were transferred to successively larger
fibronectin-coated flasks/vessels. To perform cell transfer, the
cells were grown to a subconfluent state of approximately 40%
confluence and were detached with 0.25% Trypsin-EDTA and replated
at a 1:3 or 1:12 dilution under the same culture conditions.
Example 2
Morphological Characterization of Cell Types
[0086] Cultured amniotic fluid-derived cells were tested for cell
surface and differentiation markers and were karyotyped. These
cells were found to be immortal or near-immortal and were named
Multipotent Amniotic Fetal Stem Cells (MAFSC).
[0087] All of >80 amniotic fluid sample harvests of 5 ml gave
rise to at least one adherent MAFSC colony and continuous culture.
The majority of sample harvests gave rise to 3-4 individual clones.
Among the individual clones, different colonies/cultures had
diverse colony morphologies, as shown in FIG. 1. Some cultures had
a flat, epihelial morphology (FIG. 1A). Others had a fibroblastic
morphology (FIGS. 1B, 1C). Both the epithelioid and the
fibroblastic classes of cultures senesced after .about.60
population doublings (PD), yielding a maximum of 10.sup.18 cells,
unless the cells were immortalized by the expression of the human
TERT (telomerase) gene that maintained the length of the cells'
telomeres. Indeed, mortal MAFSC cultures have been immortalized at
low (PD 15-25) transfer numbers by infection with an amphotropic
high titer retroviral vector expressing the human TERT gene. MAFSC
cultures immortalized with TERT have not senesced after >220
population doublings. Thus, the TERT-modified MAFSC cultures were
immortal, though only after genetic modification which may not be
the advantaged way to derive human stem cell strains.
[0088] About half of the amniotic fluid samples gave rise to MAFSC
clones/cultures that behaved like immortal cell lines, as shown in
FIGS. 2A and 2B. These cultures grew vigorously, with a doubling
time of 28 hours. When confluent, the cells piled up in
multilayered fashion and numerous round, semi-detached cells grew
on top of a swirling, non-contact-inhibited layer of cells. These
aggressive cultures expressed the telomerase gene/protein. The
cells were cloneable into single cell clones and are non-senescing.
These vigorously growing MAFSC lines expressed very high levels of
a set of cell surface determinants known to be present on
non-differentiated human Embryo Stem Cells (hES) and expressed a
set of surface determinants known to be associated with
non-differentiated human Mesenchymal Stem Cells (MSC). MAFSC cells
did not express markers characteristic of hematopoietic cells, e.g.
CD45 and CD34, see FIGS. 3 and 4, which show flow cytometry
examples of one such vigorous MAFSC line, #111a.
Example 3
FACS Analysis of MAFSC Cells
[0089] Cells were prepared for FACS analysis by trypsinizing to
remove them from the tissue culture flask, washing in buffer, HBSS,
2% BSA, 0.1% sodium azide, then resuspended in 100 .mu.L of the
same buffer. For intracellular antigens (i.e. Oct-4), the cells
were fixed and permeablized using Beckman-Coulter IntraPrep
reagents, as suggested by the manufacturer. Primary antibodies
specific for the indicated cell-surface or intracellular marker
were added at a 1:10 dilution and incubated for 30 minutes at room
temperature, then washed. For samples using primary antibodies that
were not fluorescently-conjugated, the cells were then resuspended
in 250 .mu.L of buffer and the appropriate fluorescent-labeled
secondary antibody was added at a 1:250 dilution and incubated for
30 minutes at room temperature. Labeled cells were washed and
resuspended in buffer or 1% paraformaldehyde for analysis by a
FACSCalibur flow cytometer. The data obtained from this analysis
were plotted as the x-axis being the number of cells analyzed per
point and the y-axis indicating the logarithm of fluorescent
intensity of the antibody-labeled cells. The fluorescence was
compared to control cells that were not labeled with antibody, to
discount any background fluorescence. The percent indicated was the
fraction of cells that were positive for the specific
antibody-labeled antigen. The level of antibody label (X-axis) is
proportional to the concentration of the specific antigen present
on the cells.
Example 4
Stem Cell Markers on MAFSC Cells
[0090] MAFSC lines expressed very high levels of a set of cell
surface determinants known to be present on non-differentiated
human Embryo Stem Cells (hES) and expressed a set of surface
determinants known to be associated with non-differentiated human
Mesenchymal Stem Cells (MSC). MAFSC cells did not express markers
characteristic of hematopoietic cells, e.g. CD45 and CD34, see
FIGS. 3 and 4, which show flow cytometry examples of one such
vigorous MAFSC line, #111a. The flow cytometry was performed as
described above in Example 3.
[0091] Mass cultures of the MAFSC cells strain 111a were
characterized by very high expression of the globoseries glycolipid
antigens SSEA3 (96%), SSEA4 (96%), the lack of expression of a
lactoseries oligosaccharide antigen, SSEA1, the expression of the
keratin sulphate-related antigens Tra-1-60 (71%) and Tra-1-81 (82%)
and the tissue non-specific alkaline phosphatase-related antigen
Tra-2-54 (63%), FIG. 3. The expression (or lack of expression,
SSEA1) of these antigens is expressly exhibited by pluropotential,
undifferentiated human embryo stem cells in which the expression of
these antigens is lost (or gained, SSEA1) by the induction of
differentiation with retinoic acid (Draper J S, Pigott C, Thompson
J A, Andrews P W. 2002 Journal of Anatomy 200: 249-258). MAFSC
cells expressed high levels of HLA Class I but not of HLA Class II,
low levels of CD 117 (c-kit ligand) and Stro-1, FIG. 3.
[0092] In addition to the embryo stem cell markers shown in FIG. 3
and discussed in Table 1, MAFSC cells (such as, for example the
MAFSC 111 a line), expressed high levels of the antigen CD13
(99.6%) aminopeptidase N, CD44 (99.7%) hyaluronic acid-binding
receptor, CD49b (99.8%) collagen/laminin-binding integrin alpha2,
and CD105 (97%) endoglin. This set of cell surface antigens is
found on human mesenchymal stem cells but not normally on human
embryo stem cells (M F Pittinger et al., Science 284:143-147, 1999;
S Gronthos et al., J Cell Physiol. 189:54-63, 2001). Hence, the
amniotic fluid-derived MAFSC cells, grown and propagated as
described here, represent a novel class of human stem cells that
combined the characteristics of hES cells and of hMSC cells and can
be expected to differentiate into many diverse directions.
[0093] The amniotic fluid-derived stem cells also expressed the
transcription factor OCT-4. The human embryonic stem cell markers
typically found on MAFSC cells are shown in Table 1 and are data
obtained for MAFSC cells, clone 111a, cultured for >40
population doublings in MAFSC culture medium. The markers typically
displayed by long term MAFSC cells are compared with the same
markers found on fresh amniotic fluid-derived cells, "AES", and
with various control cells, the human embryonic carcinoma cell line
NTERA-4; amniotic-fluid-derived, long-term fibroblasts, MY-TERT,
immortalized by the human telomerase reverse transcriptase gene,
TERT; and normal human foreskin fibroblasts, HFF. Expression of the
various markers is further described in co-pending U.S. Patent
Application filed Aug. 13, 2004, entitled, "Multipotent Amniotic
Fetal Stem Cells."
1 TABLE 1 Embryonic Stem Cell Markers NTERA-4 Embryonic MY-Tert HFF
Marker MAFSC 111a "AES" Carcinoma Immortal Amnio- Foreskin (%
Positive) Cultured Amniocytes Fresh Amniocytes Line fibroblasts
Fibroblasts SSEA-1 3.3 n.d.* 3.8 2 1.5 SSEA-3 96 19.4 9.1 17.3 2.3
SSEA-4 96 39.8 96.8 67.8 8.2 Tra-1-60 71 48.9 99.8 4.9 1.7 Tra-1-81
82 42 99.6 2.8 1.7 Tra-2-54 63 30.4 99.2 16.1 27.9 Oct-4 39 12.7 99
2.6 3.4 *n.d. = not determined
Example 5
Cryopreservation and Banking of Fresh Amniocentesis-Derived and of
Cultured MAFSC Cells
[0094] Both fresh amniocentesis-derived cells and cultured MAFSC
cells were cryopreserved for banking purposes. Samples of amniotic
fluid ranging from 2 to 5 ml were harvested. The cells were
centrifuged to remove excess amniotic fluid. The cells were then
frozen in medium containing 10% dimethyl sulfoxide and 25% fresh,
filtered (0.10 micron) amniotic fluid (DMSO/AF freezing medium).
Alternatively, the cells were grown to produce MAFSC cultures,
which were then frozen as above. The fresh amniocentesis-derived
cells and cultured MAFSC cells were frozen in DMSO/AF freezing
medium in a controlled-rate liquid nitrogen freezer at 1.degree.
C./min. Frozen samples were stored under liquid nitrogen in
freezing ampoules.
Example 6
Establishment of a Universal Stem Cell Bank Composed of
Transplantable Amniocentesis-Derived Stem Cells
[0095] To assure the availability of stem cells transplantable into
all or into a majority of potential recipients, stem cells will be
banked that possess a range of genetic characteristics and that
display a range of antigens, for example a range of human leukocyte
antigens (HLA antigens). Following widely recognized and
universally applied transplantation methodologies in human bone
marrow transplantation and in human organ transplantation, matching
of ten HLA specificities is generally sufficient to achieve full
and problem-free transplantations. The development of an
amniocyte-derived stem cell bank facilitates the cryopreservation
of hundreds or of thousands of lines possessing finely-mapped
transplantation specificities for the accomplishment of routine
stem cell transplantation into a majority of potential
subjects.
Example 7
Linkage of Cell "Bank" to Searchable Database
[0096] The cryopreserved cells may be listed in a searchable
database. Information such as genetic background of the stored
cells, familial data, date of submission, suspected or known
genetic diseases of individuals or relatives, etc., may be
included, for example. When an individual has a medical problem
that can be alleviated or reversed by administering cells derived
from the thawed cryopreserved cells, the database can be searched
for pertinent information. A suitable sample can then be chosen for
thawing, cell proliferation, differentiation, and administration to
the individual.
Example 8
Cell Recovery Procedures and Viability of Thawed Cells
[0097] Cell thawing was done rapidly in a 37.degree. C. water bath
resulting in the recovery of >99% of the frozen AF and MAFSC
cells. Thawed cells were then grown by being immediately diluted
10-fold to reduce the concentration of DMSO to <1%. The percent
viability of the cells was determined. When the amniotic fluid
cells are properly frozen, as is the state of the art, and they are
rapidly thawed, there is essentially no cell death: viability is
close to 99%, the cells' growth behavior in MAFSC cultures is also
not affected by cryopreservation and thawing.
Example 9
Measurements of the Viability of the Cryopreserved Cells
[0098] The viability of cryopreserved cells was equal to that of
fresh amniotic fluid cells. A typical fresh 2nd trimester
amniocentesis sample had a viablility of 30-50%, depending on the
exact number of weeks of the pregnancy: earlier harvests were more
highly viable. At later times in the pregnancy, e.g. 35 weeks, the
viability of freshly removed amniotic cells is about 10%, however
the absolute number of viable cells in a sample is roughly equal to
that found in 16-18 week amniocentesis harvests, namely
1-2.times.10.sup.4 cells/ml. At later times in the pregnancy more
dead cells accumulate, while the absolute number of live cells
stays similar.
[0099] The time between the harvesting of the amniotic cells to the
time of seeding the cells in MAFSC culture medium or to the time of
cryopreserving the cells was important. A delay of 24 hours in
seeding/growing or in cryopreserving the cells reduced the
probability of success in growing MAFSC stem cells by .about.90%.
Hence, speedy cryopreservation of amniotic fluid samples, within
2-4 hours of amniocentesis drawing, was crucial in the success of
growing MAFSC stem cells from the sample. Interestingly, the
concentration of viable cells did not diminish significantly by
delaying the amniotic fluid sample procedures (seeding or freezing)
by keeping the samples at room temperature in their natural fluid.
The number of resultant stem cells, however, was significantly
reduced, by .about.90%. Therefore, the MAFSC stem cells in an
amniotic fluid sample were more fragile than the bulk of the cells
in the harvest.
Example 10
Use of Revived Cells to Differentiate into Various Cell Types
[0100] Cryopreserved cells were revived, cultured, and
differentiated into various cell types, such as neural cells,
adipogenic cells, and chondrogenic cells, as described in
co-pending U.S. Patent Application filed Aug. 13, 2004, entitled,
"Multipotent Amniotic Fetal Stem Cells," which is incorporated by
reference herein in its entirety.
Example 11
[0101] Treatment of Diseases Using Transplantation of
Differentiated MAFSCs
[0102] Cryopreserved MAFSCs that have been thawed from a cell bank
system or MAFSC stem cells that have been grown from a
cryopreserved amniotic fluid cell sample may be differentiated into
various tissues, as needed. In one example, MAFSC stem cells can be
differentiated into pancreatic beta cells that secrete insulin
under the control of glucose prevalence. Beta cells or beta cells
enveloped into pancreatic islets with or without scaffolding can be
implanted at a suitable site in diabetic patients. In this way, the
glucose responsive beta cells or reconstructed pancreatic islets
can control the level of glucose in the diabetic patient. The
availability of syngenetic ("own") amniotic cells or the genetic
matching of amniotic fluid-derived islet cells to a diabetic's
transplantation antigen status thus facilitates the regulation of
glucose concentration without the danger of ersatz-pancreatic
rejection by the diabetic recipient.
Example 12
Use of Thawed MAFSCs to Promote Bone Marrow Regeneration
[0103] Following MAFSC cell differentiation into the cells of
interest, differentiated MAFSCs can be transplanted into a patient
in need of treatment to promote bone marrow regeneration. MAFSC
cells of suitable transplantation genotype, either the fetal-donor
of origin of the cryopreserved amniotic cells or a transplantation
antigen-matched recipient patient, are differentiated in vitro by
state of the art methods into hematopoietic stem and/or progenitor
cells, for example by methods similar to those described by Carotta
et al., "Directed differentiation and mass cultivation of pure
erythroid progenitors from mouse embryonic stem cells", Blood 2004,
May 27, prepublished online, DOI 10.1182. MAFSC cells
differentiated into specific hematopoietic stem/progenitor cell
types can then be used for transplantation by infusion into
recipients in need of hematopoietic cell transplantation. The
cryopreservation of millions of samples of amniotic fluid cells and
the potential generation of multiple MAFSC stem cell lines of
different transplantation specificities facilitates the
preparation, by differentiation, of suitable hematopoietic
stem/progenitor cells as a general bone marrow-transplantation
resource. Genetically and antigenically matched hematopoietic
stem/progenitor cell populations will induce minimal
transplantation complications in the transplantation of patients
requiring hematopoietic cell transplantation.
Example 13
Genetic Modification of MAFSCs to Express the TERT Gene to Alter
MAFSC Properties
[0104] MAFSCs or MAFSC-derived cells may also be genetically
modified either before or after the banking period, using any
suitable means, such as cell transfection procedures. The nucleic
acid molecule can be stably integrated into the genome of the host
MAFSC cell, or the nucleic acid molecule and can also be present as
an extrachromosomal molecule, such as a vector or plasmid. One
example of a useful genetic modification of a MAFSC cell is the
insertion of the "TERT" gene (human telomerase reverse
transcriptase, GenBank Accession No. NM.sub.--003219). A vector
suitable for mammalian transfection is prepared, containing a
selectable marker gene and the gene encoding "TERT", operably
linked to a suitable promoter sequence. The vector is used to
stably transfect a mammalian MAFSC cell. Stably transfected cells
are then selected using the selection agent corresponding to the
selectable marker gene. Expression of TERT is then confirmed using
antibody-based detection procedures. MAFSC Cells that are able to
express this TERT sequence can be significantly expanded.
Example 14
Use of an Amniotic Fluid-Derived Cell Cell Banking and Recording
System to Find Samples of Similar Type for Matching Purposes
[0105] An individual in need of stem cells or stem cell products
for treatment of a disease is identified. Type identifiers of the
individual are identified. The database of amniotic fluid-derived
cell banked samples is searched for donors with similar type
identifiers. Further analysis is performed to confirm the
similarity. The amniotic fluid-derived cell sample is retrieved,
and the cells are revived. The cells are treated so as to expand
and differentiate into the desired cell type, and are subsequently
transplanted to the individual in need of treatment. The success of
the treatment is ascertained at daily to monthly intervals. Using
this method, the disease is successfully treated.
[0106] It will be appreciated that no matter how detailed the
foregoing appears in text, the invention can be practiced in many
ways. As is also stated above, it should further be noted that the
use of particular terminology when describing certain features or
aspects of the present invention should not be taken to imply that
the broadest reasonable meaning of such terminology is not
intended, or that the terminology is being re-defined herein to be
restricted to including any specific characteristics of the
features or aspects of the invention with which that terminology is
associated. Thus, although this invention has been described in
terms of certain preferred embodiments, other embodiments which
will be apparent to those of ordinary skill in the art in view of
the disclosure herein are also within the scope of this invention.
Accordingly, the scope of the invention is intended to be defined
only by reference to the appended claims and any equivalents
thereof. All documents cited herein are incorporated herein by
reference in their entireties.
Sequence CWU 1
1
2 1 4015 DNA Homo sapiens 1 gcagcgctgc gtcctgctgc gcacgtggga
agccctggcc ccggccaccc ccgcgatgcc 60 gcgcgctccc cgctgccgag
ccgtgcgctc cctgctgcgc agccactacc gcgaggtgct 120 gccgctggcc
acgttcgtgc ggcgcctggg gccccagggc tggcggctgg tgcagcgcgg 180
ggacccggcg gctttccgcg cgctggtggc ccagtgcctg gtgtgcgtgc cctgggacgc
240 acggccgccc cccgccgccc cctccttccg ccaggtgtcc tgcctgaagg
agctggtggc 300 ccgagtgctg cagaggctgt gcgagcgcgg cgcgaagaac
gtgctggcct tcggcttcgc 360 gctgctggac ggggcccgcg ggggcccccc
cgaggccttc accaccagcg tgcgcagcta 420 cctgcccaac acggtgaccg
acgcactgcg ggggagcggg gcgtgggggc tgctgctgcg 480 ccgcgtgggc
gacgacgtgc tggttcacct gctggcacgc tgcgcgctct ttgtgctggt 540
ggctcccagc tgcgcctacc aggtgtgcgg gccgccgctg taccagctcg gcgctgccac
600 tcaggcccgg cccccgccac acgctagtgg accccgaagg cgtctgggat
gcgaacgggc 660 ctggaaccat agcgtcaggg aggccggggt ccccctgggc
ctgccagccc cgggtgcgag 720 gaggcgcggg ggcagtgcca gccgaagtct
gccgttgccc aagaggccca ggcgtggcgc 780 tgcccctgag ccggagcgga
cgcccgttgg gcaggggtcc tgggcccacc cgggcaggac 840 gcgtggaccg
agtgaccgtg gtttctgtgt ggtgtcacct gccagacccg ccgaagaagc 900
cacctctttg gagggtgcgc tctctggcac gcgccactcc cacccatccg tgggccgcca
960 gcaccacgcg ggccccccat ccacatcgcg gccaccacgt ccctgggaca
cgccttgtcc 1020 cccggtgtac gccgagacca agcacttcct ctactcctca
ggcgacaagg agcagctgcg 1080 gccctccttc ctactcagct ctctgaggcc
cagcctgact ggcgctcgga ggctcgtgga 1140 gaccatcttt ctgggttcca
ggccctggat gccagggact ccccgcaggt tgccccgcct 1200 gccccagcgc
tactggcaaa tgcggcccct gtttctggag ctgcttggga accacgcgca 1260
gtgcccctac ggggtgctcc tcaagacgca ctgcccgctg cgagctgcgg tcaccccagc
1320 agccggtgtc tgtgcccggg agaagcccca gggctctgtg gcggcccccg
aggaggagga 1380 cacagacccc cgtcgcctgg tgcagctgct ccgccagcac
agcagcccct ggcaggtgta 1440 cggcttcgtg cgggcctgcc tgcgccggct
ggtgccccca ggcctctggg gctccaggca 1500 caacgaacgc cgcttcctca
ggaacaccaa gaagttcatc tccctgggga agcatgccaa 1560 gctctcgctg
caggagctga cgtggaagat gagcgtgcgg gactgcgctt ggctgcgcag 1620
gagcccaggg gttggctgtg ttccggccgc agagcaccgt ctgcgtgagg agatcctggc
1680 caagttcctg cactggctga tgagtgtgta cgtcgtcgag ctgctcaggt
ctttctttta 1740 tgtcacggag accacgtttc aaaagaacag gctctttttc
taccggaaga gtgtctggag 1800 caagttgcaa agcattggaa tcagacagca
cttgaagagg gtgcagctgc gggagctgtc 1860 ggaagcagag gtcaggcagc
atcgggaagc caggcccgcc ctgctgacgt ccagactccg 1920 cttcatcccc
aagcctgacg ggctgcggcc gattgtgaac atggactacg tcgtgggagc 1980
cagaacgttc cgcagagaaa agagggccga gcgtctcacc tcgagggtga aggcactgtt
2040 cagcgtgctc aactacgagc gggcgcggcg ccccggcctc ctgggcgcct
ctgtgctggg 2100 cctggacgat atccacaggg cctggcgcac cttcgtgctg
cgtgtgcggg cccaggaccc 2160 gccgcctgag ctgtactttg tcaaggtgga
tgtgacgggc gcgtacgaca ccatccccca 2220 ggacaggctc acggaggtca
tcgccagcat catcaaaccc cagaacacgt actgcgtgcg 2280 tcggtatgcc
gtggtccaga aggccgccca tgggcacgtc cgcaaggcct tcaagagcca 2340
cgtctctacc ttgacagacc tccagccgta catgcgacag ttcgtggctc acctgcagga
2400 gaccagcccg ctgagggatg ccgtcgtcat cgagcagagc tcctccctga
atgaggccag 2460 cagtggcctc ttcgacgtct tcctacgctt catgtgccac
cacgccgtgc gcatcagggg 2520 caagtcctac gtccagtgcc aggggatccc
gcagggctcc atcctctcca cgctgctctg 2580 cagcctgtgc tacggcgaca
tggagaacaa gctgtttgcg gggattcggc gggacgggct 2640 gctcctgcgt
ttggtggatg atttcttgtt ggtgacacct cacctcaccc acgcgaaaac 2700
cttcctcagg accctggtcc gaggtgtccc tgagtatggc tgcgtggtga acttgcggaa
2760 gacagtggtg aacttccctg tagaagacga ggccctgggt ggcacggctt
ttgttcagat 2820 gccggcccac ggcctattcc cctggtgcgg cctgctgctg
gatacccgga ccctggaggt 2880 gcagagcgac tactccagct atgcccggac
ctccatcaga gccagtctca ccttcaaccg 2940 cggcttcaag gctgggagga
acatgcgtcg caaactcttt ggggtcttgc ggctgaagtg 3000 tcacagcctg
tttctggatt tgcaggtgaa cagcctccag acggtgtgca ccaacatcta 3060
caagatcctc ctgctgcagg cgtacaggtt tcacgcatgt gtgctgcagc tcccatttca
3120 tcagcaagtt tggaagaacc ccacattttt cctgcgcgtc atctctgaca
cggcctccct 3180 ctgctactcc atcctgaaag ccaagaacgc agggatgtcg
ctgggggcca agggcgccgc 3240 cggccctctg ccctccgagg ccgtgcagtg
gctgtgccac caagcattcc tgctcaagct 3300 gactcgacac cgtgtcacct
acgtgccact cctggggtca ctcaggacag cccagacgca 3360 gctgagtcgg
aagctcccgg ggacgacgct gactgccctg gaggccgcag ccaacccggc 3420
actgccctca gacttcaaga ccatcctgga ctgatggcca cccgcccaca gccaggccga
3480 gagcagacac cagcagccct gtcacgccgg gctctacgtc ccagggaggg
aggggcggcc 3540 cacacccagg cccgcaccgc tgggagtctg aggcctgagt
gagtgtttgg ccgaggcctg 3600 catgtccggc tgaaggctga gtgtccggct
gaggcctgag cgagtgtcca gccaagggct 3660 gagtgtccag cacacctgcc
gtcttcactt ccccacaggc tggcgctcgg ctccacccca 3720 gggccagctt
ttcctcacca ggagcccggc ttccactccc cacataggaa tagtccatcc 3780
ccagattcgc cattgttcac ccctcgccct gccctccttt gccttccacc cccaccatcc
3840 aggtggagac cctgagaagg accctgggag ctctgggaat ttggagtgac
caaaggtgtg 3900 ccctgtacac aggcgaggac cctgcacctg gatgggggtc
cctgtgggtc aaattggggg 3960 gaggtgctgt gggagtaaaa tactgaatat
atgagttttt cagttttgaa aaaaa 4015 2 1132 PRT Homo sapiens 2 Met Pro
Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15
His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20
25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe
Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp
Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser
Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys
Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu
Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr
Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu
Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly
Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150
155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu
Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His
Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp
Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro
Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser
Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro
Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His
Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270
Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275
280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His
His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp
Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His
Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser
Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg
Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met
Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr
Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395
400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg
405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys
Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp
Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro
Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu
Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg
Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys
His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser
Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520
525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe
530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg
Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn
Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln
Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg
Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg
Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro
Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640
Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645
650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg
Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp
Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala
Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val
Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr
Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys
Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His
Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765
Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770
775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn
Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe
Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val
Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu
Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe
Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp
Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys
Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890
895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu
900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly
Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu
Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile
Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly
Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys
Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln
Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005
Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln
1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser
Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala
Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala
Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His
Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085
Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser
1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala
Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr
Ile Leu Asp 1125 1130
* * * * *