U.S. patent application number 10/655275 was filed with the patent office on 2005-03-10 for entrapped stem cells and uses thereof.
Invention is credited to Conn, Bryan, Rubin, Albert L., Smith, Barry, Stenzel, Kurt.
Application Number | 20050053586 10/655275 |
Document ID | / |
Family ID | 34226098 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053586 |
Kind Code |
A1 |
Conn, Bryan ; et
al. |
March 10, 2005 |
Entrapped stem cells and uses thereof
Abstract
The invention relates to the stem cells, embryonic stem cells in
particular. It has been found that, when these stem cells are
entrapped such that their proliferation is inhibited, they produce
material which inhibits the proliferation of other, non-entrapped
cells, including stem cells and neoplastic and/or
hyperproliferative, but otherwise normal cells. It has also been
found that entrapped cancer cells will produce material which
inhibits the proliferation of stem cells. Further, it has been
found that the entrapment of the stem cells inhibits their
differentiation and thus the entrapment process can serve as a
long-term storage device for maintaining the undifferentiated state
of at least a portion of the entrapped cells.
Inventors: |
Conn, Bryan; (New York,
NY) ; Smith, Barry; (New York, NY) ; Rubin,
Albert L.; (Engelwood, NJ) ; Stenzel, Kurt;
(New York, NY) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
34226098 |
Appl. No.: |
10/655275 |
Filed: |
September 4, 2003 |
Current U.S.
Class: |
424/93.7 ;
435/366 |
Current CPC
Class: |
A61K 35/13 20130101;
C12N 5/0012 20130101; A01N 1/0231 20130101; A01N 1/0205 20130101;
A61P 35/00 20180101; C12N 5/0606 20130101; A61K 35/545 20130101;
C12N 2502/02 20130101; A61P 43/00 20180101; C12N 5/0693 20130101;
C12N 2533/76 20130101 |
Class at
Publication: |
424/093.7 ;
435/366 |
International
Class: |
A61K 048/00; C12N
005/08 |
Claims
We claim:
1. A composition of matter comprising a sample of stem cells
entrapped in a biocompatible, selectively permeable structure,
wherein entrapment of said sample of stem cells inhibits
proliferation of at least a portion of the entrapped stem
cells.
2. The composition of matter of claim 1, wherein said stem cells
are mammalian stem cells.
3. The composition of matter of claim 2, wherein said mammalian
stem cells are human stem cells.
4. The composition of matter of claim 2, wherein said mammalian
stem cells are murine stem cells.
5. The composition of matter of claim 1, wherein said stem cells
are embryonic stem cells.
6. The composition of matter of claim 1, wherein said stem cells
are entrapped in agarose.
7. The composition of matter of claim 6, wherein said agarose is
coated with agarose.
8. The composition of matter of claim 1, wherein said stem cells
are entrapped in a mixture of agarose and collagen, or a mixture of
agarose and gelatin.
9. The composition of matter of claim 8, wherein said mixture of
agarose and collagen or said mixture of agarose and gelatin is
coated with agarose.
10. A process of inhibiting proliferation of at least a portion of
a population of stem cells, comprising entrapping said stem cells
in a biocompatible, selectively permeable structure which induces
said stem cells to produce a factor which inhibits their
proliferation.
11. The process of claim 10, wherein said stem cells are mammalian
stem cells.
12. The process of claim 11, wherein said mammalian stem cells are
human stem cells.
13. The process of claim 11, wherein said mammalian cells are
murine stem cells.
14. The process of claim 10, wherein said stem cells are embryonic
stem cells.
15. The process of claim 10, wherein said stem cells are entrapped
in agarose.
16. The process of claim 15, wherein said agarose is coated with
agarose.
17. The process of claim 10, wherein said stem cells are entrapped
in a mixture of agarose and collagen or a mixture of agarose and
gelatin.
18. The process of claim 17, which said mixture of agarose and
collagen or mixture of agarose and gelatin is coated with
agarose.
19. A method for inhibiting proliferation of at least a portion of
a non-entrapped cell population, comprising culturing said cell
population in the presence of the composition of matter of claim
1.
20. The method of claim 19, wherein said cell population is a stem
cell population.
21. The method of claim 19, wherein said cell population is from a
species different from the species of origin of the cells entrapped
in said composition of matter.
22. The method of claim 19, wherein said cell population is from
the same species as is the species of origin of the cells entrapped
in said composition of matter.
23. The method of claim 19, wherein said cell population is a
cancer cell population.
24. The method of claim 19, wherein said cell population is a
mammalian cell population.
25. The method of claim 19, wherein said cell population is a
hyperproliferative cell population.
26. The method of claim 24, wherein said mammalian cell population
is a human cell population.
27. The method of claim 24, wherein said mammalian cell population
is a murine cell population.
28. A method for inhibiting proliferation of at least a portion of
a cell population comprising administering the composition of
matter of claim 1 to a subject in need thereof, in an amount
sufficient and at a site sufficient to inhibit proliferation of at
least a portion of said cell population.
29. The method of claim 28, wherein said cell population is a stem
cell population.
30. The method of claim 28, wherein said cell population comprises
neoplastic cells.
31. The method of claim 28, wherein said cell population is a
hyperproliferative cell population.
32. The method of claim 28, wherein cells entrapped in said
composition of matter are from a species different from said
subject.
33. The method of claim 28, wherein cells entrapped in said
composition of matter are from the same species as said
subject.
34. The method of claim 33, wherein said cells are autologous to
said subject.
35. The method of claim 28, wherein said subject is a human.
36. The method of claim 33, wherein said subject is a human.
37. A method for inhibiting proliferation of a non-entrapped cell
population, comprising culturing the composition of matter of claim
1, in a medium for a time sufficient to permit diffusion of a cell
proliferation inhibiting material into said medium, and contacting
said medium to said non-entrapped cell population for a time
sufficient to inhibit proliferation of said non-entrapped cell
population.
38. The method of claim 37, wherein said non-entrapped cell
population is a stem cell population.
39. The method of claim 37, wherein said non-entrapped cell
population and the cells entrapped in said composition of matter
are from different species.
40. The method of claim 37, wherein said non-entrapped cell
population and the cells entrapped in said composition of matter
are from the same species.
41. The method of claim 38, wherein said stem cell population is a
mammalian cell population.
42. The method of claim 41, wherein said mammalian cell population
is a human cell population.
43. The method of claim 41, wherein said mammalian cell population
is a murine cell population.
44. The method of claim 37, comprising contacting said medium to a
subject in need of inhibition of proliferation of a cell
population.
45. The method of claim 44, wherein said cell population is a stem
cell population.
46. The method of claim 44, wherein said cell population comprises
neoplastic cells.
47. The method of claim 44, wherein cells entrapped in said
composition of matter are from a species different from said
subject.
48. The method of claim 44, wherein cells entrapped in said
composition of matter are from the same species as said
subject.
49. The method of claim 48, wherein said cells are autologous to
said subject.
50. A method for inhibiting proliferation of a non-entrapped
population of stem cells, comprising culturing said stem cells in
the presence of a composition of matter which comprises a sample of
cancer cells entrapped in a biocompatible, selectively permeable
structure, wherein said sample of cancer cells produces material
which inhibits proliferation of said stem cells.
51. The method of claim 50, wherein said stem cells are mammalian
stem cells.
52. The method of claim 50, wherein said stem cells are embryonic
stem cells.
53. The method of claim 50, wherein said mammalian stem cells are
human stem cells.
54. The method of claim 50, wherein said mammalian stem cells are
murine stem cells.
55. The method of claim 50, wherein said stem cells and cancer
cells originate from the same species.
56. The method of claim 50, wherein said stem cells and cancer
cells originate from different species.
57. A method for inhibiting proliferation of non-entrapped stem
cells, comprising culturing a composition of matter which comprises
a sample of cancer cells entrapped in a biocompatible, selectively
permeable structure, wherein said sample of cancer cells produces
material which inhibits proliferation of stem cells and which
diffuses into a culture medium, and contacting said culture medium
to said stem cells.
58. The method of claim 57, wherein said stem cells are mammalian
stem cells.
59. The method of claim 57, wherein said stem cells are embryonic
stem cells.
60. The method of claim 58, wherein said mammalian stem cells are
human stem cells.
61. The method of claim 58, wherein said mammalian stem cells are
murine stem cells.
62. The method of claim 57, wherein said stem cells and cancer
cells originate from the same species.
63. The method of claim 57, wherein said stem cells and cancer
cells originate from different species.
64. A composition of matter comprising a sample of stem cells
entrapped in a biocompatible, selectively permeable structure,
wherein entrapment of said sample of stem cells causes at least a
portion of the entrapped stem cells to remain in an
undifferentiated state.
65. A process of inhibiting differentiation of at least a portion
of a population of stem cells, comprising entrapping said stem cell
in a biocompatible, selectively permeable structure which induces
said stem cells to produce a factor which causes such portion to
remain in an undifferentiated state.
66. A method for inhibiting differentiation of at least a portion
of a population of stem cells, comprising entrapping said stem
cells in a biocompatible, selectively permeable structure, wherein
such entrapment causes at least a portion of said stem cells to
remain in an undifferentiated state.
Description
FIELD OF THE INVENTION
[0001] This invention relates to entrapped cells, such as stem
cells. The entrapped cells, when cultured in the entrapment
material, produce a product which, when it is in contact with other
non-entrapped, freely growing cells in vitro or in vivo, inhibits
their proliferation. Further, the entrapment of the stem cells acts
to inhibit the proliferation of at least some of the entrapped stem
cells, and may inhibit the differentiation of at least a portion of
the entrapped stem cells.
BACKGROUND AND PRIOR ART
[0002] Entrapment of biological materials, such as cells, is a
technique that has been used for various ends. Exemplary of the
patent literature in this area are U.S. Pat. No. 6,303,151 (Asina,
et al.); U.S. Pat. No. 6,224,912 (Asina, et al.); U.S. Pat. No.
5,888,497 (Jain, et al.); U.S. Pat. No. 5,643,569 (Jain, et al.),
and U.S. Pat. No. RE38,027 (Jain, et al.), all of which are
incorporated by reference in their entirety. This family of related
patents shows that cancer cells and islets can be entrapped in a
biocompatible matrix, such as agarose, agarose/collagen mixtures,
and agarose/gelatin mixtures, and then be coated with agarose. The
resulting, entrapped cells produce materials which, inter alia,
diffuse out of the permeable biocompatible matrices in which they
are retained, and have useful biological properties. In the case of
islets, insulin is produced. In the case of cancer cells, material
diffuses from the matrix, and this material has an effect on the
growth and proliferation of cancer cells. As review of the '912 and
'151 patents, cited supra, will show, this effect crosses species,
i.e., entrapped or encapsulated cancer cells from a given species
produce material that inhibits the growth and/or proliferation of
cancer cells from other species, as well as the species from which
the cancer cells originated.
[0003] Additional examples of entrapment techniques include, e.g.,
U.S. Pat. No. 5,227,298 (Weber, et al.); U.S. Pat. No. 5,053,332
(Cook, et al.); U.S. Pat. No. 4,997,443 (Walthall, et al.); U.S.
Pat. No. 4,971,833 (Larsson, et al.); U.S. Pat. No. 4,902,295
(Walthall, et al.); U.S. Pat. No. 4,798,786 (Tice, et al.); U.S.
Pat. No. 4,673,566 (Goosen, et al.); U.S. Pat. No. 4,647,536
(Mosbach, et al.); U.S. Pat. No. 4,409,331 (Lim); U.S. Pat. No.
4,392,909 (Lim); U.S. Pat. No. 4,352,883 (Lim); and, U.S. Pat. No.
4,663,286 (Tsang, et al.). All of these references are incorporated
by reference.
[0004] Entrapment does not always result in a positive impact on
the entrapped cells. For example, see Lloyd-George, et al., Biomat.
Art. Cells & Immob. Biotech., 21(3):323-333 (1993); Schinstine,
et al., Cell Transplant, 41(1):93-102 (1995); Chicheportiche, et
al., Diabetologica, 31:54-57 (1988); Jaeger, et al., Progress In
Brain Research, 82:41-46 (1990); Zekom, et al., Diabetologica,
29:99-106 (1992); Zhou, et al., Am. J. Physiol., 274:C1356-1362
(1998); Darquy, et al., Diabetologica, 28:776-780 (1985); Tse, et
al., Biotech. & Bioeng., 51:271-280 (1996): Jaeger, et al., J.
Neurol., 21-469-480 (1992); Hortelano, et al., Blood,
87(12):5095-5103 (1996): Gardiner, et al., Transp. Proc.,
29:2019-2020 (1997). All of these references are incorporated by
reference.
[0005] None of the references discussed supra deals with the class
of cells known as stem cells, including embryonic stem cells.
[0006] One definition of stem cells, advanced by Reya, et al.,
Nature, 414:105-111 (2001), incorporated by reference, refers to
stems cells as cells which have the ability to perpetuate
themselves through self renewal and to generate mature cells of
particular tissues via differentiation. One can obtain different
types of stem cells, including neural, hematolymphoid, myeloid, and
other types of stem cells from various organs. These all have
potential to develop into specific organs or tissues. Certain stem
cells, such as embryonic stem cells, are pluripotent, in that their
differentiation path has not been determined at all, and they can
develop into various organs and tissues.
[0007] The discussions of the various therapeutic uses to which
stem cells may be put are well known, and need not be discussed
here. It is worth mentioning, as it bears on the invention
described herein, that stem cells are very uncommon, their
purification and separation from other cell types is laborious and
difficult, and stem cells will differentiate into mature cell
unless treated in some way to prevent this.
[0008] It has now been found that entrapment procedures, in line
with those disclosed by Jain et al. and Iwata et al., Journ.
Biomedical Material and Res., 26:967 (1992) affect stem cells in a
very desirable way. To elaborate, entrapped stem cells produce
materials which inhibit proliferation of various cell types,
including stem cells and cancer cells. The effect of this material
crosses species lines. Further, it has been found that stem cells,
when entrapped as is described herein, retain their differentiating
abilities, including their pluripotentiality, for an indefinite
period of time.
[0009] These features, as well as others, will be seen in the
disclosure which now follows.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE 1
[0010] Two different murine embryonic stem (ES) cell lines (i.e.,
ES-D3 and SCC-PSA1, which are both publicly available) were
obtained from the American Type Culture Collection ("ATCC").
[0011] Both lines were grown under standard culture conditions,
which included growth as a monolayer, atop "STO" embryonic
fibroblast feeder cells. These were also obtained from the ATCC.
The stem cells were cultured in DMEM medium that had been
supplemented with 100% ES-Qualified fetal bovine serum, leukemia
inhibitory factor (LIF), and P-mercaptoethanol (collectively,
"Medium A"). The cells, which were cryopreserved when received,
were thawed, and established as cultures after at least 3 passages
before being cultured as described, supra.
[0012] After tlree days, the ES cells were 70-80% confluent, and
were trypsinized and then entrapped in agarose beads, coated with
agarose, in accordance with U.S. Pat. Nos. 6,303,151; 6,224,912;
and, 5,888,497, all of which are incorporated by reference. In
brief, however, Sigma XII agarose was used, at an initial
concentration of about 1.0%. A 100 .mu.l aliquot of this agarose
solution was added to 34 .mu.l of cell suspension. The resulting
beads contained 2.0.times.10.sup.5.+-.1.5.times- .10.sup.4 murine
embryonic stem cells. The beads were given a second coat of
agarose, at a concentration of about 5.0%. The beads were cultured
in medium as described supra, except no LIF or viable STO feeder
cells were present ("Medium B").
[0013] The viability of cells in the beads over time was assessed,
via standard histochemical and microscopic examination, as well as
standard MTT assays, using cells removed from beads or maintained
in the beads, at various points in time.
[0014] It was observed that entrapped stem cells increase their
metabolic activity when first coated. This is followed by a
decrease in activity, as cells die via apoptosis, reaching their
lowest point of metabolic activity around day 21. After this low
point, however, surviving cells slowly proliferate, and total
metabolic activity was seen to gradually increase up to day 35 post
entrapment and beyond. This parallels observations on entrapped
cancer cells.
[0015] Morphologically, there was a significant difference between
the colonies formed within the inner layer of agarose of the bead
by the cancer cells and those formed by the stem cells. Although
both types of colonies are ovoid in shape, those formed by the
cancer cells are characterized by an outer zone of viable cells
(generally two to three cells in thickness) with a central zone of
eosiniphilic cellular debris. The colonies formed by the stem
cells, on the other hand, are fully occupied by viable cells and
there is no central zone of cellular debris.
EXAMPLE 2
[0016] In these experiments, the inhibitory effect of stem cells on
the proliferation of other stem cells was tested.
[0017] Ten-week-old agarose/agarose beads containing stem cells
(SCC-PSA1 cells) were tested for viability using the MTT assay,
discussed supra, and were cultured in Medium B discussed in example
1, for 6 days. After 6 days, the medium had been conditioned by the
entrapped stem cells. It is therefore called the Stem-cell
Conditioned Medium (SCM).
[0018] After these 6 days, the SCM was transferred to 6 well plates
that contained fresh SCC-PSA1 cells. These plates each contained
9.times.10.sup.5 STO feeder cells, which were covered with
1.5.times.10.sup.4 SCC-PSA1 cells. The STO cells had been treated
with mitomycin C to prevent proliferation. There were three
controls, i.e., wells which contained Medium B (an unconditioned
medium), and three wells that contained the SCM.
[0019] After 3 days, the contents of all wells were trypsinized,
and total cells were counted, using standard methods. The raw count
was adjusted to account for the 9.times.10.sup.5 feeder cells. The
results follow:
1 Average Cells After Percent Total Standard subtracting Inhibition
Test Article Cells/Well Deviation STO (of SCC cells) Control Medium
1.43 .times. 10.sup.6 .+-.9.9 .times. 10.sup.4 5.27 .times.
10.sup.5 SCM (w/SCC) 1.19 .times. 10.sup.6 .+-.3.6 .times. 10.sup.4
2.90 .times. 10.sup.5 44.9%
[0020] A similar experiment was carried out, with the following
results:
2 Average Cells After Percent Total Standard subtracting Inhibition
Test Article Cells/Well Deviation STO (of SCC cells) Control Medium
3.09 .times. 10.sup.6 .+-.1.7 .times. 10.sup.5 1.41 .times.
10.sup.6 SCM (w/SCC) 2.36 .times. 10.sup.6 .+-.9.5 .times. 10.sup.4
6.88 .times. 10.sup.5 51.4%
[0021] Further, the effect was not cell-line specific, as is
demonstrated by the following results, where ES-D3 cells were added
to the medium:
3 Average Cells After Percent Total Standard subtracting Inhibition
(of Test Article Cells/Well Deviation STO ES-D3 cells) Control
Medium 1.27 .times. 10.sup.6 .+-.1.1 .times. 10.sup.5 3.67 .times.
10.sup.5 SCM(w/SCC) 1.14 .times. 10.sup.6 .+-.7.6 .times. 10.sup.4
2.37 .times. 10.sup.5 35.5%
EXAMPLE 3
[0022] Example 2 showed that the proliferation inhibitory effect of
the stem cells was not cell line specific. In the experiments
described herein, the entrapped stem cells were tested for their
ability to inhibit the proliferation of cancer cells.
[0023] In these experiments, RENCA tumor cells were used. A total
of 15,000 tumor cells were seeded per well. SCM (conditioned either
with SCC-PSAI or ES-D3), as described supra, was used, as was the
control medium (Medium B), also as described.
[0024] With respect to the SCM, the conditioning took place over 5
days. The assay was run over a period of 32 weeks. The inhibition
of the RENCA cells was determined by fixing the cells with 100%
methanol, followed by staining with neutral red, lysis with SDS,
and scanning with a spectrophotometer to measure the amount of
neutral red in the cell lysate, which is proportional to the number
of cells per well.
[0025] The results are summarized in the following two tables,
which represent work with ES-D3, and SCC-PSA1 stem cells,
respectively. The results for weeks 1-3 correlate with the results
discussed in example 1, i.e., death of the entrapped stem cells,
reaching a low point on day 21, followed by regeneration.
4 Week 1 3 12 16 20 24 28 32 % Inhibition of -2.1% -8.8% 39.0%
24.4% 25.0% 20.9% 34.9% 31.5% RENCA Cells by SCM (w/ES-D3) Week 1 3
9 12 16 20 24 28 32 % Inhibition of -10.0% 8.9% 21.0% 40.4% 32.8%
22.5% 36.6% 38.0% 35.1% RENCA Cells by SCM (w/SCC-PSA1)
EXAMPLE 4
[0026] In the preceding experiments, the ability of entrapped stem
cells to inhibit proliferation of stem cells and cancer cells was
tested, and proven. These next experiments were designed to
determine if entrapped cancer cells could inhibit the proliferation
of stem cells.
[0027] Stem cells were plated and cultured in the same way as was
described, supra. RENCA cell containing beads, prepared as
described in U.S. Pat. Nos. 6,303,151; 6,224,912; and, 5,888,497
were cultured in Medium B to condition it, for 5 days. This RENCA
Conditioned Medium (RCM) was then added to plated stem cells, and
the stem cells were counted after 3 days. The results, which
follow, present data for ES-D3 cells first, and then SCC-PSA1
cells:
5 Average Cells After Total Standard subtracting Test Article
Cells/Well Deviation STO Percent Inhibition (of ES-D3) Control 1.69
.times. 10.sup.6 .+-.1.15 .times. 10.sup.4 7.93 .times. 10.sup.5
Medium RCM 1.42 .times. 10.sup.6 .+-.8.7410.sup.4 5.23 .times.
10.sup.5 34.0% Percent Inhibition (of SCC-PSA1) Control 1.25
.times. 10.sup.6 .+-.8.08 .times. 10.sup.4 3.47 .times. 10.sup.5
Medium RCM 1.05 .times. 10.sup.6 .+-.4.04 .times. 10.sup.4 1.47
.times. 10.sup.5 57.7%
[0028] These results indicate that the entrapped cancer cells did
inhibit the proliferation of stem cells.
EXAMPLE 5
[0029] One issue with stem cell research is the fact that, by their
nature, stem cells differentiate. As it is difficult to secure stem
cells and keep them from differentiating in the first place, it
would be desirable to have a methodology available by which stem
cells could be kept in their undifferentiated state, for as long a
period as possible.
[0030] To this end, stem cells were entrapped as described in
example 1, supra. The resulting structures were stored in Medium B
described supra, and were tested over a period of more than two
years.
[0031] Over this two-year period, stem cells were released from the
entrapment structures and cultured under standard conditions
(including STO co-cultures and LIF media additive). In all cases,
the released cells established a traditional stem cell monolayer
that proliferated in a non-differentiated manner, but maintained
the capability to spontaneously differentiate. This demonstrates
that the entrapment of stem cells can maintain their
non-differentiated phenotypes for greater than two years in the
absence of the traditionally required inhibitors of differentiation
(e.g., STO and LIF).
[0032] Notwithstanding this fact, if the cells do not receive the
required materials after a short period of time, they do begin
differentiation.
[0033] The foregoing examples describe the invention, which
includes, inter alia, compositions of matter which can be used to
produce material which suppresses proliferation of cells, such as,
but not being limited to, cancer cells and stem cells. These
compositions comprise stem cells, such as embryonic stem cells,
entrapped in a selectively permeable material to form a structure
which restricts the proliferation of the entrapped cells. As a
result of their being restricted, the cells produce unexpectedly
high amounts of material which suppresses proliferation of other
cells. The restricted cells produce more of the material than
comparable, non-restricted cells.
[0034] The material used to make the structures of the invention
may include any biocompatible matter which restricts the growth of
stem cells, thereby inducing them to produce greater amounts of
cell proliferation growth-suppressing material. The structure has a
suitable pore size such that the above material can diffuse to the
external environment, and such that it can prevent products or
cells from the immune system of the host from entering the
structure and causing the rejection of the cells or otherwise
impair their ability to survive and continue to produce the desired
material. The materials used to form the structure will also be
capable of maintaining viable (proliferation-restricted, but
surviving) cells both in vitro and in vivo, preferably for periods
of up to several years, by providing for the entrance of proper
nutrients, and elimination of cellular waste products, and a
compatible physico-chemical intrastructural environment. The
resulting structures provide an environment suitable for the
extended study of stem cells and their various differentiation,
transcription and nuclear factors. Results therefrom can be used to
direct the desired differentiation of other stem cells. The
materials used to prepare the structure is preferably well
tolerated when implanted in vivo, most preferably for the entire
duration of implantation in the host.
[0035] A non-limiting list of materials and combinations of
materials that might be utilized includes alginate-poly-(L-lysine);
alginate-poly-(L-lysine)-alginate;
alginate-poly-(L-lysine)-polyethylenei- mine; chitosan-alginate;
polyhydroxylethyl-methacrylate-methyl methacrylate;
carbonylmethylcellulose; K-carragenan; chitosan;
agarose-polyethersulphone-hexadi-methirine-bromide (Polybrene);
ethyl-cellulose; silica gels; and combinations thereof.
[0036] The structures which comprise the compositions of matter may
take many shapes, such as a bead, a sphere, a cylinder, a capsule,
a sheet or any other shape which is suitable for implantation in a
subject, and/or culture in an in vitro milieu. The size of the
structure can vary, depending upon its eventual use, as will be
clear to the skilled artisan.
[0037] The structures of the invention are selectively permeable,
such that nutrients may enter the structure, and so that the
proliferation-inhibiting material as well as cellular waste may
leave the structure. For in vivo use, it is preferred that the
structures prevent the entry of products or cells of the immune
system of a host which would cause the rejection of the cells, or
otherwise impair the ability of the cells to produce the
proliferation-suppressive material.
[0038] "Entrapped" as used herein means that the cells are
contained within a structure which prevents their escape to the
environment surrounding the structure, be that an in vitro or in
vivo environment. Notwithstanding the inability to escape
therefrom, the cells are within a structure which both permits
entry of molecules such as water, nutrients, and so forth, and
permits the passage from the structure of waste materials and
molecular products produced by the cells. The structure in which
the cells are contained thus supports the continued
viability/survival of the cells for long periods of time. It may
also, depending on the nature of the structure/material, cause the
cells contained within it to alter their behavior, including, but
not limited to, such behavior as proliferation, state of
differentiation and/or phenotypic expression. By inhibiting
differentiation, one de facto has a storage device useful for
maintaining stem cells as stem cells. Exemplary, but non-exclusive,
means of entrapping the cells include encapsulating them, encasing
them, enclosing them, or otherwise surrounding them on all sides
with some permeable material. Via the entrapment, the proliferation
of the entrapped stem cells is inhibited. Further, there are
situations where at least a portion of the population that is
entrapped does not undergo any differentiation as well.
[0039] Another aspect of the invention includes compositions which
are useful in suppressing cell proliferation. The compositions are
prepared by culturing restricted cells as described supra in an
appropriate culture medium, followed by recovery of the resultant
conditioned medium. Concentrates can then be formed from the
conditioned medium.
[0040] The invention is not limited to any particular type of stem
cell species; any stem cell type may be used in accordance with the
invention. Exemplary types of cells which can be used are human or
murine stem cells, as well as stem cells from other species,
especially mammalian species. Embryonic stem cells are especially
preferred, but stem cells obtained from various organs and/or organ
systems may be used as well.
[0041] As will be clear from this disclosure, a further aspect of
the invention is therapeutic methods for treating individuals
suffering from cell proliferation disorders such as polycystic
kidney disease, hypertrophic tissue reaction (including scar
formation), autoimmune disease, lympho-proliferative disorders,
polycythemia vera, as well as both benign and malignant cell
neoplasia. When used in a therapeutic context, as will be
elaborated upon infra, the type of cell restricted in the structure
need not be the same type of cell that is causing the disorder from
which the individual is suffering, although it can be. One such
method involves inserting at least one of the structures of the
invention into the subject, in an amount sufficient to cause
suppression of cell proliferation in the subject. Preferably, the
subject is a human being, although it is applicable to other
animals, such as domestic animals, farm animals, or any type of
animal.
[0042] The composition of the present invention can be used as
primary therapy in the treatment of various cell proliferative
disorders, and as an adjunct treatment in combination with other
therapies. For example, in neoplastic disorders, such as cancer,
patients may be treated with compositions and methods described
herein, in conjunction with radiation therapy, chemotherapy, or
treatment with other biologically active materials such as
cytokines, anti-sense molecules, steroid hormones, gene therapy,
and the like. Additionally, the compositions and methods of the
invention can be used in conjunction with surgical procedures to
treat disorders such as cancer, e.g., by implanting the structures
after resection of a tumor to prevent regrowth and metastases.
Cancers which are present in an inoperable state may be rendered
operable by treatment with the anti-proliferative compositions of
the invention. The excess proliferation of cells that are not
needed or desirable for proper organ system function, but are not
neoplastic, such as that of polycythemia vera or polycystic kidney
disease, may also be treated by this means. Hyperproliferative
disorders, such as polycythermia vera and polycystic kidney
disease, involve cells that exhibit excess proliferation but
generate otherwise normal (i.e., non-neoplastic or transformed)
cells. Such disorders, resulting in numerous cells that are not
needed or desirable for proper organ function, may also be treated
by these means. Additionally, conditions which are characterized by
hyperproliferative, normal cells, such as hypertrophic scars, can
also be treated in this way. In conditions such as this one, normal
cells, i.e., fibroblasts have proliferated beyond what is necessary
for healing, but unlike neoplasias, they are not characterized by
further, ongoing, unregulated proliferation. Other conditions
characterized by this phenomenon well known to the skilled artisan,
and need not be set forth here.
[0043] The compositions of the invention can also be used
prophylactically in individuals at risk for developing cell
proliferation disorders, subjects who show the presence of
individual risk factors, a family history of the disorder
generally, family history of a specific type (e.g., breast cancer),
and exposure to occupational or other problematic materials. For
prophylaxis against cancer, e.g., a prophylactically effective
amount of the structures of the invention are administered to the
individual upon identification of one or more risk factors.
[0044] As indicated by the examples, supra, the antiproliferative
effect is not limited by the type of cell used, nor by the species
from which the stem cell originated. Hence, one can administer
structures which contain stem cells of a first type to a subject of
a different species. For example, murine stem cells may be
restricted in the structure of the invention, and then be
administered to a human. Of course, the structures may contain stem
cells from the same species as is being treated. Still further, the
stem cell may be taken from the individual to be treated, entrapped
and restricted, and then administered to the same individual.
[0045] Processes for making the structures of the invention are
also a part of the invention.
[0046] Other facets of the invention will be clear to the skilled
artisan, and need not be set out here.
[0047] The terms and expression which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expression of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
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