U.S. patent application number 11/486635 was filed with the patent office on 2007-02-22 for compositions of placentally-derived stem cells for the treatment of cancer.
Invention is credited to Thomas E. Ichim.
Application Number | 20070041954 11/486635 |
Document ID | / |
Family ID | 37193805 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070041954 |
Kind Code |
A1 |
Ichim; Thomas E. |
February 22, 2007 |
Compositions of placentally-derived stem cells for the treatment of
cancer
Abstract
Disclosed are preparations of placentally-derived stem cells and
compositions useful for the treatment of cancer. Said stem cells
and compositions function through inducing a "guided
differentiation" program in cancer cells, thereby reducing
malignancy. Further extension of the invention pertains to
augmenting ability of administered cells to induce differentiation
through the co-administration of known differentiation inducing
agents. Within the context of this disclosure, methods for inducing
host responses to cancer are also described.
Inventors: |
Ichim; Thomas E.; (San
Diego, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
37193805 |
Appl. No.: |
11/486635 |
Filed: |
July 13, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60699579 |
Jul 14, 2005 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/372 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; A61K 35/50 20130101; C12N 5/0605 20130101;
C12N 5/0694 20130101; A61K 35/44 20130101 |
Class at
Publication: |
424/093.7 ;
435/372 |
International
Class: |
A61K 35/14 20070101
A61K035/14; C12N 5/08 20060101 C12N005/08 |
Claims
1. A composition comprising placentally-derived tissue extract and
cells, capable of inducing the differentiation of a substantially
neoplastic cell into a cell with benign characteristics.
2. The composition of claim 1, wherein the placental tissue is
human.
3. The composition of claim 1, wherein said neoplastic cell
possesses one or more of the characteristics selected from the
following: invasiveness, ability to metastasize, ability to
suppress immune response, ability to proliferate past the Hayflick
limit.
4. The composition of claim 3, wherein said neoplastic cell is
selected from the group of a cancer consisting of: a soft tissue
sarcoma, a lymphoma, a cancer of the brain, an esophageal cancer, a
uterine cancer, a cancer of the cervix, a bone cancer, a lung
cancer, a cancer of the endometrium, a bladder cancer, a breast
cancer, a cancer of the larynx, a cancer of the colon/rectum, a
stomach cancer, a cancer of the ovary, a pancreatic cancers, an
adrenal gland cancer and a prostate cancer.
5. The composition of claim 1, wherein said cell with benign
characteristics lacks one or more of the following: invasiveness,
ability to metastasize, ability to suppress immune response, and
ability to proliferate past the Hayflick limit.
6. The composition of claim 1, wherein the placental tissue is
processed through collagenase or other cell dissociating methods,
followed by purification of single cells.
7. The composition of claim 6, wherein said purification of single
cells is performed using Percoll gradient of 0.3 mg/ml or other
density gradients.
8. The composition of claim 1, wherein said placentally-derived
cells are cord blood stem cells.
9. The composition of claim 1, further comprising a compound
capable of inducing cellular differentiation.
10. The composition of claim 9, wherein said compound capable of
inducing cellular differentiation is selected from the group
consisting of: dihydroxyvitamin D3, retinoic acid, or valproic
acid.
11. The composition of claim 9, wherein said compound capable of
inducing cellular differentiation is a histone deacetylase
inhibitor.
12. The composition of claim 11, wherein said histone deacetylase
inhibitor is selected from the group consisting of: trichostatin A,
a short-chain fatty acid, a hydroxamate, a cyclic tetrapeptide, or
a benzamide.
13. The composition of claim 1, wherein said composition is an
adjuvant to a chemotherapeutic drug.
14. A method of reducing malignancy of a tumor in a patient
comprising: a. extracting placental tissue obtained from a healthy
mother; b. homogenizing said tissue and obtaining a single cell
suspension; c. purifying CD34+ cells; and d. administering the
CD34+ cells to a patient in need thereof.
15. The method of claim 14, wherein a differentiation inducing
agent is co-administered with the CD34+ cells.
16. The method of claim 14, wherein said CD34+ cells are expanded
in vitro prior to administration.
17. The method of claim 16, wherein expansion is performed by
culture in media conditioned with cytokines and/or growth factors
selected from the group consisting of: leukemia inhibitory factor,
IL-1 through IL-13, IL-15 through IL-17, IL-19 through IL-22,
granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), macrophage colony
stimulating factor (M-CSF), erythropoietin (Epo), thrombopoietin
(Tpo), Flt3-ligand, B cell activating factor, artemin, bone
morphogenic protein factors, epidermal growth factor (EGF), glial
derived neurotrophic factor, lymphotactin, macrophage inflammatory
proteins, myostatin, neurturin, nerve growth factors, platelet
derived growth factors, placental growth factor, pleiotrophin,
stern cell factor, stem cell growth factors, transforming growth
factors, tumor necrosis factors, Vascular Endothelial Cell Growth
Factors, and fibroblast growth factors, FGF-acidic and basic
fibroblast growth factor.
18. The method of claim 14, wherein one or more genes is
transfected into the CD34+ cells prior to administration.
19. The method of claim 18, wherein said one or more transfected
genes are associated with inhibition of tumor growth.
20. The method of claim 19, wherein said one or more transfected
genes are selected from the group consisting of: IFN-g, IL-2, IL12,
IL-15, IL-18, IL-23.
21. The method of claim 14, wherein a chemotherapeutic agent is
co-administered with the CD34+ cells.
22. The method of claim 21, wherein said chemotherapeutic agent is
selected from the group consisting of: methotrexate, vincristine,
adriamycin, cisplatin, non-sugar containing
chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,
doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA,
valrubicin, carmustaine and poliferposan, MM1270, BAY 12-9566, RAS
famesyl transferase inhibitor, famesyl transferase inhibitor, MMP,
MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,
Hycamtin/Topotecan, PKC412, Valspodar/PSC833,
Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070,
BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853,
ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP
845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,
Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,
Temodal/Temozolomide, Evacet/liposomal doxorubicin,
Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,
Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,
SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609
(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT
(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU
enhancer, Campto/Levamisole, Camptosar/Irinotecan,
Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,
Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,
Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU
79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal
doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin,
Daunorubicin HCl, Estramustine phosphate sodium, Etoposide
(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide,
Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a,
Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),
Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),
Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl,
Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen
citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine
(m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM),
Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal
bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin),
Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine
sulfate.
23. A cell free extract capable of inducing differentation in
cancer cells, the cell free extract produced by the process
comprising: a. extracting human placenta; b. homogenizing said
placenta, resulting in a homogenate; C. centrifuging said
homogenate in order to free the supernatant of cellular debris; d.
purifying differentiation associated molecular weight fractions;
and e. administering said fractions to a patient in need
thereof.
24. The cell free extract of claim 23 wherein the
differentiation-inducing fraction purified is of molecular weight
of 10-10,000 Daltons.
25. The cell free extract of claim 23 wherein the
differentiation-inducing fraction purified is of molecular weight
of 300-3,000 Daltons.
26. A method of stimulating an immune response to cancer cells by
administration of the cell free extract of claim 23, together with
an appropriate immunological adjuvant.
27. A method of prophylactically treating a cancer patient by
administration of the cell free extract of claim 23 to a patient in
need thereof.
28. A method of inhibiting progression of a preneoplastic lesion to
neoplasia by administration of the cell free extract of claim
23.
29. A cell free extract capable of inducing differentiation in
cancer cells, the cell free extract produced by the process
comprising: a. extracting bone marrow stem cells or unpurified bone
marrow nucleated cells; b. homogenizing said cells, resulting in a
homogenate; c. centrifuging said homogenate in order to free the
supernatant of cellular debris; d. purifying differentiation
associated molecular weight fractions; and e. administering said
fractions to a patient in need thereof.
30. A differentiation inducing composition, suitable for
administration to a patient in need thereof, derived from placental
tissue, the composition produced by the process comprising: a.
extracting human placenta; b. homogenizing said placenta, resulting
in a homogenate; c. centrifuging said homogenate in order to free
the supernatant of cellular debris; d. purifying the cell
free-debris through adsorption on a C-18 column; and e. filter
sterilizing said composition.
31. The composition of claim 30, wherein the homogenized portion of
the placenta consists substantially of chorionic tissue.
32. The composition of claim 30, wherein a histone deacetylase
inhibitor is added directly to the composition.
33. A method of inducing differentiation and reprogramming of
distal cancer lesions through an intratumoral administration of the
composition described in claim 30.
34. A method of substantially increasing efficacy of the
composition of claim 30 by administration in a liposomal
complex.
35. A differentiation inducing composition, comprising a
substantially purified therapeutic composition prepared through the
following steps: (a) preparing a supernatant; (b) subjecting the
supernatant to solid phase extraction to produce a product; (c)
subjecting the product from step (b) to gel filtration to produce a
product; (d) subjecting the product from step (c) to anion exchange
fast phase liquid chromatography to produce a product; (e)
subjecting the product from step (d) to amino-high performance
liquid chromatography to produce a product; and (f) subjecting the
product from step (e) to reverse phase high performance liquid
chromatography to produce purified differentiation inducing
factor.
36. The composition of claim 35, wherein said supernatant is
prepared by culturing cord-blood derived stem cells to produce a
factor-containing supernatant.
37. The composition of claim 35, wherein said supernatant is
prepared through centrifugation of lysed placental extract.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Application No. 60/699,579, which was filed on
Jul. 14, 2005, the disclosure of which is hereby incorporated by
reference in its entirety.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The invention disclosed relates generally to the fields of
cancer therapy. More particularly, it relates to the process of
cancer cell differentiation into cells of reduced malignancy, or
lacking of malignancy. More specifically, the invention pertains to
the utilization of cord-blood and placentally derived stem cells
for the stimulation of cancer cell differentiation.
[0004] 2. Description of Related Art
[0005] In the field of cellular biology, the definition of "stem
cell" is dependent on function more than on morphology. The
characteristic function of a stem cell is the ability to both
self-renew and to differentiate. Stem cells differentiate upon need
of the host in response to a wide variety of stimuli, these include
the need for increased erythrocytes due to high altitude, or the
need for augmented numbers of neutrophils in response to a
bacterial infection [1]. Additionally, stem cells can be
artificially induced to replicate through various means including
administration of cytokines, growth factors, and small peptides
[2]. A wide variety of tissue-specific stem cells have been
identified, these include liver [3], skin [4], renal [5],
pancreatic [6], gastric [7] and neuronal [8].
[0006] There are many similarities between neoplastically
transformed tissue and stem cells in the sense that both express
embryonic-like features, including the ability to migrate
(metastasize), ability to suppress immune responses, and ability to
proliferate. The sole difference resides in the fact that stem
cells are highly regulated in terms of growth and differentiation,
whereas cancer cells are not. At the molecular level, the more
aggressive a cancer is, the more de-differentiated and "stem
cell-like" it becomes. Specifically, regions of DNA that are
transcriptionally silenced in mature tissue, become active when the
tissue turns cancerous. For example the enzyme telomerase is needed
for cells to escape proliferative senescence (ie multiple past the
Hayflick limit). Stem cells possess active telomerase, in
similarity to cancer cells. In contrast, as cells differentiate
into specialized tissue, telomerase expression becomes silenced
epigenetically through synergistic involvement of DNA methylation
and histone deacetylation [9]. Conversely, tumor cells selectively
silence, again through epigenetic mechanisms, genes that stop the
tumor from being neoplastic. For example it is known that in
prostate cancer tumor suppressor genes become silenced by
methylation [10]. In fact, this is a common phenomena seen in a
wide variety of histologically-differing tumors [11].
[0007] Stem cells actively secrete differentiation inducing
factors, both known and unknown, that are capable of inducing
epigenetic reprogramming in cells. In U.S. Patent Publication No.
2003/0211603 (Earp et al., hereby incorporated by reference in its
entirety) disclose that embryonic stem cells possess the ability to
reprogram differentiated cells to take a "de-differentiated"
phenotype. Although the subject matter did not indicate potential
application to cancer cells, such an application would be
counterintuitive since more dedifferentiated tumors would be
expected to possess increased malignancy. The invention disclosed
in this patent is based on the unique and unanticipated discovery
that such "reprogramming" of cancer cells does not lead to
increased malignancy but instead induces a differentiation program
which results in formation of normal, or normal-like
progenitors.
[0008] Cord blood is a rich source of hematopoietic stem cells,
capable of reconstituting the hematopoietic lineage in NOD-SCID
mice. The potential utilization of cord blood stem cells, such as
CD34+ cells, has led to the cryopreservation of such cells for more
than a decade [12]. Indeed, the clinical use of cord blood derived
stem cells is becoming increasing acceptable as an alternative to
bone marrow transplant, or cytokine mobilized peripheral blood stem
cell transplant for a wide variety of diseases. This is in part
because of a reduced incidence of graft versus host reaction using
cord blood stem cells in comparison with other sources [13].
Indeed, this was observed in pediatric leukemia patients lacking
suitable major histocompatibility complex (MHC) matched donors
[14], as well as in transplantation of patients with certain
anemias or hematopoietic stem cell disorders such as
beta-thalassemia major [15].
[0009] Placental extracts have also been previously described in
the art. For example, a low molecular weight fraction of placental
tissue is known to be immunosuppressive as reported by Chauaot.
Additionally, the use of placenta in cosmetics is well known.
Therefore, placenta and cord-blood are two accepted sources of
biomaterial that is innocuous for human use.
SUMMARY OF THE INVENTION
[0010] The invention described in the present disclosure teaches
methods of generating compositions therapeutically useful for
treatment of cancer patients. While chemotherapy and radiation
therapy induce severe side effects through collateral damage to
non-cancerous cells and organs, the present invention utilizes the
ability of cancer cells to be "reprogrammed" into benign,
non-cancerous progeny. The use of cellular differentiation therapy
has been previous attempted in cancer patients; however, the
majority of such differentiation inducers have been synthetic
chemicals administered at unnaturally high doses.
[0011] In the present invention, the inventor teaches the
utilization of stem cells to guide the differentiation of cancer
cells into non-malignant cells. For example, it is disclosed that
the administration of cord blood stem cells to a cancer patient is
demonstrated to have some anticancer effect. This effect is
magnified if certain derivatives from the cord blood stem cells are
systemically administered. Said derivatives include the low
molecular weight fraction that can be purified through column
chromatography. Other methods of separation include high
performance liquid chromatography, Fas Q liquid chromatography, or
on-line mass spectrometry.
[0012] A simpler method of concentrating the differentiation
inducing abilities of the cord blood stem cells is through purified
the cell culture supernatant of said cells through solid phase
adsorption such as through the use of a C-18 columns. The
therapeutic composition can also be produced in a more convenient
manner through utilization of placenta lysate concentrated by
C-18.
[0013] Noted within the disclosure is that cancer cells treated
with differentiation inducing agents possess an inhibited
proliferative index. This is attributed to the fact that the
differentiation inducing composition endows the neoplastic cells
with a more benign phenotype (i.e. reduced proliferation).
[0014] The present disclosure is not intended to be limiting in any
way and can be practiced with other techniques known to one skilled
in the art. For example, co-administration of placental extract
with differentiation inducers known in the art is within the scope
of the invention.
[0015] Additionally, it is known that differentiated cells express
antigens not found in immature progeny. U.S. Patent Publication No.
2004/0185563, hereby incorporated by reference in its entirety,
describes a method of increasing efficacy of immunotherapy by
induction of neoantigens on cancer cells through differentiation by
administration of histone deacetylase inhibitors. An application of
the invention disclosed includes the utilization of stem cell
derivatives for increasing the immunogenicity of tumor tissue. This
would result in sensitization to conventionally used immune
stimulatory anticancer approaches including but not limited to
cancer vaccines, cytokine therapy, and non specific immune
stimulators such as BCG, CpG, or beta glucan.
[0016] In some embodiments, a composition comprising
placentally-derived tissue extract and cells is provided, which is
capable of inducing the differentiation of a substantially
neoplastic cell into a cell with benign characteristics. The
placental tissue can be human. The neoplastic cell can possess one
or more of the following characteristics: invasiveness, ability to
metastasize, ability to suppress immune response, ability to
proliferate past the Hayflick limit. The neoplastic cell can be
chosen from, for example, the group of cancers consisting of: a
soft tissue sarcoma, a lymphoma, a cancer of the brain, an
esophageal cancer, a uterine cancer, a cancer of the cervix, a bone
cancer, a lung cancer, a cancer of the endometrium, a bladder
cancer, a breast cancer, a cancer of the larynx, a cancer of the
colon/rectum, a stomach cancer, a cancer of the ovary, a pancreatic
cancers, an adrenal gland cancer and a prostate cancer. The cell
with benign characteristics can be, for example, a cell that lacks
one or more of the following: invasiveness, ability to metastasize,
ability to suppress immune response, and ability to proliferate
past the Hayflick limit. The placental tissue may be processed, for
example, through collagenase or other cell dissociating methods,
followed by purification of single cells. For example, the cellular
purification can be performed using Percoll gradient of 0.3 mg/ml
or other density gradients. The placentally-derived cells can be,
for example, cord blood stem cells, mesenchymal stem cells, or
other placentally derived stem cells. The placentally-derived cells
and tissue extract can be administered, for example, to a patient
in need thereof together with a compound capable of inducing
cellular differentiation. The compound capable of inducing cellular
differentiation can be, for example, dihydroxyvitamin D3, retinoic
acid, valproic acid, or a histone deacetylase inhibitor. The
histone deacetylase inhibitor can be, for example, trichostatin A,
a short-chain fatty acid, a hydroxamate, a cyclic tetrapeptide, or
a benzamide. The composition can be used as an adjuvant to a
chemotherapeutic drug.
[0017] In additional embodiments, a method of reducing malignancy
of a tumor in a patient is provided, by extracting placental tissue
obtained from a healthy mother, homogenizing said tissue and
obtaining a single cell suspension, purifying CD34+ cells, and
administering the (CD34+ cells to a patient in need thereof. The
CD34+ cells can be, for example, co-administered with a
differentiation-inducing agent. The CD34+ cells can be, for
example, expanded in vitro prior to administration. The expansion
can be performed by culture in media conditioned with cytokines
and/or growth factors selected from the following: leukemia
inhibitory factor, IL-1 through IL-13, IL-15 through IL-17, IL-19
through IL-22, granulocyte macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage
colon) stimulating factor (M-CSF), erythropoietin (Epo),
thrombopoietin (Tpo), Flt3-ligand, B cell activating factor,
artemin, bone morphogenic protein factors, epidermal growth factor
(EGF), glial derived neurotrophic factor, lymphotactin, macrophage
inflammatory proteins, myostatin, neurturin, nerve growth factors,
platelet derived growth factors, placental growth factor,
pleiotrophin, stem cell factor, stem cell growth factors,
transforming growth factors, tumor necrosis factors, Vascular
Endothelial Cell Growth Factors, and fibroblast growth factors,
FGF-acidic and basic fibroblast growth factor. In additional
embodiments, one or more genes can be transfected into the CD34+
cells prior to administration. The transfected genes can be
associated with inhibition of tumor growth. The transfected genes
can be selected from the group consisting of: IFN-g, IL-2, IL12,
IL-15, IL-18, IL-23.
[0018] A chemotherapeutic agent can also be co-administered with
the CD34+ cells. The chemotherapeutic agent can be chosen from, for
example, methotrexate, vincristine, adriamycin, cisplatin,
non-sugar containing chloroethyinitrosoureas, 5-fluorouracil,
mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline,
Meglamine GLA, valrubicin, carmustaine and poliferposan, MM1270,
BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase
inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, Cl-994,
TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833,
Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070,
BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853,
ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP
845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,
Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,
Temodal/Temozolomide, Evacet/liposomal doxorubicin,
Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,
Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,
SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609
(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT
(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU
enhancer, Campto/Levamisole, Camptosar/Irinotecan,
Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,
Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,
Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU
79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal
doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin,
Daunorubicin HCl, Estramustine phosphate sodium, Etoposide
(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide,
Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a,
Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),
Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),
Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl,
Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen
citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine
(m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM),
Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal
bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin),
Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine
sulfate.
[0019] In an additional embodiment, a cell free extract capable of
inducing differentation in cancer cells is provided, which is
produced by extracting human placenta; homogenizing said placenta,
resulting in a homogenate; centrifuging said homogenate in order to
free the supernatant of cellular debris; purifying differentiation
associated molecular weight fractions; and administering said
fractions to a patient in need thereof. The purified,
differentiation-inducing fraction can have, for example, a
molecular weight of 10-10,000 Daltons, or a molecular weight of
300-3,000 Daltons. The cell-free extract can be used in a method of
stimulating an immune response to cancer cells by administration of
the extract, together with an appropriate immunological adjuvant.
In additional embodiments, a method of prophylactically treating a
cancer patient is provided by administration of the cell free
extract a patient in need thereof. In an additional embodiment, a
method of inhibiting progression of a preneoplastic lesion to
neoplasia is provided, by administration of the cell free extract.
The cell free extract can, for example, be derived from bone marrow
stem cells or unpurified bone marrow nucleated cells as an
alternative to cord blood or other placentally-derived cells.
[0020] In an additional embodiment, a differentiation inducing
composition, suitable for administration to a patient in need
thereof, and derived from placental tissue is provided, by
extracting human placenta, homogenizing the placenta, resulting in
a homogenate, centrifuging the homogenate in order to free the
supernatant of cellular debris, purifying the cell free-debris
through adsorption on a C-18 column; and filter sterilizing the
composition. The homogenized portion of the placenta may consist
substantially of, for example, chorionic tissue. A histone
deacetylase inhibitor can be added directly to the composition. In
additional embodiments, a the composition can be administered
intratumorally in a method of inducing differentiation and
reprogramming of distal cancer lesions. In additional embodiments,
a method of substantially increasing efficacy of the composition is
provided, by administration in a liposomal complex. A substantially
purified therapeutic composition can be prepared, for example, by
preparing a supernatant, subjecting the supernatant to solid phase
extraction to produce a product, subjecting the resulting product
to gel filtration to produce a product, subjecting the resulting
product to anion exchange fast phase liquid chromatography to
produce a product, subjecting the resulting product to amino-high
performance liquid chromatography to produce a product, and
subjecting the resulting product to reverse phase high performance
liquid chromatography to produce purified differentiation inducing
factor. The supernatant can be prepared, for example, by culturing
cord-blood derived stem cells to produce a factor-containing
supernatant, or by centrifugation of lysed placental extract.
DESCRIPTION OF FIGURES
[0021] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
becomes better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0022] FIG. 1 is a bar graph demonstrating the induction of HL-60
differentiation in vitro by co-culture with cord blood nucleated
cells.
[0023] FIG. 2 is a bar graph demonstrating the induction of HL-60
differentiation in vitro by co-culture with cord blood CD34+ stem
cells.
[0024] FIG. 3 is a bar graph demonstrating the induction of HL-60
differentiation by culture with placental extracts.
[0025] FIG. 4 is a bar graph demonstrating the induction of HL60
differentiation using cord blood extracts.
[0026] FIG. 5 is a bar graph demonstrating the inhibition of HL-60
proliferation by culture with placental extracts.
[0027] FIG. 6 is a bar graph demonstrating the inhibition of LNCaP
proliferation by culture with placental extracts.
[0028] FIG. 7 is a bar graph demonstrating the inhibition of PC-3
proliferation by culture with placental extracts.
[0029] FIG. 8 is a bar graph demonstrating the inhibition of Lewis
Lung Carcinoma cell proliferation by culture with placental
extracts.
[0030] FIG. 9 is a bar graph demonstrating the inhibition of lung
metastasis using placental extract in the Lewis Lung Carcinoma
murine model of lung cancer.
[0031] FIG. 10 is a table showing the inhibition of Lewis Lung
Carcinoma growth using placental extract.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Without intending to be limited by theory, it is also
hypothesized that factors present in the culture of cord blood stem
cells have the ability to induce a differentiation program in
cancer cells in effect, to begin a reprogramming process that
endows the cell an ability to make progeny with benign,
noncancerous, or semicancerous phenotype. Simultaneously or
subsequently, the cell encounters growth conditions or agents that
promote differentiation down another differentiation pathway. These
ideas are placed here for the interest of the skilled artisan, and
do not need to be understood to put the invention into practice.
The claimed invention is not restricted by mechanism of action, and
is limited only by the features explicitly stated, as interpreted
by one skilled in the art.
[0033] The invention described teaches methods of inducing the
differentiation of cancer cells in a host in need thereof through
administration of cells or factors derived from said cells that
possess stem cell like properties. Specifically, the
differentiation-inducing capabilities of the compositions described
are useful for the treatment of breast cancer, skin cancer, bone
cancer, prostate cancer, liver cancer, lung cancer, brain cancer,
cancer of the larynx, gallbladder, pancreas, rectum, parathyroid,
thyroid, adrenal, neural tissue, head and neck, colon, stomach,
bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of
both ulcerating and papillary type, metastatic skin carcinoma,
osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma,
giant cell tumor, small-cell lung tumor, islet cell tumor, primary
brain tumor, acute and chronic lymphocytic and granulocytic tumors,
hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma,
pheochromocytoma, mucosal neuronms, intestinal ganglloneuromas,
hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's
tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical
dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,
soft tissue sarcoma, malignant carcinoid, topical skin lesion,
mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic
and other sarcoma, malignant hypercalcemia, renal cell tumor,
polycythermia vera, adenocarcinoma, glioblastoma multiforma,
malignant melanomas, and epidermoid carcinomas.
[0034] As described herein, cells with stem cell activity, such as
cord blood cells, can be derived from human placental tissue. Thus,
the terms "placentally derived tissues" and "placentally derived
cells" can include cord blood cells.
[0035] In some embodiments, cells with stem cell like activity are
purified from human placenta using antibody mediated cell depletion
followed by positive selection. Methods of depleting lineage
positive cells are well known in the art and include the use of
antibodies to the lineage antigens (CD5, CD45R (B220), CD11b,
anti-Ly-6G (Gr-1), 7-4, and Ter-119) followed by complement
depletion. A less damaging protocol involves utilization of
magnetically bound antibodies and depletion by use of magnetic
separation. This protocol is well known in the art and is
commercially available using the Magnetic Activated Cell Sorting
(MACS) technology offered by Miltenyi Biotech. Further purification
of cells with stem cell activity can be performed by selection for
the stem cell antigen CD34. Selection can be accomplished again by
magnetic separation using the "positive selection" method, or
alternatively by fluorescent activated cell sorting (FACS). Other
methods of purifying cells with stem cell activity include use of
Wheat Germ Agglutinin, or selection for low density profile using a
chemical gradient such as Percoll.
[0036] Methods for expansion of stem cell populations have been
described. For example, U.S. patent application Ser. No. 11/353,692
filed on Feb. 14, 2006, entitled METHOD FOR EXPANSION OF STEM
CELLS, describes a method of increasing the growth of stem cells by
mixing the stem cells with a growth medium that has been
conditioned by an incubation with placental tissue. This method
increases the expansion of the stem cell population. This patent
application is incorporated by reference herein in its
entirety.
[0037] Cells with stem cell activity are recognized by ability to
reconstitute severe combined immunodeficiency mice with
multilineage human blood populations as determined by the species
specific marker CD45. Other methods of assessing the "stem cell"
ability of purified cord blood cells includes plating said cells in
methylcellulose in the presence of differentiation-inducing factors
and assessing for production of multilineage colonies. For example,
plating in the presence of G-CSF would induce formation of
granulocytes, plating in the presence of EPO would induce
erythrocyte formation, and plating in the presence of
thrombopoietic would induce colonies consisting of
megakaryocytes.
[0038] Upon purification of a stem cell population, the cells can
be administered directly into a cancer patient through parenteral
introduction, comprising intramuscular, intravenous, peritoneal,
subcutaneous, intradermal, as well as topical administration. A
preferred method of administration is intravenous, however, various
compositions of the cells can be derived by one skilled in the art.
For example, when treating a patient with melanoma, the purified
cord blood stem cells can be lyzed and admixed with transdermal
carrying agents for effective direct delivery. Transdermal carriers
include systems such as iontophoretic and sonophoretic systems,
thermosetting gels, and prodrugs. For enhanced uptake, absorption
promoters may be utilized. The absorption promoters may be selected
in particular, from propylene glycol, hexylene glycol, propylene
glycol dipelargonate, glyceryl monoethyl ether, diethylene glycol,
monoglycerides, monooleate of ethoxylated glycerides (with 8 to 10
ethylene oxide units), Azone (1-dodecylazacycloheptan-2-one),
2-(n-nonyl)-1,3-dioxolane, isopropylmyristate, octylmyristate,
dodecyl-myristate, myristyl alcohol, lauryl alcohol, lauric acid,
lauryl lactate, terpinol, 1-menthol, d-limonene,
.beta.-cyclodextrin and its derivatives or surfactants such as
polysorbates, sorbitan esters, sucrose esters, fatty acids, bile
salts, or alternatively lipophilic and/or hydrophilic and/or
amphiphilic products such as poly-glycerol esters,
N-methylpyrrolidone, polyglycosylated glycerides and cetyl lactate.
The absorption promoter preferably represents from 5 to 25% of the
weight of the composition. Further description of absorption
promoters appears in U.S. Pat. No. 6,538,039, which is hereby
incorporated by reference in its entirety.
[0039] In terms of administrating the cells through the intravenous
route, several modifications can be made to the cells to increase
efficacy, this includes transfection with suicide genes, immune
stimulatory genes, or genes which encode pro-drug activating
enzymes. Additionally, the cord blood stem cells may be labeled in
order to detect presence of tumor cells.
[0040] Typical administration of cells is performed based on the
therapeutic need of the patient. In a standard embodiment, cord
blood stem cells are infused intravenously in a physiologically
suitable solution such as albumin with saline. Cell concentrations
may range from 10.sup.3-10.sup.9 CD34+ cells/kg. Frequency may
include weekly, daily, or depending on status of tumor growth as
determined by biomarkers, imaging or patient general medical
condition.
[0041] Another embodiment deals with using placental extracts as a
heterogeneous starting point for preparation of a pharmaceutical
agent. This embodiment uses full term, healthy, pathogen free
chorions obtained from deliveries or cesarean sections deliveries.
Many methods exist for preparation of cell lysate. Cell lysis
procedures were extensively described in US Patent Publication No.
2003/0211603, hereby incorporated by reference in its entirety.
Procedures described therein are modified for cord blood stem
cells. For example, cell lysate or extract can be prepared from
cord blood stem cell cultures or bone marrow stem cell cultures.
Before lysing, the cells typically are allowed to recover after the
last passage by culturing for about 2-3 days in standard medium
until the culture is at least about 50% confluent. The cells can be
lysed directly in the culture dish, for example, by replacing the
medium with a solubilizing liquid, or by repeated freeze-thawing.
Alternatively, the cells can be resuspended from the culture
surface before lysis, for example, by brief collagenase digestion,
or by scraping. The resuspended cells are collected, for example,
by centrifugation, and then lysed by adding a suitable solvent, by
freeze-thawing, by shearing through a narrow-gauge needle or in a
tissue grinder, by sonicating, by mechanical homogenization, or by
any other suitable method. In certain circumstances, subcellular
organelles can be removed or enriched, or membrane fractions can be
prepared, according to standard methods. Techniques in
subfractionating cells to produce cell components and extracts can
be found in Storrie et al., Meth. Enzymol. 182:203, 1990; and in
Subcellular Fractionation: A Practical Approach (Grahan &
Rickwood, eds., Oxford, 1997), both of which are hereby
incorporated by reference in their entirety. Viscosity of the cell
extract caused by long-chain nucleic acids can be reduced by
treating with DNAse, or other appropriate nucleases. Non-ionic
detergents with a high critical micelle concentration (such as
sodium deoxycholate) can be removed by dialysis. Other detergents
(such as Triton.TM. X-100, octyl glucoside, or Nonidet.TM.-P40) can
be removed, for example, on adsorbent beads or chromatography
columns. Large particulates can be removed, for example, by
centrifugation or microfiltration. As another option, high
molecular weight solutes can be concentrated from the clarified
extract (for example, by microfiltration, salt precipitation,
column chromatography, or lyophilization).
[0042] In accordance with a further embodiment, a method is
provided for preparing a purified cancer differentiation inducing
factor comprising the steps of
[0043] (a) culturing cord-blood derived stem cells to produce a
factor-containing supernatant;
[0044] (b) subjecting the supernatant to solid phase extraction to
produce a product;
[0045] (c) subjecting the product from step (b) to gel filtration
to produce a product;
[0046] (d) subjecting the product from step (c) to anion exchange
fast phase liquid chromatography to produce a product;
[0047] (e) subjecting the product from step (d) to amino-high
performance liquid chromatography to produce a product; and
[0048] (f) subjecting the product from step (e) to reverse phase
high performance liquid chromatography to product purified
differentiation inducing factor.
[0049] Bone marrow, either fetal or adult, human, or from other
animals may also be used as a source of stem cells for purification
of the differentiation inducing composition. For example, human
fetal bones may be dissected from 21- to 24-week-old fetuses
obtained by elective abortion with approved consent (i.e., from
Anatomic Gift Foundation, White Oak, Ga.). To purify human HSCs, BM
cell suspensions are prepared by flushing split long bones with
RPMI 1640 containing 2% heat-inactivated FCS (Gemini Bio-Products,
Inc, Calabasas, Calif.). Low-density (<1.077 g/mL) mononuclear
cells are isolated (Lymphoprep; Nycomed Pharma, Oslo, Norway) and
washed twice in staining buffer (SB) consisting of Hanks'balanced
salt solution (HBSS) with 2% heat-inactivated FCS and 10 mmol/L
HEPES. Samples are then incubated for 10 minutes with 1 mg/mL
heat-inactivated human gammaglobulin (Gamimune; Miles Inc, Elkhart,
Ind.) to block Fe receptor binding of mouse antibodies. Fluorescein
isothiocyanate (FITC)-labeled CD34 monoclonal antibodies (MoAbs)
and phycoerythrin (PE)-labeled thy-1 MoAbs are then added at 0.5 to
1 .mu.g/10.sup.6 cells in 0.1 to 0.3 mL SB for 20 minutes on ice.
Control samples are incubated in a cocktail of FITC-labeled and
PE-labeled isotype-matched MoAbs. Cells are washed twice in SB,
resuspended in SB containing 1 .mu.g/mL propidium iodide (Molecular
Probes Inc, Eugene, Oreg.), and sorted using the tri-laser
fluorescence-activated cell sorter (FACS) MoFlo (Cytomation, Inc,
Fort Collins, Colo.). Live cells (ie, those excluding propidium
iodide) are always greater than 95%. Sort gates are set based on
the mean fluorescence intensity of the isotype control sample.
Cells are collected in 12- or 24-well plates in RPMI 1640
containing 10% FCS and 10 mmol/L HEPES, counted, and reanalyzed for
purity in every experiment. Typically, 450,000 to 500,000 CD34+
thy-1.sup.+ cells are obtained from a single donor. MoAbs for CD34
are purchased from Becton Dickinson (Mountain View, Calif.). MoAbs
for thy-1 and isotype controls are purchased from Pharmingen (San
Diego, Calif.).
[0050] Using both the placental source described above or the bone
marrow source of differentiation-inducing activity, concentration
of this activity can be performed using solid phase extraction.
C.sub.18 cartridges (Mini-Spe-ed C18-14%, S.P.E. Limited, Concord
ON) are prepared by washing with 10 ml of methanol followed by 10
ml 18 megaohm/cm deionized-distilled water. Up to 100 ml of
supernatants of cultured cord blood stein cells are passed through
each cartridge before elution. After washing the cartridges with 5
ml of deionized-distilled water, material adsorbed to the C.sub.18
cartridge is eluted with 3 ml methanol, evaporated under a stream
of nitrogen, redissolved in a small volume of methanol, and stored
at 4.degree. C. Before testing the eluate for differentiation
inducing activity in vitro, the methanol is evaporated under
nitrogen and replaced by culture medium.
[0051] C.sub.18 cartridges can be used to adsorb small hydrophobic
molecules from the cord blood stem cell culture supernatant,
allowing the elimination of salts and other polar contaminants.
EXAMPLES
[0052] The following examples are offered to illustrate, but not to
limit, the claimed invention.
1. CD34+ Cell Purification
[0053] Cord blood was obtained from consenting mothers undergoing
cesarean delivery of healthy, full-term infants, and low-density
(less than 1.077 g/ml) cells were isolated by centrifugation on
Ficoll-Hypaque. CD34.sup.+ cell-enriched populations (65-98%
CD34.sup.+ cells) were obtained by removal of lineage
marker-positive cells using a column (n=2); and positive (EasySep)
selection using magnetic beads (n=1; StemCell Technologies Inc.).
Cells were stimulated overnight for in vivo experiments and for 48
hours for in vitro experiments at densities less than or equal to
2.times.10.sup.5 cells/ml in Iscove's medium supplemented with 1%
BSA, 10 .mu.g/ml bovine pancreatic insulin, and 200 .mu.g/ml human
transferrin (BIT; StemCell Technologies Inc.), 10.sup.-4 mol
2-mercaptoethanol, 2 mM glutamine, 100 ng/ml FL (Immunex Corp.),
100 ng/ml SF, 50 ng/ml Tpo (Genentech Inc.), and 100 ng/ml hIL-6
(provided by S. Rose-John, Christian-Albrechts University, Kiel,
Germany). The following day, the cells were pelleted, resuspended
in fresh growth factor-supplemented medium with 5 .mu.g/ml
protamine sulfate and 0.5.times.10.sup.8 to 5.times.10.sup.8
infectious units/ml (MOI=9-140; 140 in experiment 1, 9 and 90 in
experiment 2), placed in a 24-well plate coated with 2
.mu.g/cm.sup.2 Retronectin (Takara Shuzo Co.) or with 5
.mu.g/cm.sup.2 fibronectin (Sigma-Aldrich), and then incubated at
37.degree. C. for 6 hours.
2. Generation of Placental Lysate
[0054] Term placentas were harvested and the chorion was
mechanically separated under sterile conditions. Placentas were
collected in ice-cold Dutch modification of RPMI 1640 (Sigma, St.
Louis, Mo.) supplemented with 10% FCS, 2 mM L-glutamine, gentamicin
(25 .mu.g/ml), and penicillin/streptomycin (100 U/ml) (complete
medium). They were incubated with occasional agitation for 20 min
at room temperature in calcium- and magnesium-free HBSS (Life
Technologies, Paisley, U.K.) containing 1 mM DTT (Sigma). Purified
cell homogenate was prepared by collagenase digestion. Briefly,
bacterial collagenase (Advance Biofactures, Lynbrook, N.Y.)
digestion was sequentially performed, 4 .mu.l of 50 mM calcium
acetate was added, followed by 3 .mu.l (3 units) of bacterial
collagenase. Reactions were then incubated for another 60 min at
37.degree. C. Heparitinase digestions (15 .mu.l) were carried out
for 90 min at 37.degree. C. in a pH 7.0 buffer consisting of 100 mM
sodium acetate, 10 mM calcium acetate, using 5 milliunits of
enzyme. In those reactions where bacterial collagenase digestion
was sequentially performed, 4 .mu.l of 5.times. collagenase buffer
(250 mM Tris-HCl, pH 7.2, 50 mM calcium acetate) was added,
followed by 3 .mu.l (3 units) of bacterial collagenase. Reactions
were then incubated for another 60 min at 37.degree. C. Purified
cells were subsequently sonicated, centrifuged at 10,000 g for 3
hours, and supernatant was collected, filter sterilized, and
quantified for protein content using the Bradfort Assay.
3. Induction of HL-60 Differentiation in vitro Using Cord Blood
Co-Culture
[0055] As demonstrated in FIG. 1, coculture of 100,000 HL-60 human
myelomonocytic leukemia cells with increasing numbers of cord blood
nucleated cells resulted in an increased proportion of HL-60 cells
differentiating into adherent monocytic cells. HL-60 cells were
plated in 96 well plates at a concentration of 100,000 cells per
well. The addition of cord blood nucleated cells was performed at
the concentration indicated in the figure. The percentage of
adherent cells was determined by a blinded observer. The fact that
the HL-60 were truly differentiating was attested to by
morphological changes (data not shown).
4. Induction of HL-60 Differentiation in vitro Using Purified Cord
Blood CD34+ Cells
[0056] As demonstrated in FIG. 2, coculture of 100,000 HL-60 human
myelomonocytic leukemia cells with increasing numbers of CD34+
purified cord blood nucleated cells resulted in an increased
proportion of HL-60 cells differentiating into adherent monocytic
cells. HL-60 cells were plated in 96 well plates at a concentration
of 100,000 cells per well. The addition of cord blood CD34+ cells
was performed at the concentration indicated in the figure. The
percentage of adherent cells was determined by a blinded observer.
The fact that the HL-60 were truly differentiating was attested to
by morphological changes (data not shown).
5. Induction of HL-60 Differentiation by Culture with Cord Blood
Extracts
[0057] Cord blood extracts were prepared by culturing nucleated
cells in Hanks media for 48 hours and the supernatant was
concentrated using Solid Phase Extraction. C.sub.18 cartridges
(Mini-Spe-ed C18-14%, S.P.E. Limited, Concord ON) were prepared by
washing with 10 ml of methanol followed by 10 ml 18 megaohm-cm
deionized-distilled water. Up to 100 ml of supernatants of cultured
cord blood stem cells are passed through each cartridge before
elution. After washing the cartridges with 5 ml of
deionized-distilled water, material adsorbed to the C.sub.18
cartridge is eluted with 3 ml methanol, evaporated under a stream
of nitrogen, redissolved in a small volume of methanol, and stored
at 4.degree. C. Before testing the eluate for differentiation
inducing activity in vitro, the methanol is evaporated under
nitrogen and replaced by culture medium. Before addition to the
media, however, the concentration of C.sub.18 extract was
quantified using the Bradfort assay. The indicated concentration of
cord blood extract was added to 100,000 proliferating HL-60 cells
per well. As shown in FIG. 3, and increased number of
differentiated HL-60 cells was observed after 48 hours.
6. Induction of HL-60 Differentiation by Placental Extracts
[0058] Placental extracts were derived and quantified as described
above. The indicated concentration of cord blood extract was added
to 100,000 proliferating HL-60 cells per well. As shown in FIG. 4,
an increased number of differentiated HL-60 cells was observed
after 48 hours. The extent of differentiation was dependent on the
concentration of placental extract added.
7. Inhibition of Cancer Cell Proliferation by Culture with
Placental Extracts
[0059] HL-60, LNCaP, PC-3 and Lewis Lung carcinoma cells
(1.times.10.sup.5/well) were plated in 96 well plates in 200 .mu.l
of RPMI 1640 (Life Technologies) supplemented with 10% FCS (Life
Technologies), 100 U/ml of penicillin (Life Technologies), and 100
.mu.g/ml of streptomycin (Life Technologies). Cells were cultured
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 for 3
days in the presence of the indicated about of placental extract,
and pulsed with 1 .mu.Ci of [.sup.3H]thymidine (Amersham Pharmacia
Biotech) for the last 16 h of culture. Cells were harvested onto
glass fiber filters, and the radioactivity incorporated was
quantitated using a Wallac Betaplate liquid scintillation counter.
Results were expressed as the mean cpm of triplicate
cultures.+-.SEM. As seen in FIGS. 5-8, a dose-dependent inhibition
of tumor cell proliferation was noted in all 3 in vitro
experimental tumor systems.
8. Inhibition of Cancer Cell Metastasis by Placental Extract
[0060] Lewis lung carcinoma (ATCC) were propagated by sequential
subcutaneous transplantation in C57BL/6 mice. A single-cell
suspension of tumor cells was prepared by mincing the tumor,
followed by passage of the suspension first through a stainless
steel mesh and then through a series of hypodermic needles of
increasing gauge. This suspension of cells was aliquoted and
frozen. For each series of injections, an aliquot of frozen tumor
cells was thawed and expanded by culturing for 3 passages in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf
serum, penicillin, and streptomycin. Be fore injection, the tumor
cells were detached by trypsinization, washed once in cold DMEM
containing 10% fetal calf serum, and once in cold serum-free
medium. Cell viability was usually greater than 85%, as determined
by trypan blue exclusion. The dorsal skin of mice was shaved 1 day
before injection. The mice were anesthetized by Metofane
inhalation, and tumor cells (500,000) trypan blue-excluding cells
in 100 .mu.L) were injected into the dorsal subcutis using a
27-gauge needle.
[0061] Three weeks after tumor inoculation quantitation of lung
metastasis was performed. Mice were killed using CO2 narcosis, and
the lungs were removed, rinsed in phosphate-buffered saline (PBS),
and placed in Bouin's fixative for at least 24 hours. The fixed
lungs were carefully separated into individual lobes with forceps,
and the number of surface metastases (appearing as white foci
against a yellow background) was counted for each lobe using a
dissecting microscope at 4.times.magnification (total metastatic
foci) or by the naked eye (large metastatic foci).
[0062] Two treatment protocols were used. In the first one, lug of
placental extract was administered 1 day prior to tumor cell
inoculation, in the second protocol 1 ug of placental extract was
administered on days -1, 1, 4, and 7 in reference to tumor cell
inoculation. As seen in FIG. 9, protocol 2 was most effective at
decreasing the amount of visible tumor foci.
9. Inhibition of Tumor Growth by Placental Extract
[0063] C57/BL6 mice were inoculated with Lewis Lung Carcinoma cells
as described in the above section. Tumor growth was quantitated by
a blinded observer using calipers. The size of the tumor is
represented in FIG. 10. As observed, the mice treated with Protocol
2 (described above) resulted in a significant inhibition of tumor
growth. Although not illustrated, the survivors had a dense,
fibrous scar-like tissue where the tumor used to be.
[0064] It will be apparent to one skilled in the art that varying
substitutions and modifications can be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0065] Those skilled in the art recognize that the aspects and
embodiments set forth herein can may be practiced separate from
each other or in conjunction with each other. Therefore,
combinations of separate embodiments are within the scope as
disclosed herein.
[0066] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0067] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions indicates the exclusion of equivalents of the
features shown and described or portions thereof. It is recognized
that various modifications are possible within the scope of the
invention disclosed. Thus, it should be understood that although
the present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the disclosure.
[0068] The following references are incorporated herein by
reference in their entireties.
REFERENCES
[0069] 1. Attar, E. C. and D. T. Scadden, Regulation of
hematopoietic stem cell growth. Leukemia, 2004. 18(11): p. 1760-8.
[0070] 2. Ema, H. and H. Nakauchi, Self-renewal and lineage
restriction of hematopoietic stem cells. Curr Opin Genet Dev, 2003.
13(5): p. 508-12. [0071] 3. Craig, C. E., et al., The
histopathology of regeneration in massive hepatic necrosis. Semin
Liver Dis, 2004. 24(1): p. 49-64. [0072] 4. Kaur, P., et al.,
Keratinocyte stem cell assays: an evolving science. J Investig
Dermatol Symp Proc, 2004. 9(3): p. 238-47. [0073] 5. Rookmaaker, M.
B., et al., Progenitor cells in the kidney: biology and therapeutic
perspectives. Kidney Int, 2004. 66(2): p. 518-22. [0074] 6.
Chaudhari, M., et al., Pancreatic stem cells: a therapeutic agent
that may offer the best approach for curing type I diabetes.
Pediatr Diabetes, 2001. 2(4): p. 195-202. [0075] 7. Brittan, M. and
N. A. Wright, Gastrointestinal stem cells. J Pathol, 2002. 197(4):
p. 492-509. [0076] 8. Martino, G., Flow the brain repairs itself:
new therapeutic strategies in inflammatory and degenerative CNS
disorders. Lancet Neurol, 2004. 3(6): p. 372-8. [0077] 9. Liu, L.,
et al., Epigenetic regulation of human telomerase reverse
transcriptase promoter activity during cellular differentiation.
Genes Chromosomes Cancer, 2004. 41(1): p. 26-37. [0078] 10.
Lodygin, D., J. Diebold, and H. Hermeking, Prostate cancer is
characterized by epigenetic silencing of 14-3-3sigma expression.
Oncogene, 2004. 23(56):9034-9041. [0079] 11. Mhawech, P., et al.,
Downregulation of 14-3-3sigma in ovary, prostate and endometrial
carcinomas is associated with CpG island methylation. Mod Pathol,
2004. 18(3):340-348. [0080] 12. de Vries, E. G., et al., The happy
destiny of frozen haematopoietic stem cells: from immature stem
cells to mature applications. Eur J Cancer, 2004. 40(13): p.
1987-92. [0081] 13. Rocha, V., G. Sanz, and E. Gluckman, Umbilical
cord blood transplantation. Curr Opin Hematol, 2004. 11(6): p.
375-385. [0082] 14. Michel, G., et al., Unrelated cord blood
transplantation for childhood acute myeloid leukemia: a Eurocord
Group analysis. Blood, 2003. 102(13): p. 4290-7. [0083] 15. Hall,
J. G., et al., Unrelated umbilical cord blood transplantation for
an infant with beta-thalassemia major. J Pediatr Hematol Oncol,
2004. 26(6): p. 382-5.
* * * * *