COMPOSITION AND METHOD FOR TREATING CELL PROLIFERATION

DISORDER

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/565,256, filed on September 29, 2017, which is herein incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Any abnormal proliferation of cells is called a tumor, which can be benign or malignant.

While a benign tumor remains confined to its original location, a malignant tumor can invade surrounding normal tissue and spread to other parts of the body. Malignant tumors are called cancer. Unregulated cell proliferation is one of the hallmarks of cancer. Signals that control normal cell growth have no or reduced effect on cancer cells such that cancer cells grow and divide in an uncontrolled manner, and invade normal tissues and organs via the circulatory or lymphatic systems. In 2015 about 90 million people worldwide had cancer and about 9 million people died from cancer.

SUMMARY OF THE INVENTION

[0003] As recognized herein, cardiac derived stromal cells can inhibit the growth of tumor cells.

Cardiac derived stromal cells can be cytotoxic to tumor cells. Cardiac derived stromal cells can treat a cell proliferation disorder. Disclosed herein are compositions, formulations, methods, and kits for treating a cell proliferation disorder.

[0004] In one aspect, disclosed herein is a method of treating a cell proliferation disorder,

comprising: administering to a subject an effective amount of a composition comprising isolated cardiac derived stromal cells, thereby reducing cell proliferation of the cell proliferation disorder in the subject. In some embodiments, the treating comprises contacting proliferating cells of the cell proliferation disorder with the composition. In some embodiments, the subject has or is suspected of having the cell proliferation disorder. In some embodiments, the subject is in need of the treating. In some

embodiments, the subject is a human subject.

[0005] In some embodiments, the cell proliferation disorder is a cancer. In some embodiments, the cancer is selected from the group consisting of synovioma, mesothelioma, brain tumor, breast cancer, triple negative breast cancer, Ewing's tumor, Wilms' tumor, cervical cancer, chordoma, colon carcinoma, esophageal cancer, liver cancer,

gastrointestinal cancer, head and neck cancer, hepatic cancer, kidney cancer, ovarian cancer, pancreatic cancer, plasmacytoma, prostate cancer, retinal cancer, skin cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas,

cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, epithelial lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, lymphocytic leukemia, large granular lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm's macroglobulinemia, heavy chain disease, lymphoblastic leukemia, T- cell leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cell leukemia, B- lymphocytic leukemia, mixed cell leukemias, myeloid leukemias, myelocytic leukemia, myelogenous leukemia, neutrophilic leukemia, eosinophilic leukemia, monocytic leukemia, myelomonocytic leukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia, promyelocytic leukemia, pancreatic carcinoma, pancreatic ductal

adenocarcinoma, and glioblastoma. In some embodiments, the subject is experiencing a recurrence or relapse of the cancer.

[0006] In some embodiments, the administering comprises transdermal administration,

intravenous, intradermal, subcutaneous, transcutaneous, intramuscular, intrathecal, intraperitoneal, intranasal, pulmonary, implanted administration, or any combination thereof. In some embodiments, the cell proliferation disorder is angiogenesis or the cell proliferation disorder is mediated by angiogenesis.

[0007] In some embodiments, the method further comprises: administering an additional cancer therapy. In some embodiments, the additional cancer therapy comprises surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, or any combination thereof.

[0008] In some embodiments, the method further comprises: prior to the administering,

obtaining the isolated cardiac derived stromal cells from myocardial tissue. In some embodiments, the method further comprises: prior to the administering, expanding the isolated cardiac derived stromal cells in culture. [0009] In some embodiments, the isolated cardiac derived stromal cells inhibit tumor cell proliferation. In some embodiments, the tumor cell comprises leukemic cell, lymphoma cell, myeloma cell, triple negative breast cancer cell, or a combination thereof. In some embodiments, the lymphoma cell is HL-60, K562 or Raji. In some embodiments, the myeloma cell is U-266, RPMI8266 or ARP-1. In some embodiments, the triple negative breast cancer cell is HIM3 p53 expressing cell line or HIM3 p53 knockout cell line. In some embodiments, the tumor cell does not proliferate after the isolated cardiac derived stromal cells are removed from the tumor cell.

[0010] In some embodiments, the isolated cardiac derived stromal cells express CD 105, CD90 and CD73. In some embodiments, the isolated cardiac derived stromal cells do not express CD45 or CD34. In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of genes of selected cytokines and cytokine receptors than bone marrow mesenchymal stem cells, wherein the selected cytokines and cytokine receptors comprises IL10RB, T FRSF11B, TGFBR2, T FRSF12A, IFNGR2, FAS, PDGFRA, CXCL16, GHR, VEGFC, CSF1R, CSF1R, T FRSF14, CCL26, MET, 1L11RA, LEPR, or any combination thereof. In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of genes of selected cell adhesion molecules than bone marrow mesenchymal stem cells, where the selected cell adhesion molecules comprises ITGB 1, VCAM1, CD99, HLA-F, HLA-E, ICAM3, ICAM2, HLA-DMA, FASC, or any combination thereof. In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of selected focal adhesion molecules than bone marrow mesenchymal stem cells, wherein the selected focal adhesion molecules comprises PIK3CD, MYLK, CC D2, LAMA5, MYLPF, ITGA1, TNC, or any combination thereof.

[0011] In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of selected micro RNA than bone marrow mesenchymal stem cells, wherein the selected micro RNA comprises miR-1, miR-lOa, miR-15a, miR-15b, miR-16-1, miR-18a, miR- 19b-l, miR-20a, miR-26a-2, miR-33a, miR-92a-l, miR-133a, miR-206, miR-374a, miR- 411, miR-424, miR-450a, miR-450b-5p, miR-503, miR-516a-3p, miR-542-3p, miR- 551b, miR-589, miR-770-5p, miR-935, or any combination thereof. In some

embodiments, the isolated cardiac derived stromal cells express one-half or less of selected micro RNA than bone marrow mesenchymal stem cells, wherein the selected micro RNA comprises miR-lOb, miR-335, miR-451, miR-479, miR-628-3p, miR-768- 3p, or any combination thereof. [0012] In some embodiments, the isolated cardiac derived stromal cells are mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are allogenic. In some embodiments, the mesenchymal stem cells are autologous. In some embodiments, the isolated cardiac derived stromal cells are isolated from myocardial tissue. In some embodiments, the isolated cardiac derived stromal cells are derived from myocardial tissue

[0013] In some embodiments, the composition further comprises a growth factor, a

differentiation factor, a regeneration factor, or any combination thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a pharmaceutically acceptable liquid medium for injection.

[0014] In another aspect, disclosed herein is a composition, comprising: (a) isolated cardiac derived stromal cells; and (b) a pharmaceutically acceptable carrier. In some

embodiments, the isolated cardiac derived stromal cells express CD 105, CD90 and CD 73. In some embodiments, the isolated cardiac derived stromal cells do not express CD45 or CD34.

[0015] In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of genes of selected cytokines and cytokine receptors than bone marrow mesenchymal stem cells, wherein the selected cytokines and cytokine receptors comprises IL10RB, T FRSF11B, TGFBR2, TNFRSF12A, IFNGR2, FAS, PDGFRA, CXCL16, GHR, VEGFC, CSF1R, CSF1R, T FRSF14, CCL26, MET, 1L11RA, LEPR, or any combination thereof. In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of genes of selected cell adhesion molecules than bone marrow mesenchymal stem cells, where the selected cell adhesion molecules comprises ITGB 1, VCAM1, CD99, HLA-F, HLA-E, ICAM3, ICAM2, HLA-DMA, FASC, or any combination thereof. In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of selected focal adhesion molecules than bone marrow mesenchymal stem cells, wherein the selected focal adhesion molecules comprises PIK3CD, MYLK, CC D2, LAMA5, MYLPF, ITGA1, TNC, or any combination thereof.

[0016] In some embodiments, the isolated cardiac derived stromal cells express 2 fold or more of selected micro RNA than bone marrow mesenchymal stem cells, wherein the selected micro RNA comprises miR-1, miR-lOa, miR-15a, miR-15b, miR-16-1, miR-18a, miR- 19b-l, miR-20a, miR-26a-2, miR-33a, miR-92a-l, miR-133a, miR-206, miR-374a, miR- 411, miR-424, miR-450a, miR-450b-5p, miR-503, miR-516a-3p, miR-542-3p, miR- 551b, miR-589, miR-770-5p, miR-935, or any combination thereof. In some

embodiments, the selected micro RNA is miR-206. In some embodiments, the isolated cardiac derived stromal cells express 500 fold or more of miR-206 than the bone marrow mesenchymal stem cells. In some embodiments, the isolated cardiac derived stromal cells express one-half or less of selected micro RNA than bone marrow mesenchymal stem cells, wherein the selected micro RNA comprises miR-lOb, miR-335, miR-451, miR-479, miR-628-3p, miR-768-3p, or any combination thereof.

[0017] In some embodiments, the isolated cardiac derived stromal cells are allogenic. In some embodiments, the isolated cardiac derived stromal cells are autologous. In some embodiments, the composition further comprises: a growth factor, a differentiation factor, a regeneration factor, or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier is a pharmaceutically acceptable liquid medium for injection. In some embodiments, the composition further comprises: at least one additional anti-cancer agent.

[0018] In some embodiments, at least a portion of the isolated cardiac derived stromal cells is viable. In some embodiments, the at least a portion is at least about 80% of a total cell number of the composition.

[0019] In some embodiments, the composition further comprises: a viscosity modifying

component, wherein the viscosity modifying component comprises a polyol, a sugar, a derivative of any of these, a salt of any of these, or any combination thereof. In some embodiments, the composition further comprises a buffer solution. In some

embodiments, the buffer solution is phosphate-buffered saline or plasmaLyte. In some embodiments, the composition further comprises a carrier protein. In some embodiments, the carrier protein is human serum albumin or a derivative thereof.

[0020] In another aspect, disclosed herein is a process, comprising: forming a composition by combining (a) isolated cardiac derived stromal cells; and (b) a pharmaceutically accepted carrier. In some embodiments, the process further comprises: adding a preservative, an anti-irritant, or a combination thereof to the composition. In some embodiments, the process further comprises: treating the composition with a solution comprising at least one selected from the group consisting of: an antibiotic, an antimycotic, and a

combination thereof. INCORPORATION BY REFERENCE

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be

incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the invention are set forth with particularity in the appended

claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0023] FIG. 1 exhibits a non-limiting illustration of a process of using cardiac derived stromal cells to treat a cell proliferation disorder.

[0024] FIG. 2 shows morphology of stromal cell lines. Stromal cells obtained from bone

marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel) are seeded in flasks, then evaluated by light microscopy (magnification 200x)

[0025] FIG. 3 displays phenotypic analysis of stromal cell lines. Flow cytometry histograms of bone marrow mesenchymal stem cells (BMMSC, first column), adipose stromal cells (second column), liver stromal cells (third column), and heart stromal cells (fourth column) are displayed with appropriate isotype control staining (open histogram) versus specific antibody staining (solid histogram) for phenotype markers of CD 105 (first row), CD90 (second row), CD73 (third row), CD45 (fourth row), and CD 34 (fifth row).

[0026] FIG. 4 depicts adipogenic differentiation potential of stromal cell lines. Stromal cells obtained from bone marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel) accumulated as intracellular lipid droplets are determined by oil red O staining, then evaluated by light microscopy (magnification 200x).

[0027] FIG. 5 shows osteogenic differentiation potential of stromal cell lines. Stromal cells obtained from bone marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel) having extracellular calcium deposition are demonstrated by positive Alizarin Red S staining, then evaluated by light microscopy (magnification 200x). [0028] FIG. 6 displays culture of HL-60 cells co-cultured on bone marrow mesenchymal stem cells for 11 days.

[0029] FIG. 7 depicts culture of HL-60 cells co-cultured on cardiac derived mesenchymal stem cells for 11 days.

[0030] FIG. 8 shows proliferation and viability of K562 cells cultured with stromal cell lines.

Panel A shows K562 cells cultured on stromal cell lines of bone marrow mesenchymal stem cells (Panel A, top left), adipose stromal cells (Panel A, top right), liver stromal cells (Panel A, bottom left), and heart stromal cells (Panel A, bottom right) as observed under a light microscope on day 14 of culture (magnification 200). Panel B shows K562 total cell number after 14 days of culture. Panel C shows K562 cell viability (shown as % viability of total cells) after 14 days of culture.

[0031] FIG. 9 displays culture of tumor cell line U266 with two different batches of bone

marrow mesenchymal stem cells or cardiac derived stromal cells conditioned media (20%).

[0032] FIG. 10 depicts culture of tumor cell line ARP-1 with two different batches of bone marrow mesenchymal stem cells or cardiac derived stromal cells conditioned media (20%).

[0033] FIG. 11 shows culture of tumor cell line RPMI-8266 with two different batches of bone marrow mesenchymal stem cells or cardiac derived stromal cells conditioned media (20%).

[0034] FIG. 12 displays comparison of microRNA levels in cardiac derived stromal cells versus bone marrow mesenchymal stem cells by array analysis and real time PCR confirmation.

[0035] FIG. 13 depicts cord blood mononuclear cells (MNCs) co-culture on stroma. Panel A depicts total cell count of cord blood cells after 14 days co-culture. Panel B depicts total colony forming units specific for granulocyte/macrophage progenitors (CFU-GM) colonies from co-cultured progeny after 14 days co-culture.

[0036] FIG. 14 shows the growth of K562 leukemia cells after 7 days. Panel A depicts total cell count of tumor cell line K562 in the absence and presence of cardiac derived stromal cells. Panel B depicts total cell count of tumor cell line K562 over concentrated media (CM) conditioned by various amount of cardiac derived stromal cells.

[0037] FIG. 15 displays the inhibitory activities of concentrated media conditioned by cardiac derived stromal cells under different treatment conditions.

[0038] FIG. 16 depicts the inhibitory activity of concentrated media conditioned by cardiac derived stromal cells before and after treatment with trypsin. [0039] FIG. 17 shows the growth of K562 leukemia cells over time in concentrated media conditioned by mesenchymal stromal cells or cardiac derived stromal cells before and after washing.

[0040] FIG. 18 displays the growth of triple negative breast cancer (TNBC) cells in the presence or absence of cardiac derived stromal cells.

[0041] FIG. 19 depicts culture of triple negative breast cancer (TNBC) cells cultured in control and co-cultured with cardiac derived stromal cells.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Disclosed herein are compositions, formulations, methods, and kits for treating a cell proliferation disorder.

Definitions

[0043] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" can be intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof can be used in either the detailed description and/or the claims, such terms can be intended to be inclusive in a manner similar to the term "comprising".

[0044] The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean about plus or minus 10%, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%) of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2- fold, of a value. Where particular values may be described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. Also, where ranges and/or subranges of values are provided, the ranges and/or subranges can include the endpoints of the ranges and/or subranges.

[0045] The term "substantially" as used herein can refer to a value approaching 100% of a given value. For example, an active agent that is "substantially localized" in an organ can indicate that about 90% by weight of an active agent, salt, or metabolite can be present in an organ relative to a total amount of an active agent, salt, or metabolite. In some cases, the term can refer to an amount that can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some cases, the term can refer to an amount that can be about 100% of a total amount.

[0046] The term "subject", "patient" or "individual" as used herein can encompass a mammal and a non-mammal. A mammal can be any member of the Mammalian class, including but not limited to a human, a non-human primates such as a chimpanzee, an ape or other monkey species; a farm animal such as cattle, a horse, a sheep, a goat, a swine; a domestic animal such as a rabbit, a dog (or a canine), and a cat (or a feline); a laboratory animal including a rodent, such as a rat, a mouse and a guinea pig, and the like. A non- mammal can include a bird, a fish and the like. In some embodiments, a subject can be a mammal. In some embodiments, a subject can be a human. In some instances, a human can be an adult. In some instances, a human can be a child. In some instances, a human can be age 0-18 years old. In some instances, a human can be age 18-130 years. In some instances, a subject can be a female. In some instances, a subject can be diagnosed with, or can be suspected of having, a condition or disease. A subject can be a patient. A subject can be an individual. In some instances, a subject, patient or individual can be used interchangeably.

[0047] The term "preventing" can mean preventing additional symptoms, ameliorating or

preventing the underlying metabolic causes of symptoms, and can include prophylaxis.

[0048] In some instances, "treat," "treating", "treatment," "ameliorate" or "ameliorating" and other grammatical equivalents can include prophylaxis. "Treat," "treating", "treatment," "ameliorate" or "ameliorating" and other grammatical equivalents can further include achieving a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit can mean eradication of the underlying disease being treated. Also, a therapeutic benefit can be achieved with the eradication of one or more of the physiological symptoms associated with the underlying disease such that an improvement can be observed in a subject notwithstanding that, in some embodiments, the subject can still be afflicted with the underlying disease.

[0049] The terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" as used herein, can refer to a sufficient amount of a compound being administered which will at least partially ameliorate a symptom of a disease or condition being treated.

[0050] The terms "compound", "agent", "active agent", or "active ingredient" can be used to refer to a drug or therapeutic as described herein. In some cases, the terms "additional compound", "additional agent", or "additional therapeutic agent" can be used

interchangeably to refer to other active compounds, agents, or therapeutics that may be used in a composition described herein.

[0051] The terms "administer," "administering", "administration," and the like, as used herein, can refer to methods that can be used to enable delivery of compounds or compositions to the desired site of biological action. These methods can include oral administration, intraduodenal administration, parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. In some instances, a subject can administer the composition in the absence of supervision. In some instances, a subject can administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician's assistant, orderly, hospice worker, etc.).

[0052] The terms "pharmaceutically acceptable salt" or simply "salt" as used herein, can refer to a salt that retains at least some of the biological effectiveness of the free acids and bases of the specified compound. In some instances, the salt can be not biologically or otherwise undesirable. In some embodiments, a compound disclosed herein can possess acidic or basic groups and therefore can react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. In some embodiments, a salt can be prepared in situ during the final isolation and purification of a compound, or by separately reacting with a purified compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.

[0053] Examples of pharmaceutically acceptable salts can include those salts prepared by

reaction of a compound disclosed herein with a mineral, organic acid or inorganic base, such salts can include, acetate, acrylate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, bisulfite, bitartrate, bromide, butyrate, butyn-l,4-dioate, camphorate, camphorsulfonate, caproate, caprylate, chlorobenzoate, chloride, citrate, cyclopentanepropionate, decanoate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hexyne-l,6-dioate, hydroxybenzoate, γ- hydroxybutyrate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isobutyrate, lactate, maleate, malonate, methanesulfonate, mandelate,

metaphosphate, methanesulfonate, methoxybenzoate, methylbenzoate,

monohydrogenphosphate, 1-napthalenesulfonate, 2-napthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, pyrosulfate, pyrophosphate, propiolate, phthalate, phenyl acetate, phenylbutyrate, propanesulfonate, salicylate, succinate, sulfate, sulfite, succinate, suberate, sebacate, sulfonate, tartrate, thiocyanate, tosylate, undeconate and

xylenesulfonate.

[0054] The terms "co-administration", "administered in combination with" and their

grammatical equivalents or the like, as used herein, can encompass administration of selected therapeutic agents to a single patient, and can include treatment regimens in which the agents can be administered by the same or different route of administration or at the same or different times. In some embodiments, a compound disclosed herein can be co-administered with other agents. These terms can encompass administration of two or more agents to an animal so that both agents and/or their metabolites can be present in the animal at the same time. They can include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents can be present. Thus, in some embodiments, a compound and another agent(s) can be administered in a single composition. In some embodiments, a compound and another agent(s) can be admixed in the composition.

[0055] The term "allogeneic" as used herein can refer to cells isolated from one subject (the donor) and transferred into the body of another subject (the recipient or host).

[0056] The term "autologous" as used herein can refer to cells that are isolated and transferred back into the same subject (the recipient or host). For example, a transplant process can be autologous when the donor and the recipient of the plurality of isolated cells are the same individual. Thus, in an autologous transplant process cells can be harvested from a subject and then returned to the same subject.

[0057] The term "stem cells" or "progenitor cells" as used herein can refer to cells that have the ability to renew themselves through mitosis as well as differentiate into various specialized cell types. The stem cells used in the invention are somatic stem cells, such as cardiac stem cells or cardiac progenitor cells.

[0058] The term "adult stem cells" as used herein can refer to stem cells that are not embryonic in origin nor derived from embryos or fetal tissues.

[0059] Stem cells can be selected to be lineage negative. The term "lineage negative" as used herein can refer to that a cell does not express antigens characteristic of specific cell lineages. Lineage negative stem cells can be selected to be c-kit positive. The term "c- kit" as used herein can refer to a receptor which is known to be present on the surface of stem cells and which is utilized in the process of identifying and separating stem cells from other surrounding cells.

[0060] The term "differentiation" as used herein can refer to the process of cell development with an increase in the level of organization or complexity of a cell or tissue,

accompanied with a more specialized function.

[0061] The terms "dose" and "dosage" as used herein can be used interchangeably to refer to an amount of an active agent or a pharmaceutical composition administered to a subject.

[0062] The term "cytotoxin" or "cytotoxic agent" as used herein can refer to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.

[0063] The term "anti-cancer agent" as used herein can refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, and immunotherapeutic agents. For example, examples of anti-cancer agent can include, but are not limited to, a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Some examples can include auristatins such as MMAE and MMAF; calicheamycins such as gamma- calicheamycin; and maytansinoids such as DM1, DM3 and DM4.

Overview

[0064] Disclosed herein are compositions, formulations, methods, and kits for treating a cell proliferation disorder. Turning now to FIG. 1, in some cases, a first subject can undergo an operation to have some cardiac tissues removed from his body, as depicted in block 101 of the illustration in FIG. 1. Stromal cells from the cardiac tissues can be isolated, expanded, and treated, as shown in block 102. Then cardiac derived stromal cells can be screened for the desired cell types with some selected biomarker or the lack thereof, selected for the presence of some receptors or the lack thereof, and formulated, as illustrated in block 103. Finally, the isolated cardiac derived stromal cells can be administered to a second subject in need thereof, as shown in block 104. The second subject can have or be suspected of having a cell proliferation disorder. The first subject can be the same as or different from the second subject. Step(s) can be added to or deleted from the steps depicted in FIG. 1.

Cancer

[0065] Tumor can be neoplastic cell growth and proliferation, whether malignant or benign, and can include both pre-cancerous and cancerous cells and tissues. The term "anti-tumor activity" can mean a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. Possible ways of showing anti-tumor activity can include demonstrating a decline in growth rate of abnormal cells that arises during therapy, reduction of tumor size, and decrease of tumor cell stability. Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other models to investigate anti -tumor activity.

[0066] Malignancy can refer to a non-benign tumor or a cancer. Cancer can include a

malignancy characterized by deregulated or uncontrolled cell growth. Normal cells can become cancerous after mutations accumulate in the various genes that control cell proliferation. For example, growth-promoting genes (e.g., the gene for the signaling protein Ras) can become super active to produce cells that are easily stimulated by growth receptors. Other cancer-related gene mutations can inactivate the genes that suppress cell proliferation or signal the need for apoptosis.

[0067] Cancer can be classified by the type of cells presumed to be the origin of the tumor, including carcinoma (derived from epithelial cells), sarcoma (derived from connective tissue), leukemia and lymphoma (derived from hematopoietic cells that mature in the blood and lymph nodes, respectively), germ cell tumor (derived from pluripotent cells), and blastoma (derived from immature precursor cells or embryonic tissue). Cancer can include primary malignant tumors and secondary malignant tumors. The primary malignant tumors can refer to tumor cells that have not migrated to sites in the subject's body other than the site of the original tumor. The secondary malignant tumors can refer to tumor cells that arise from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.

[0068] Metastasis can be the spread of cancerous cells to new areas of the body. A metastatic cancer can be the cancer that has spread from the primary site of origin (where the caner started) into different areas of the body. Metastasis can be a terminal stage of cancer, when cancerous cells enter the bloodstream or the lymphatic system to spread to a new location, where the cancerous cells begin to divide and lay the foundation for secondary tumor. The dispersed tumors can be called metastatic tumors. Common places for metastases to occur can be the lungs, liver, brain and the bones. Thus, cancer can include primary malignant tumors and secondary malignant tumors. The primary malignant tumors can refer to tumor cells that have not migrated to sites in the subject's body other than the site of the original tumor. The secondary malignant tumors can refer to tumor cells that arise from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.

Stromal Cells

[0069] Stromal cells can refer to cells that make up certain types of connective tissue

(supporting tissue that surrounds other tissues and organs). Stromal cells can be of direct or indirect (hematopoietic) mesenchymal origin and can encompasses different cell populations residing in the connective tissue. They share the ability to produce the macromolecular components of the extracellular matrix and organize them in appropriate spatial assembly. They can provide a proper three-dimensional scaffold and stimulate the growth and differentiation of parenchymal precursors leading to tissues and organs.

[0070] Cardiac stromal cells can be a cell population in the heart and can be obtained from

biopsies, such as right ventricle endomyocardial biopsies of subjects, or other types of surgeries. Cardiac stromal cells can be expanded ex vivo, analyzed, harvested, frozen, and stored. Cardiac stromal cells can maintain the architecture of the heart. Cardiac fibroblasts can be cardiac stromal cells that are responsible for the formation and renewal of extracellular matrix (ECM). In the normal adult heart, cardiac fibroblasts can proliferate slowly. Isolated cardiac fibroblasts can grow slowly in culture and can undergo senescence rapidly.

[0071] Mesenchymal stromal cells (MSCs) can be supportive cells found in many tissues,

including, but not limited to bone marrow, lung, liver, amniotic fluid, adipose, placental, umbilical, and vascular tissues. MSCs can be expanded ex vivo. MSCs can possess angiogenic and/or immunomodulatory properties. MSCs can be cryopreserved.

[0072] MSCs can be characterized by their multipotential capacity to differentiate into

osteoblasts, chondrocytes, myocytes, and adipocytes and by a panel of surface markers which can distinguish these cells from endothelial, hematopoietic, and monocyte like cells. For example, MSCs can be positive for CD44, CD73, CD90, and CD105 and negative for hematopoietice (e.g., CD45, or lineage markers), endothelial (CD31, von Willebrand factor) and macrophage (CD1 lb/MAC-1) markers. MSC can be immune privileged because they can lack cell surface expression of certain major

histocompatibility complex (MHC) class II and costimulatory-type molecules (i.e., CD80/CD86). A human adult heart can comprise cardiac mesenchymal stromal cells.

[0073] In self-renewing organs, stromal cells can reside in specific niches that constitute the microenvironment in which tissue-specific progenitor cells can be maintained in a quiescent state. After receiving activation signals, progenitor cells can proliferate and migrate to the sites of injury where they differentiate and acquire the mature phenotype. Tissue-specific progenitor cells niche homeostasis can be regulated by the division of progenitor cells.

[0074] MSCs can represent the major stromal cell population in the bone marrow. Bone marrow MSCs can support maintenance of hematopoietic stem cells (HSC). The proliferation and differentiation of HSCs can be controlled by a group of proteins called hematopoietic growth factors (HGFs), which are produced in part by bone marrow MSCs. The HSCs can reside in the bone marrow in close proximity to stromal cells which can provide the "stem cell niche". Deficiencies in the microenvironment at a cellular or molecular level can result in abnormal cell production leading to anemia, leukemia or embryonic lethality. In addition, the bone marrow can be a primary site of metastasis for many tumors including breast cancer and the site of relapse for many blood cancers Bone marrow stromal cells can be supportive of tumor cell proliferation.

[0075] Stromal cell morphology

[0076] Stromal cells can be obtained from bone marrow, adipose, liver and heart tissues using various methods, including, for example, a collagenase digestion protocol. Collected stromal cells can be cultured in flasks in alpha modified Minimum Essential Medium (MEM) with 20% fetal calf serum (FCS), and then undergo morphology evaluation by light microscopy. FIG. 2 shows morphology of evaluated stromal cell lines from bone marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel). As shown in FIG. 2, stromal cells from each tissue can have similar spindle-shaped, fibroblastic morphology similar to those observed for bone marrow MSCs. Furthermore, the observed morphology can be maintained after multiple passages of the cells.

[0077] Stromal cell phenotypes

[0078] The Mesenchymal and Tissue Stem Cell Committee of the International Society for

Cellular Therapy has established the phenotype of MSCs as CD105+, CD90+, CD73+, CD45- and CD34- (Cytotherapy (2006) Vol. 8, No. 4, 315-317, which is entirely incorporated herein by reference for all purposes). Stromal cell lines from difference tissue sources can be analyzed by flow cytometry for the expression of markers. FIG. 3 displays phenotypic analysis of stromal cell lines. Flow cytometry histograms

(representing three independent experiments) of bone marrow mesenchymal stem cells (BMMSC, first column), adipose stromal cells (second column), liver stromal cells (third column), and heart stromal cells (fourth column) can display with appropriate isotype control staining (open histogram) versus specific antibody staining (solid histogram) for phenotype markers of CD 105 (first row), CD90 (second row), CD73 (third row), CD45 (fourth row), and CD 34 (fifth row), shown in FIG. 3. Each of the stromal cell lines can have surface expression of CD105, CD90 and CD73, with levels of expression varying slightly between cell lines. All tested MSCs can be negative for expression of

hematopoietic cell markers CD45 and CD34. The results from FIGs. 2 and 3 demonstrate that morphologically and phenotypically, stromal cells from various tissues can have identical morphological and phenotypic expression.

[0079] Stromal cell differential potential

[0080] One of the hallmarks of MSCs can be their ability to differentiate into non-hematopoietic cells such as adipocytes, osteocytes, or chondrocytes. The ability of clonally expanded cells to form two or more of these three distinct cell types can be a reliable functional screen to identify MSCs and distinguish them from pre-adipocytic, pre-osteoblast, or pre- chondrocytic cells.

[0081] Osteogenic differentiation of MSCs can be induced in vitro by treating a MSCs culture with a pro-osteogenic cocktail, such as, for example, a differentiation medium

comprising dexamethasone, ascorbic acid-w-phosphate and beta-glycerophosphate. Mineralized deposits weeks after treatment can be analyzed by staining with Alizrin-Red S solution C or silver nitrate for von Kossa Staining, showing the calcium phosphate deposits. In addition, osteogenic differentiation can be accompanied by the expression of genes coding for, such as, for example, osterix, cbfal, osteopontin, osteocalcin, or bone sialoprotein, either at the RNA level or at the protein level.

[0082] Chondrogenic potential can be achieved by forcing aggregation of about 200, 000 to about 300,000 MScs in a chondrogenic medium to generate a micromass pellet culture comprising dexamethasone, ascorbic acid phosphate and insulin-transferrin-selenium medium supplement (ITS+ supplement), which consists of bovine insulin, transferrin, selenous acid, linoleic acid and bovine serum albumin. Further additives to the medium can include sodium pyruvate, proline, L-glutamine and TGF-betal, a growth factor possibly involved in chondrogenesis in vivo. There can be an upregulation of

chondrogenic markers such as collagen II, collagen XI, aggrecan, perlecan and syndecan during the treatment. After two to three weeks in culture, micromass pellets can be fixed and embedded for sectioning and subsequent staining with Safranin-O, Toluidine blue or Alcian blue to highlight acid mucopolysaccharides, glycosaminoglycans and

proteoglycans respectively.

[0083] Adipogenic potential can be induced following treatment of MSCs with a medium

supplemented with dexamethasone, isobutylmethylxanthine, insulin and a PPARgamma agonist (e.g., BRL 49653) with about twice weekly medium changes for about three weeks. Alternatively, MSCs can be exposed to three cycles of a treatment which alternates three days of culture in induction medium followed by two days in

maintenance medium. In the latter instance, the induction medium can contain indomethacin instead of BRL 49653 and the maintenance medium can contain insulin. The appearance of adipocytes containing lipid-filled droplets can be demonstrated by staining with oil red O or by real-time PCR (RT-PCR) detection of adipsin, adipocyte acid-binding protein (aP2) and PPAR gamma expression. Quantitative data can be obtained by enzymatic dosage of the glycerol-3 -phosphate dehydrogenase, a marker of the mature adipocyte. Furthermore, the quantitation of adipocytes can be accomplished by flow cytometry in a MSC culture stained with the lipophilic dye Nile Red.

[0084] Myogenic potential can be demonstrated when treating MSCs with the demethylating agent 5-azacytidine to form muscle cells. In addition, a zenogeneic in vitro model in which MSCs can be co-cultured with murine skeletal myocytes can be relied upon to evaluate myogenic potentials of MSCs. Similarly cardiomyocytes can be induced from MSCs as well after treatment with 5-azacytidine in vivo.

[0085] As shown in FIG. 4, stromal cells obtained from bone marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel) accumulated as intracellular lipid droplets can be determined by oil red O staining, then evaluated by light microscopy. Stromal cell lines from all tested tissue sources can display adipogenic potential.

[0086] FIG. 5 shows osteogenic differentiation potential of stromal cell lines. Stromal cells obtained from bone marrow (top left panel), adipose tissue (top right panel), liver (bottom left panel) and heart (bottom right panel) having extracellular calcium deposition can be demonstrated by positive Alizarin Red S staining, and then evaluated by light microscopy. Stromal cell lines from all tested tissue sources can display osteogenic potential.

[0087] Accordingly, cardiac derived stromal cells can have similar morphology and phenotype to stromal cells obtained/derived from bone marrow, adipose and liver tissues. Further, cardiac derived stromal cells can have differential potential for adipogenic and osteogenic lineages, similar to stromal cells obtained/derived from bone marrow, adipose and liver tissues.

Cardiac derived stromal cells inhibit tumor cell proliferation

[0088] Inhibition of tumor cell proliferation

[0089] Cultured cardiac derived stromal cells can inhibit proliferations of tumor cell lines.

Within a week of co-culture with cardiac derived stromal cells, the viability of tumor cells can be less than about 20% and the remaining tumor cells can fail to proliferate. In contrast, tumor cells cultured in the absence of a stromal layer or cultured on bone marrow MSCs can proliferate and maintain viability greater than 99%. Similar results can be obtained when using different cardiac derived stromal cell lines and with a range of tumor cell lines, including, for example, leukemic cells, lymphoma cells (HL-60, K562 and Raji), and myeloma cells (U-266, RPMI8266 and ARP-1). FIG. 6 displays culture of HL-60 cells co-cultured on bone marrow mesenchymal stem cells for 11 days. FIG. 7 displays culture of HL-60 cells co-cultured on cardiac derived mesenchymal stem cells for 11 days. As shown in FIGs. 6 and 7, growth of the tumor cell lines can be inhibited by co-cultured cardiac derived stromal cells when compared with those co- cultured bone marrow mesenchymal stem cells.

[0090] Inhibition specificity when compared with other stromal cells

[0091] Among the stromal cells obtained from different tissues, cardiac derived stromal cells can demonstrate specific inhibition of tumor cell proliferation while other stromal cell lines lack such specificity. The growth of the myeloid tumor cell line K562 can be compared on stromal cells from various tissues. As shown in panel A of FIG. 8, the K562 cells can proliferate on bone marrow, adipose and liver stromal cells, but not on the cardiac derived stromal cells.

[0092] Panel B of FIG. 8 shows K562 total cell number after 14 days of culture in different stromal cell cultures. Error bars represent standard error of mean (N = 6; * denotes p<0.05). Heart stromal cells can produce reduced total co-culture tumor cells when compared with stromal cells from bone marrow, adipose and liver. [0093] In addition, the viability of the K562 cells co-cultured on the cardiac derived stromal cells can be low compared to the other stromal cells (panel C of FIG. 8), with less than 40% viable cells. In contract, bone marrow, adipose and liver stromal cells can result in about 80%) to about 90% viable K562 cells under similar conditions after 14 days of culture. Error bars represent standard error of mean (N = 6; * denotes p<0.05).

[0094] The effect of the cardiac derived stromal cells can be produced using 20 ^χ concentrated media (CM) conditioned by cardiac derived stromal cells for 7 days. The cardiac derived stromal cells CM can decrease the proliferation of K562 cells in a dose dependent manner. For example, in 3 days of culture, control cultures of K562 cells can contain about 1.1 x 10 ⁶ cells/mL while addition of 10%> cardiac derived stromal cells CM can result in about 3.7 ^χ 10 ⁵ cells/mL (about 32% of control) with both sets of cultures seeded with the same number of K562 cells. Cultures containing 20% cardiac derived stromal cells CM can result in about 2.0 ^χ 10 ⁵ cells/mL (about 18% of control). In addition, the cultures containing 20% cardiac derived stromal cells CM can contain about 68%) viable cells compared to about 93% viable cells in the control cultures.

[0095] The effect of cardiac derived stromal cells CM can be produced in several myeloma cell lines, including, for example, U266, A P-1 and RPMI-8266 (shown in FIG. 9-11, respectively). Control cultures containing media conditioned by bone marrow MSCs can result in cell proliferation while the cultures with cardiac derived stromal cells CM can result in inhibition of cell growth and decreased viability. The extent of inhibition with CM can be less than that obtained with co-culture on the cardiac derived stromal cells.

[0096] Differential gene expression analysis

[0097] Global gene array analysis and microRNA array analysis can be performed on both bone marrow MSCs and cardiac derived stromal cells. The bone marrow MSCs and cardiac derived stromal cells can be culture expanded and then RNA prepared for microarray analysis. The analysis results can demonstrate distinct gene patterns between these two sources of stromal cells. For example, analysis results can demonstrate distinct cytokine and cytokine receptor patterns. Table 1 displays that cytokines and cytokine receptors can be expressed at 2 fold or higher levels in cardiac stromal cells compared to bone marrow MSCs. These data can suggest that stromal cells in different tissues can secrete differing cytokines and express different cytokine receptors consistent with local control of tissue specific stem cells and progenitor cells through cytokines.

[0098] Cardiac derived stromal cells can also express increased gene levels of cell adhesion molecules and focal adhesion molecules with an increase in a number of genes associated with endothelial, vascular and muscle cells. Table 2 displays that cell adhesion molecules can be expressed at 2 fold or higher levels in cardiac stromal cells compared to bone marrow MSCs. Table 3 displays that focal adhesion molecules can be expressed at 2 fold or higher levels in cardiac stromal cells compared to bone marrow MSCs. The increased expression of myosin genes and laminin alpha 5 can be consistent with cardiac expression compared to bone marrow derived cells.

99] Table 1

[00101] Table 3

[00102] microRNA array

[00103] microRNA (miRs) can be 21-23 nucleotide non-coding RNA molecules, which can modulate the stability and/or translational efficiency of messenger RNAs (mRNA). miRs can target multiple transcripts and individual transcripts can be subject to multiple miR regulation. For example, miR can involve in pluripotency maintenance, cell proliferation and differentiation, epithelial to mesenchymal transition, senescence, and apoptosis. miR also can promote cell to cell phenotypic conversion and reprogram adult cell into pluripotent stem cells.

[00104] miRs can be up regulated or down regulated by more than 2 fold in cardiac

derived stromal cells compared to bone marrow MSCs (Tables 4 and 5). Table 4 shows microRNAs expressed at higher levels by 2 or more fold in cardiac derived stromal cells compared to bone marrow MSCs. Table 5 shows microRNAs expressed at lower levels in cardiac derived stromal cells by 2 or more fold compared to bone marrow MSCs. Among the up regulated miRs three (miR-1, miR- 133a and miR-206) can have functions in myogenesis and cardiomyocyte development, and two (miR-20a and miR-26a) can have functions in stem cell differentiation. In addition, miR-206, which can be up regulated by more than one thousand fold in cardiac derived stromal cells. miR-206 can be a muscle specific miR that can promote muscle differentiation and regulate connexin 43 expression during skeletal muscle development. The higher expression of miR-206, miR-1 and miR- 133a can be confirmed by real time PCR as shown in FIG. 12, with higher expression of each miR in cardiac derived stromal cells compared to bone marrow

MSCs.

[00105] Table 4

miR Fold Up

miR-1 63

miR- 10a 11

miR-15a 3 miR-15b 6

miR-16-1 16

miR-18a 24

miR-19b-l 15

miR-20a 16

miR-26a-2 7

miR-33a 26

miR-92a-l 137

miR-133a 57

miR-206 1181

miR-374a 4

miR-411 2

miR-424 10

miR-450a 2

miR-450b-5p 37

miR-454 3

miR-503 35

miR-516a-3p 2

miR-542-3p 3

miR-551b 5

miR-589 5

miR-770-5p 2

miR-935 213

)106] Table 5

miR Fold Down

miR-lOb 9

miR-335 13

miR-451 13

miR-479 8

miR-628-3p 9

miR-768-3p 2

[00107] In some cases, the isolated cardiac derived stromal cells express at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, or at least 1, 100- fold of miR-206 when compared with mir-206 expressed by the bone marrow

mesenchymal stem cells.

[00108] Inhibition specificity when compared with normal cells

[00109] To characterize the specificity of the inhibitory effect of the cardiac derived

stromal cells over tumor cells, human cord blood cells as a source of normal hematopoietic cells can be co-cultured on cardiac derived stromal cells as well as on other stromal cell. After 14 days of culture, maximum proliferation can be enumerated with bone marrow MSCs (BMMSC, mean 7.11 x 10 ⁶ ) (panel A of FIG. 13). Similar proliferation can be seen in co-culture with adipose stromal cells (AS, mean 3.14 10 ⁶ ) and liver stromal cells (LS, mean 4.01 x 10 ⁶ ). However, the proliferation in heart stromal cells (HS) can be less than that in bone marrow MSCs, adipose stromal cells, or liver stromal cells.

[00110] To test for the functional output induced by proliferation on stromal cells, cell progeny in colony forming assays specific for granulocyte/macrophage progenitors (CFU-GM) can be assayed. Cord blood progeny co-cultured on bone marrow MSCs can yield 5.65 ^χ 10 ⁴ CFU-GM colonies (panel B of FIG. 13) while adipose stromal cells can proliferate 3.31 ^χ 10 ⁴ total CFU-GM colonies. Liver stromal cells can induce

proliferation of more CFU-GM colonies (7.81 x 10 ⁴ CFU-GM) compared with bone marrow MSCs. Though cardiac derived stromal cells can induce minimal total cell proliferation in co-cultures, proliferation of CFU-GM colonies (6.38 x 10 ⁴ CFU-GM colonies) in cardiac derived stromal cells can be similar to those in bone marrow MSCs.

[00111] Since cardiac derived stromal cells can have inhibitory effects on their co- cultures with tumor cells and cord blood cells compared to other stromal lines co- cultures with tumor cells and cord blood cells, whether this effect is permanent or temporary in long-term culturing can be tested. Towards this end, the hematopoietic cells can be removed from cardiac derived stromal cells co-culture after 14 days, and be further cultured in media with growth factors but without the presence of stroma. A steady increase of total viable cells in culture can be observed, suggesting that the inhibitory effect of cardiac derived stromal cells on cord blood cells can be cytostatic but reversible upon the removal of cardiac derived stromal cells. On the other hand, co- cultured K562 cells can be removed from cardiac derived stromal cells after 14 days, and be further cultured in media with growth factors but without the presence of stroma. No increase in total K562 cells can be observed. But in similar stromal cell removal experiments of K562 cells co-cultured on bone marrow MSC, adipose stromal cells and liver stromal cells, after the stromal cells removal K562 cells can proliferate similarly to the control K562 cells.

[00112] Dose-dependent inhibition

[00113] K562 leukemia cells can be cultured on cardiac derived stromal cells (CStrCs) in different media. Panel A of FIG. 14 shows that in the absence of cardiac derived stromal cells (-CStrCs), K562 leukemia cells can proliferate to a much larger extent than in the presence of cardiac derived stromal cells (+CStrCs). Similarly, panel B of FIG. 14 shows that in the absence of concentrated media conditioned by cardiac derived stromal cells (-CStrCs-CM), K562 leukemia cells can proliferate to a much larger extent than in the presence of concentrated media conditioned by cardiac derived stromal cells (+CStrCs- CM 10% and +CStrCs-CM 20%). Furthermore, the inhibitory activity by CStrCs-CM can be dose dependent since the higher percentage of cardiac derived stromal cells (+CStrCs-CM 20%) can lead to higher levels of inhibition than the lower percentage of cardiac derived stromal cells (+CStrCs-CM 10%). A concentrated media conditioned by about 20% cardiac derived stromal cells is denoted as +CStrCs-CM 20% in FIG. 14.

[00114] Combined with the data shown in FIGs. 9, 10 and 11, the inhibitory activity of concentrated media conditioned by cardiac derived stromal cells (CStrC-CM) can be shown over tumor cells lines including K562, U266, ARP-1 and RPMI-8266. In contrast, media conditioned by bone marrow mesenchymal stromal cells (MSC-CM1 and MSC- CM2 in FIGs. 9-11) do not exhibit inhibitory effect on the corresponding tumor cell lines.

[00115] Biochemical characterization

[00116] The concentrated media conditioned by cardiac derived stromal cells can be

characterized in many ways. In some cases, FIG. 15 shows that the concentrated media conditioned by cardiac derived stromal cells (shown as CM in FIG. 15) can behave differently against tumor cell lines depending on the storage/reaction conditions of the concentrated media conditioned by cardiac derived stromal cells. As shown in FIG. 15, CM without any additional treatment (Normal CM) can exhibit inhibitory activity against tumor cell lines against the control (tumor cell lines without CM). CM going through three freeze/thaw cycles can still exhibit inhibitory activity against tumor cell lines. However, heating at 60 °C or 80 °C can decrease the inhibitory activity against tumor cell lines.

[00117] In some cases, FIG. 16 shows that when the concentrated media conditioned by cardiac derived stromal cells are treated with trypsin, the concentrated media conditioned by cardiac derived stromal cells can lose the inhibitory activity against tumor cell lines. Trypsin is a serine protease that can be found in the digestive system of some

vertebrates. Trypsin can hydrolyze proteins at the carboxyl side of the amino acids lysine or arginine. Therefore, at least in some cases, the inhibitory activity against tumor cell lines may be protein-based or protein-related. Mechanism of inhibition

[00118] FIG. 17 shows the results of K562 cell viability in the concentrated media

conditioned by the bone marrow mesenchymal stromal cells (control) or by the cardiac derived stromal cells (CM Treated) when the cells are cultured without washing or with washing. Specifically, when the K562 cells are incubated with concentrated media (the three bars on the left in FIG. 17), the concentrated media conditioned by bone marrow mesenchymal stromal cells (control) can promote tumor cell proliferation to a much higher level than the concentrated media conditioned by cardiac derived stromal cells (CM Treated). These cell samples can be washed and cultured for a further 7 days (the three bars on the right in FIG. 17) before measuring the cell numbers. FIG. 17 shows that the K562 cells can fail to proliferate after washing and further culture. Hence, the inhibitory activity of the cardiac derived stromal cells may be cytotoxic.

[00119] Molecules responsible for the inhibitory activity

[00120] In some cases, the cardiac derived stromal cells can exhibit the inhibitory activity against tumor cells through the molecules secreted by or associated with the cardiac derived stromal cells. In some cases, micro RNA (miR), exosomes, and extracellular vesicles, either secreted by or associated with the cardiac derived stromal cells, can be responsible for the inhibitory activity against tumor cells. In some cases, the molecule that is responsible for the inhibitory activity is miR-206. miR-206 can inhibit the proliferation of tumor cells when transfected into tumor cell lines. In some cases, tumor cells, such as triple negative breast cancer cells (T BC cells), down regulate miR-206 when compared with non-tumor cells (e.g., non-TNBC cells). In some cases,

downregulation of miR-206 can promote the invasion and angiogenesis of TNBC.

Composition

[00121] Disclosed herein are compositions for treating a cell proliferation disorder. The composition can comprise: (a) a plurality of isolated cardiac derived stromal cells; and (b) a pharmaceutically acceptable carrier. In some cases, the composition can further comprise another anti-cancer agent. In some cases, the plurality of isolated cardiac derived stromal cells can be derived from human cardiac cells.

[00122] In some cases, the composition can further comprise a physiological buffer, such as, for example, phosphate-buffered saline (PBS) or plasmaLyte. In some cases, the composition can further comprise a carrier protein, such as, for example, human serum albumin (HSA). [00123] PlasmaLyte can be a family of balanced crystalloid solutions with multiple different formulations available worldwide according to regional clinical practices and preferences. It can closely mimic human plasma in its content of electrolytes, osmolality, and pH. It can also have additional buffer capacity and contain anions such as acetate, gluconate, and even lactate that are converted to bicarbonate, C0 ₂ , and water. For example, in the U.S. a plasmaLyte injection solution can be a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL of the solution can contain 526 mg of sodium chloride; 502 mg of sodium gluconate; 368 mg of sodium acetate trihydrate; 37 mg of potassium chloride; and 30 mg of magnesium chloride. The solution can be adjusted with a sodium hydroxide solution to about pH 7.4, or within the range from about pH 6.5 to about pH 8.0.

[00124] Isolated cardiac stromal cells

[00125] The composition can comprise a plurality of isolated cardiac stromal cells. In some cases, the plurality of cardiac stromal cells can be isolated from tissue specimens obtained from a subject. The tissue specimens can be minced, further dissociated, and digested according to a collagenase digestion protocol. Collected cardiac stromal cells can be placed in appropriate culture medium and expanded. After about two weeks, about three weeks, about four weeks, or about more than four weeks, the expanded cardiac stromal cells can be collected by centrifugation. Other methods of isolating cardiac stromal cells can be possible.

[00126] Isolated cardiac derived stromal cells can be linage negative. Lineage negative cardiac derived stromal cells can be isolated by various means, including but not limited to, removing lineage positive cells by contacting the cardiac derived stromal cell population with antibodies against lineage markers and subsequently isolating the antibody-bound cells by using an anti-immunoglobulin antibody conjugated to magnetic beads and a biomagnet. Alternatively, the antibody -bound lineage positive cardiac derived stromal cells may be retained on a column containing beads conjugated to antiimmunoglobulin antibodies. The cells not bound to the immunomagnetic beads can represent the lineage negative cardiac derived stromal cell fraction and can be isolated.

[00127] For instance, cells expressing markers of the cardiac lineage (e.g. markers of vascular cell or cardiomyocyte commitment) can be removed from cardiac derived stromal cell populations to isolate lineage negative cardiac derived stromal cells.

Markers of the vascular lineage can include, but are not limited to, GATA6 (SMC transcription factor), Etsl (EC transcription factor), Tie-2 (angiopoietin receptors), VE- cadherin (cell adhesion molecule), CD62E/E-selectin (cell adhesion molecule), alpha- SM-actin (a-SMA, contractile protein), CD31 (PECAM-1), vWF (carrier of factor VIII), Bandeiraera simplicifolia and Ulex europaeus lectins (EC surface glycoprotein-binding molecules). Markers of the myocyte lineage can include, but are not limited to, GATA4 (cardiac transcription factor), Nkx2.5 and MEF2C (myocyte transcription factors), and alpha-sarcomeric actin (a-SA, contractile protein).

[00128] In some cases, the cardiac derived stromal cells can be screened to isolate those cells that express c-kit, the stem cell surface marker and the receptor for stem cell facto. Examples of such isolation can include, but are not limited to, various processes of cell sorting, such as fluorescence activated cell sorting (FACS), magnetic cell sorting, and affinity chromatography, or the like.

[00129] In some cases, the cardiac derived stromal cells can be screened to isolate those cells that express a specific receptor or those that do not express a specific receptor.

[00130] In some cases, the cardiac derived stromal cells can be exposed to one or more cytokines. Suitable concentrations of the one or more cytokines can include a

concentration of about 0.1 to about 500 ng/mL, about 10 to about 500 ng/mL, about 20 to about 400 ng/mL, about 30 to about 300 ng/mL, about 50 to about 200 ng/mL, or about 80 to about 150 ng/mL. In some cases, the concentration of one or more cytokines can be about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 ng/mL.

[00131] Non-limiting examples of cytokines can include hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), Activin A, Bone Morphogenic Protein 2, Bone Morphogenic Protein 4, Bone Morphogenic Protein 6, Cardiotrophin-1, Fibroblast Growth Factor 1, Fibroblast Growth Factor 4, Flt3 Ligand, Glial -Derived Neurotrophic Factor, Heparin, Insulin-like Growth Factor-II, Insulin-Like Growth Factor Binding Protein-3, Insulin-Like Growth Factor Binding Protein-5, Interleukin-3, Interleukin-6, Interleukin-8, Leukemia Inhibitory Factor, Midkine, Platelet-Derived Growth Factor AA, Platelet-Derived Growth Factor BB, Progesterone, Putrescine, Stem Cell Factor,

Stromal -Derived Factor-1, Thrombopoietin, Transforming Growth Factor-a,

Transforming Growth Factor-.beta. l, Transforming Growth Factor-.beta.2, Transforming Growth Factor-.beta.3, Vascular Endothelial Growth Factor, Wntl, Wnt3a, and Wnt5a, and the functional variants thereof. [00132] In some cases, the plurality of isolated cardiac derived stromal cells can comprise an autologous cardiac derived stromal cell. In some cases, the plurality of isolated cardiac derived stromal cells can comprise a heterologous cardiac derived stromal cell. In some cases, at least a portion of the plurality of isolated cardiac derived stromal cells can be viable. In some cases, the viable portion of the plurality of isolated cardiac derived stromal cells can be at least about 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight relative to a total weight of a the plurality of cardiac derived stromal cells.

[00133] Other components

[00134] In some cases, the composition can further comprise a cellular cryopreservative.

In some cases, the cellular cryopreservative can comprise a sucrose, a trehalose, a starch, a derivative of any of these, or any combination thereof.

[00135] In some cases, the composition can further comprise an antibiotic and/or

antiseptic ulcer agent. Immunosuppressive treatment (e.g., corticosteroids, radiation therapy, chemotherapy), or other cancer treatment can be combined with the

compositions disclosed herein.

[00136] In some cases, the composition can further comprise a pharmaceutically

acceptable salt.

[00137] In some cases, the composition can further comprise at least one anti-cancer

agent, including but not limited to, an alkylating agent, a metabolic antagonist, an alkaloid anti-cancer agent, an antibiotic anti-cancer agent, an antibody, and a platinum drug. In some cases, the anti-cancer agent can be an alkylating agent, a metabolic antagonist, a microtubule inhibitor, an antibiotic anti-cancer agent, a topoisomerase inhibitor, a platinum drug, a molecular targeted drug, a hormone agent, and a biological drug. Examples of the alkylating agent can include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine. Examples of the metabolic antagonist include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, and

mercaptopurine. Examples of the microtubule inhibitor include an alkaloid anti-cancer agent (e.g., vincristine) and a taxan anti-cancer agent (e.g., docetaxel and paclitaxel). Examples of the antibiotic anti-cancer agent include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and zinostatin stimalamer. Examples of the topoisomerase inhibitor include CPT-11, irinotecan, nogitecan (topoisomerase I inhibitors), and etoposide and sobuzoxane (topoisomerase II inhibitors). Examples of the platinum drug include cisplatin, nedaplatin, oxaliplatin, and carboplatin. Examples of the hormone agent include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, predonisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane. Examples of the biological drug include interferon a, interferon β, interferon γ, interleukin 2, ubenimex, and dry BCG. Examples of the molecular targeted drug include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temcirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tosituzumab, bortezomib, gemutuzumab- ozogamicin, ibritumomab-ozogamicin, ibritumomab tiuxetan, tamibarotene, and tretinoin. In addition to the above specified molecular targeted drugs, there may be included the following molecular targeted drugs: angiogenesis-targeted inhibitors such as human epidermal growth factor receptor 2 inhibitor, epidermal growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epidermal growth factor tyrosine kinase inhibitor, mTOR inhibitor, and endothelial growth factor receptor 2 inhibitor (a-VEGFR- 2 antibody); tyrosine kinase inhibitors such as MAP kinase inhibitor; cytokine-targeted inhibitors, proteasome inhibitor, and antibody-anti-cancer agent formulations. These inhibitors can also include corresponding antibodies. In addition to the aforementioned drugs, the following pharmaceuticals can be used in combination: thalidomide, everolimus, elplat, ABI-007, ixabepilon, miriplatin, lapatinib, pemetrexed, cladribine, liposomal doxorubicin, Z-100, hycamtin, vandetanib, ZD4054, anastrozole,

GSK1572932A, pazopanib, denosmab, S-l, motesanib, trastuzumab, enzastaurin, immucyst, NIK-333, axitinib, bostinib, E7080, soblidotin, degarelix, fluvestrant, zoladex, cediranib, eribulin, TSU-68, TAC-101, TAS-108, NK911, NK105, erlotinib, LBH589, MK-0457, tamibarotene, lenalidomide, BNP1350, AZD0530, AZD1152, AZD2281, AZD4877, ABT-869, ONO-4538, OTS102, KW-0761, ARQ197, ofatumab, AMG655, TAK-700, TAK-683, TAK-448, CBP501, TAK-285, TAK-593, MLN8054, MLN4924, pertuzumab, R1507, NK012, BIBF1120, BIBW2992, Patupilone, MK-2461, CP751,871, PF-00299804, satraplatin, CMC-544, YM155, GPI21016, and YHO-13351. Among these anti-cancer agents, an alkylating agent, a metabolic antagonist, a microtubule inhibitor, an antibiotic anti-cancer agent, a topoisomerase inhibitor, a platinum drug, a molecular targeted drug, which have cytotoxic activities are particularly preferred. Some specific anti-cancer agents can include gemcitabine, 5-FU, CPT-11, etoposide, cisplatin, oxaliplatin, paclitaxel, docetaxel, dacarbazine, doxorubicin, bevacizumab, cetuximab, anti-endothelial growth factor receptor 2 inhibiting antibody, and epidermal growth factor tyrosine kinase inhibitor.

[00138] In some cases, additional agents can be co-administered with the composition disclosed herein or administered separately. In some cases, the anti-cancer drug can be formed into a drug formulation, with a pharmacologically acceptable carrier, through mixing, dissolution, granulation, tabletizing, emulsification, encapsulation,

lyophilization, etc.

[00139] In some cases, the composition can be combined with other cancer therapies, including but not limited to surgical operation, radiotherapy (including gamma knife treatment, cyber knife treatment, boron neutron capture therapy, and proton radiation therapy /heavy ion therapy), MR-guided focused ultrasound surgery, cryotherapy, radio frequency ablation, ethanol-injection, and artery embolization.

[00140] In some cases, the pharmaceutical acceptable carrier can be a solvent or

dispersing medium comprising: water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, or vegetable oils.

[00141] In some cases, the composition can comprise a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for the compositions.

[00142] In some cases, the composition can comprise additives which enhance the

stability, sterility, and isotonicity of the compositions, including but not limited to, antimicrobial preservatives, antioxidants, chelating agents, and buffers. For example, antibacterial and antifungal agents can include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, the composition can include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Any vehicle, diluent, or additive used in the composition can be compatible with the isolated cardiac derived stromal cells.

[00143] In some cases, the compositions can be administered to the subject in an

injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents. Examples of the compositions can include liquid preparations for parenteral, subcutaneous, intradermal, intramuscular, transdermal, transcutaneous, intrathecal, intraperitoneal, intranasal, pulmonary, intravenous, implanted administration, or any combination thereof. Such compositions can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is entirely incorporated herein by reference for all purposes, may be consulted to prepare suitable preparations, without undue experimentation.

[00144] In some cases, the compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions can be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.

[00145] In some cases, viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent, including but not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener can depend upon the agent selected.

[00146] In some cases, a pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions. Preservatives include, but are limited to, benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride. A suitable concentration of the preservative can be from about 0.02% to about 2% based on the total weight of the composition/dosage.

[00147] In some cases, the compositions of this invention can be prepared by mixing the ingredients following generally accepted procedures. For example, isolated cardiac derived stromal cells can be resuspended in an appropriate pharmaceutically acceptable carrier. Then the mixture can be adjusted to the final concentration and viscosity by the addition of water or thickening agent. In some cases, a buffer can be added to control pH or an additional solute can be added to control tonicity. Generally the pH can be from about 3 to about 7.5. Dosages of the composition for humans or other mammals can be determined without undue experimentation by the skilled artisan.

[00148] In some cases, the composition can comprise a concentration of isolated cardiac derived stromal cells from about 2 ^χ 10 ⁴ cells to about 2 ^χ 10 ⁸ cells, from about 2 ^χ 10 ⁵ cells to about 2 ^χ 10 ⁷ cells, from about 2 ^χ 10 ⁶ cells to about 1 ^χ 10 ⁷ cells. In some cases, the composition can comprise a concentration of isolated cardiac derived stromal cells from about 1 ^χ 10 ⁴ cells/mL to about 1 ^χ 10 ⁸ cells/mL, from about 1 ^χ 10 ⁵ cells/mL to about 1 x 10 ⁷ cells/mL, or from about 1 ^χ 10 ⁶ cells/mL to about 5 ^χ 10 ⁶ cells/mL.

Formulation

[00149] Disclosed herein are formulations for treating a cell proliferation disorder. The plurality of isolated cardiac derived stromal cells and the pharmaceutically acceptable carrier can be selected such that the composition can exhibit physical properties (e.g., deformability, pliability, viscosity, consistency, stability, etc.) that can be desired for the particular application. For example, by varying the relative amounts of the components, the final compositions can be formulated to be injectable or sprayable.

[00150] In some cases, the composition can be in unit dose form. The composition can be formulated as a liquid, an aerosol, an aerosolized liquid, foam, a cream, a gel, an ointment, or any combination thereof. In some cases, the formulation can be an injectable liquid form. In some cases, the formulation can be a unit dosage injectable form (e.g., solution, suspension, emulsion). In some cases, the formulation can include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.

[00151] In some cases, the formulation can further comprise a physiological buffer, such as, for example, phosphate-buffered saline (PBS) or plasmaLyte. In some cases, the composition can further comprise a carrier protein, such as, for example, human serum albumin (HSA).

[00152] PlasmaLyte can be a family of balanced crystalloid solutions with multiple

different formulations available worldwide according to regional clinical practices and preferences. It can closely mimic human plasma in its content of electrolytes, osmolality, and pH. It can also have additional buffer capacity and contain anions such as acetate, gluconate, and even lactate that are converted to bicarbonate, C0 ₂ , and water. For example, in the U.S. a plasmaLyte injection solution can be a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL of the solution can contain 526 mg of sodium chloride; 502 mg of sodium gluconate; 368 mg of sodium acetate trihydrate; 37 mg of potassium chloride; and 30 mg of magnesium chloride. The solution can be adjusted with a sodium hydroxide solution to about pH 7.4, or within the range from about pH 6.5 to about pH 8.0.

[00153] Cryopreservative?,

[00154] The formulations can further comprise one or more cryopreservatives.

Cryopreservation can preserve cells or whole tissues by cooling them to low sub-zero temperatures (e.g., lower than -90 °C). At such low temperatures, biological activities, including those biochemical reactions leading to cell death, can be effectively stopped or slowed. Methods can be used to preserve cells obtained from mammals, including humans. For example, cryopreservation of cells in a medium containing from about 20% to about 90%) by weight of fetal bovine serum (FBS) and from about 10%> to about 20% by weight of dimethyl sulfoxide (DMSO) as a cryopreservative can yield viable cells in cells recovery stage upon thawing. In some cases, controlled slow rate of freezing can minimize the formation of intracellular ice-crystals, which can contribute to frozen cell damage during the cryopreservation procedure. Rapid thawing of frozen cells at 37 °C can also improve viable cell recoveries.

[00155] To prepare the isolated cells for cryopreservation, the cells can be suspended in a culture medium comprising a cryopreservative before being dispensed into the cryopreservation receptacle. The cryopreservative can minimize the deleterious effects of cryopreservation, such as formation of intracellular ice. Suitable cryopreservatives can comprise fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), polyethylene glycol, amino acids, polysaccharides (e.g., dextran, glucan, arabinogalactans, etc.), isopropyl alcohol, propanediol, glycerol, propylene glycol, sucrose, sodium glutamate, sorbitol, polyol (e.g. mannitol), trehalose, or a combination thereof. Other suitable

cryopreservatives include CRYYOSTOR™ cryopreservation media, such as CS5 (5% DMSO), CSIO (10% DMSO), and CS2/DLITE® (2% DMSO), available from BIOLIFE SOLUTIONS®. The resulting suspension of cells for cryopreservation can have a viable cell concentration of from about 1 million cells/mL to about 20 million cells/mL, about 2 million cells/mL to about 19 million cells/mL, about 3 million cells/mL to about 18 million cells/mL, about 4 million cells/mL to about 17 million cells/mL, about 5 million cells/mL to about 16 million cells/mL, about 6 million cells/mL to about 15 million cells/mL, about 7 million cells/mL to about 15 million cells/mL, about 8 million cells/mL to about 15 million cells/mL, about 9 million cells/mL to about 15 million cells/mL, from about 10 million cells/mL to about 15 million cells/mL, about 11 million cells/mL to about 15 million cells/mL, about 11 million cells/mL to about 14 million cells/mL, or about 11 million cells/mL to about 13 million cells/mL. For subcellular fractions the viable concentration for cryopreservation can range from about 1 mg/mL to about 200 mg/mL, about 2 mg/mL to about 190 mg/mL, about 3 mg/mL to about 180 mg/mL, about 4 mg/mL to about 170 mg/mL, about 5 mg/mL to about 160 mg/mL, about 6 mg/mL to about 150 mg/mL, about 7 mg/mL to about 130 mg/mL, about 8 mg/mL to about 100 mg/mL, about 9 mg/mL to about 70 mg/mL, from about 10 mg/mL to about 50 mg/mL, or about 15 mg/mL to about 25 mg/mL. The resulting suspensions can then be dispensed into the pellet-forming receptacle for cryopreservation.

[00156] Cells can be frozen within about 1 hour, about 2 hours, about 4 hours, 6 hours, about 9 hours, about 12 hours, about 15 hours, about 20 hours, about 24 hours, about 28 hours, about 32 hours, or about 36 hours after cells/tissues/organ harvesting. However, a longer or shorter period of time between the isolation of cells and the subsequent cryopreservation is possible, depending upon the cell preparation desired. For example, cells can be cryopreserved immediately after isolation, or as soon as reasonably possible after isolation (i.e., within 1 hour or less). Alternatively, cells may be cryopreserved after about 48 hours or longer after the isolation.

Kits

[00157] Disclosed herein are kits for treating a cell proliferation disorder. Kits useful in the methods of the disclosure comprise components useful in any of the compositions, formulations and methods described herein. The kits can for example, include necessary buffers and/or reagents for treating a cell proliferation disorder. In some cases, a kit can be a pharmaceutical pack. The kit can comprise a therapeutic agent suitable for treating a cell proliferation disorder and a set of instructions for administration of the therapeutic agent to a subject in need thereof. In some cases, a kit can also comprise a therapeutic agent suitable for treating a cell proliferation disorder and a packaging material that contains the cell proliferation disorder.

Methods

[00158] Disclosed herein are methods of administering the composition to a subject, for example, a human subject in need thereof. The subject can have or be suspected of having a condition. In some embodiments, the condition comprises a cell proliferation disorder, such as a cancer.

[00159] The cardiac derived stromal cells of the present disclosure can be used to treat a cell proliferation disorder. In some cases, the present disclosure provides a method to treat a cell proliferation disorder, comprising: administering to a subject an effective amount of a composition comprising isolated cardiac derived stromal cells.

Process

[00160] Disclosed herein are processes for forming a composition, a formulation, or a kit.

The process can comprise forming a composition, a formulation, or a kit for treating a cell proliferation disorder.

[00161] Isolating cells

[00162] Cells may be isolated from a number of sources, including, for example, from cardiac tissue. The isolated cells can be autologous cells, obtained by biopsy from the subject intended to be the recipient. The isolated cells can be allogenic cells, obtained from a subject that is a different individual but within the same species as the recipient.

[00163] Cells may be isolated using techniques known to those skilled in the art. For example, the tissue may be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation may be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase and dispase. Mechanical disruption may also be accomplished by a number of methods including, but not limited to, scraping the surface of the organ, the use of grinders, blenders, sieves, homogenizers, pressure cells, or in sonicators. Preferred cell types include stromal cells, such as mesenchymal stem cells (MSCs). The preferred cells can be MSCs isolated from cardiac tissues.

[00164] Once the tissue has been reduced to a suspension of individual cells, the

suspension may be fractionated into subpopulations from which the cells elements may be obtained. This also may be accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counterstreaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting.

[00165] Cell fractionation may also be desirable, for example, when the fraction displays a specific receptor or the lack thereof. A cell population may be sorted to separate desired cells from undesired cells. The desired cells, isolated from one or more sorting techniques, may then be used for treating a cell proliferation disorder.

[00166] Processing cells

[00167] Isolated cells may be cultured in vitro to increase the number of cells available for the intended treatment. If an immunological response occurs in the subject after treating with the isolated cardiac derived stromal cells, the subject may be treated with immunosuppressive agents such as, cyclosporin or FK506, to reduce the likelihood of rejection.

[00168] Isolated cells may be transfected with generic materials. Useful genetic material may be, for example, genetic sequences which are capable of reducing or eliminating an immune response in the host. For example, the expression of cell surface antigens such as class I and class II histocompatibility antigens may be suppressed. This may allow the transplanted cells to have reduced chance of rejection by the host. In addition, transfection could also be used for gene delivery.

[00169] Isolated cells can be expanded ex vivo. The cells can be expanded ex vivo prior to forming the composition. The cells can be expanded ex vivo after forming the composition. [00170] Isolated cells may be normal or genetically engineered to provide additional or normal function. Methods for genetically engineering cells with retroviral vectors, polyethylene glycol, or other methods known to those skilled in the art may be used. These include using expression vectors which transport and express nucleic acid molecules in the cells. (See Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), which is entirely incorporated herein by reference for all purposes.)

[00171] Vector DNA is introduced into prokaryotic or cells via conventional

transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A

Laboratory Manual, 3nd Edition, Cold Spring Harbor Laboratory press (2001), which is entirely incorporated herein by reference for all purposes), and other laboratory textbooks.

[00172] The process can comprise a cryopreservation process. The process can comprise a cryopreservation process where cells or any other biological samples are preserved by cooling to a low temperature. For example, the cryopreservation can be carried out at -80 °C using solid carbon dioxide or at -196 °C using liquid nitrogen. The

cryopreservation process can protect biological tissue from freezing damage due to ice formation.

[00173] The cryopreservation can be carried out by contacting the material to be frozen with a cryoprotectant. In some cases, the cryoprotectant can be glycols (e.g., alcohols containing at least two hydroxyl groups). For example, the cryoprotectant can be ethylene glycol, propylene glycol, or glycerol. In some cases, the cryoprotectant can be Dimethyl sulfoxide (DMSO). In some cases, the cryoprotectant can be a sugar. For example, the cryoprotectant can be sucrose or trehalose. In some cases, the

cryoprotectant can be glucose. In some cases, the cryoprotectant can be a polyol. For example, the cryoprotectant can be maltitol, sorbitol, xylitol, erythritol, or isomalt.

[00174] The process can comprise a lyophilization process. The lyophilization process can be a freeze-drying process. The lyophilization process can be a dehydration process that freezes the material and then reduces the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase. The lyophilization process can comprise a pretreatment, freezing, primary drying, or secondary drying step. The pretreatment step can comprise concentrating the product, formulation revision, decreasing a high-vapor-pressure solvent, or increasing the surface area. The freezing step can comprise placing the material in a freeze-drying flask and/or rotating the flask in a bath, which can be cooled by mechanical refrigeration, dry ice in aqueous methanol, liquid nitrogen, or any combinations thereof. In some cases, the freezing temperatures can be from about -50 °C to -80 °C. The primary drying phase can comprise lowing the pressure (e.g., using a partial vacuum) and/or suppling heat to the material. The secondary drying phase can comprise removing unfrozen water molecules.

[00175] The cryopreserved and/or lyophilized composition can be stable for at least about 1 hour, for example, at least about 1 hour, about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, about 24 hours, 36 hours, 48 hours, 3 days, 4, days, 5 days, 6 days, 7 days, about 2 weeks, 3 weeks, 4 weeks, 1 month, about 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, about 2 years, 3 years, 4 years, or 5 years. In some cases, the stability of the composition can be determined by carrying out an activity assay at a predetermined temperature, for example, at room temperature, at 4 °C, at -78.5 °C, or at - 196 °C. In some cases, the activity assay is an assay quantifying the therapeutic activity of the composition.

[00176] Making a formulation

[00177] The process can make a formulation for treating a cell proliferation disorder. The formulation can have from about 1 million to about 50 million cells (e.g., isolated cardiac derived stromal cells). The cells can be incubated under standard culturing conditions, such as, for example, 37 °C, 5% C0 ₂ , for a period of time.

[00178] In some cases, the formulation can be made by mixing individual ingredients, either sequentially or randomly.

EXAMPLES

Example 1 - method of cardiac stromal cells isolation and culture

[00179] Cardiac derived stromal cells are enzymatically isolated from auricle fragments or biopsy samples obtained from a subject by using 3 mg/mL collagenase (Sigma- Aldrich). The isolated cells are cultured in standard growth medium composed of Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) supplemented with 20% fetal bovine serum (FBS, Sigma- Adrich), 10 ng/mL basic Fibroblasts Growth Factor (bFGF, Millipore), 10,000 U/mL Penicillin/Streptomycin (Invitrogen), 20 mM L- Glutamine (Sigma-Aldrich).

[00180] Alternatively, human cardiac derived stromal cells are isolated from right atrial appendage specimens collected from patients during coronary artery bypass surgery according to a collagenase digestion protocol. Briefly, after removing fat tissues, atrial appendage specimens are extensively washed with phosphate-buffered saline (PBS) and mechanically minced into small pieces. Tissues are then digested with 0.075% collagenase I (Invitrogen) at 37 °C for 30 min. Then a high-density stromal vascular fraction is collected by centrifugation. The cell pellets are washed with phosphate- buffered saline and filtered through a 100 μπι nylon mesh to remove cellular debris. A cell count is performed. The cells are then plated and incubated overnight at 37 °C. Then the cells are washed with PBS to remove non-adherent cells. The cells are maintained at 37 °C in a 5% C0 ₂ -containing, humidified atmosphere in Dulbecco's Modified Eagle's medium (DMEM) containing 10% FBS. The medium is changed every 3 or 4 days. When the cells reach about 80% confluence, cells are harvested and plated into new flasks at an initial cell density of 1 ^χ 10 ⁴ cells/mL, and cultured in a-Minimum Essential Medium (a-MEM, Sigma-Aldrich) containing 10% FBS.

[00181] Human cardiac derived stromal cells can be propagated in Ham's F12 medium

(Gibco) supplemented with 10% FBS (Sigma- Adrich), 20 ng/mL of recombinant human bFGF (Milipore), 0.2 mmol/L of L-glutamine (Gibco), 0.005 U/mL of human erythropoietin (Invitrogen), and 100 U/mL of penicillin/streptomycin (Invitrogen). Cell lines are maintained under standard incubation conditions at 37 °C with 5% atmospheric C0 ₂ and passaged using TrypLE (Thermo Fisher) when approaching from about 70% to about 90% confluence.

Example 2— micro RN A profiling

[00182] Total RNA are extracted from cells using TRIzol reagent (Invitrogen) in

accordance to the manufacturer's instructions. Total RNA concentration and purity are evaluated by a NanoDrop 1000 spectrophotometer (Thermo Scientific), while RNA integrity is assessed with an Experion electrophoresis system and RNA High Sense Analysis Kit (Bio-Rad). High quality RNAs, with A260/A280 and A260/A230 ratios >1.8 and a RQI>9.5/10, are used for subsequent investigations. Comparative miR expression profiling is carried out using the TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystems), according to the manufacturer's instructions, using a 7900TH Real Time PCR System (Applied Biosystems).

[00183] Prior to the analysis, probes are renamed and reannotated according to miRBase Release 20 (www.mirbase.org). Up to 360 target sequences unique to human miRs are analyzed, omitting probes for transfer RNA (tRNA), small nucleolar RNA (snoRNA), and misannotated sequences. Expression analysis and quality control of TaqMan Arrays are performed using the ExpressionSuite Software vl .0.3 (Applied Biosystems). All Ct values reported as greater than 40 or as not detected were changed to 40 and considered a negative call. Raw expression intensities of target miRs are normalized for differences in the amount of total RNA added to each reaction using the mean expression value of all expressed miRs in a given sample. Relative quantitation of miR expression is performed using the comparative Ct method (AACt). MiRs are deemed as non-informative and filtered out when the percentile of negative calls exceeded 6 (20% of the samples).

Experiment 3 - isolation of CD105+ and CD34- cells

[00184] After obtaining about 90% confluence of cardiac derived stromal cell are

incubated with antibodies directed against human antigens CD 105 and CD34. The population of CD105+ and CD34- cells is separated using a BD FACSAria™ III cell sorter (BD Biosciences). The cells from the first to third passages are collected.

[00185] Isolation of cells with other desired biomarkers can be conducted similar to the procedure described above by using the appropriate antibodies and sorting processes. Experiment 4— tumor cell proliferation

[00186] For proliferation assays using co-cultured cells, stromal cells are seeded at about 5.0 10 ³ cells/well in 96-well plates in DMEM containing 1% FBS. After about 12 h, tumor cells are added (5.0 ^χ 10 ³ cells/well) to cultured stromal cells in plates. After an additional 24 h, cells are fixed by incubation in PBA containing about 5.4%

formaldehyde and about 0.8% glutaraldehyde at 12-h time points. After two washes with PBS, 100 μΙ_, 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-Gal) solution (2 mg/mL) is added to each well. Cells are then incubated at 37 °C in a humidified atmosphere containing 5% C0 ₂ in the dark for 12 h. Then absorbance at 595 nm is measured using an E-Max precision microplate reader (Molecular Devices)

[00187] Cell proliferation assays can also be performed in the absence of direct contact between stromal cells and tumor cells. Stromal cells are seeded at a density of about 5.0 x 10 ⁴ cells/well in 24-well plates in DMEM containing 1% FBS. After 12-h incubation, wells are covered with Cell Disks (Sumitomo Bakelite) that serve as a bulkhead, and tumor cells (5.0 ^χ 10 ⁴ cells/well) are added to the cell disks. After an additional 48 h, cells are lysed by the addition of lysis buffer (0.5% Triton X-100, 2 mol/L NaCl in PBS) at 12-h time points, and 100 [iL 2 mg/mL X-Gal solution is combined with 15 μΙ_, of the cell suspension. The absorbance at 595 nm of the resulting solution is then measured as described above. [00188] To assess cell proliferation in the presence of conditioned media from stromal cells, media are collected from stromal cells (2 ^χ 10 ⁶ cells) cultured in 10 mL DMEM containing 1% FBS in a culture dish (10 cm in diameter) for 48 h. The media are clarified by centrifugation (1,000 g, 5 min), and the resulting supernatant is used as conditioned media. Tumor cells are seeded at a density of 5.0 ^χ 10 ³ cells/well in 96-well plates and cultured in DMEM containing 1% FBS for 12 h. Subsequently, the media are replaced with either conditioned media or DMEM containing 1% FBS. Then 10 μΙ_, Alamar Blue assay solution (Biosource International) is added to the wells at 12-h time points, and the plates are incubated at 37 °C. Fluorescence is measured using a

Fluoroskan Ascent CF apparatus (Labsystems) with excitation set to 544 nm and emission set to 590 nm.

Experiment 5 - treating a subject

[00189] Jane is diagnosed to have breast cancer with a mean tumor size of about 2.0 cm based on mammography. Doctor Jack prescribes a dose of the composition comprising the isolated cardiac derived stromal cells. Cryopreserved populations of isolated cardiac derived stromal cells that have been stored at -80 °C are thawed on ice, and their viability is determined prior to the administration to Jane. Cell viability in selected cell population is above 90%. An amount of cells according to the prescription is mixed with a pharmaceutical carrier and a thickening agent (sterile water) to become a suspension for injection. Cells are then administered to Jane via intravenous injection. Dose is about 1 x 10 ⁶ cells. Weekly injections of the same dose continue for six month. Tests show that the tumor size becomes 1.0 cm based on mammography.

[00190] The injection can be local injection at the site of the tumor or suspected tumor.

For example, a breast cancer patient, such as Jane, can undergo mastectomy. During the surgery, a dose of the composition comprising the isolated cardiac derived stromal cells can be injected around the site of the tumor which has been surgically removed. The residual tumor cells after the mastectomy can be killed by the administered composition. For subjects who have or are suspected of having blood born cancers, IV injections can be performed using the disclosed composition. For subjects who have or are suspected of having leukemia or lymphoma, injections to the bone marrow can be performed using the disclosed composition.

Example 6— anti proliferative effects of cardiac derived stromal cells on triple negative breast cancer (TNBC) cells [00191] Breast cancer is the second leading cause of deaths in women after lung cancer and it is the most common cancer among women worldwide (about 23% of all new cancer cases). Triple negative breast cancer (TNBC) refers to the breast cancer phenotype which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), as assessed by immunohistochemistry (IHC), and which also lacks overexpression of HER2 as assessed by IHC or its gene amplification as assessed by fluorescent in situ hybridization technique. An estimated 1 million cases of breast cancer are diagnosed annually worldwide. Of these, approximately 170,000 (about 12%-20%) are of the triple-negative (ER-/PR-/HER2-) phenotype. Of these TNBC cases, about 75% are "basal-like". Regarding the molecular complexity of TNBC, six subtypes of TNBC have been identified, including, basal -like (BL1 and BL2), immunomodulatory (EVI), a mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR) subtypes. TNBC has a poor prognostic factor for disease-free survival (DFS) and overall survival (OS). There is no proven effective specific targeted therapy available for TNBC treatment.

[00192] These negative results for ER, PR and HER2 may indicate that the growth of the breast cancer is not supported by the hormones estrogen and progesterone, nor by the presence of overexpressed HER2 receptors. As a result, triple negative breast cancer may not respond to hormonal therapies, such as tamoxifen or aromatase inhibitors, or other therapies that target HER2 receptors, such as Herceptin (or trastuzumab). Tamoxifen and Herceptin are two of the effective medications for treating breast cancers. Additionally, TNBC may intrinsically resist or eventually acquire the resistance to chemotherapies.

[00193] MicroRNA 206 (miR-206) may be downregulated in TNBC when compared to non-TNBC cell lines and tissues. In addition, the decreased levels of miR-206 may be inversely correlated with the expression levels of VEGF. Furthermore, the forced expression of miR-206 in the miR-206 mimic-transfected TNBC cells may downregulate the VEGF, MAPK3, and SOX9 expression levels. The miR-206 mimics may inhibit TNBC breast cell invasion and angiogenesis. Accordingly, miRNA-206 may be involved in TNBC invasion and angiogenesis.

[00194] As described above, although the human cardiac derived stromal cells (CStrCs) may appear to have an identical or similar morphology and phenotype compare with those of bone marrow (BM) derived mesenchymal stem cells (MSCs), a global gene array analysis and a micro RNA array to compare human cardiac derived stromal cells (CStrCs) and BM-MSCs can show the differences. As described above, this analysis can demonstrate distinct gene expression patterns between these two sources of stromal cells. In some cases, as shown in Tables 4 and 5, the analysis showed that there are 26 miRs up regulated and 6 miRs down regulated by more than 2-fold in CStrCs when compared to BM MSC. In some cases, miR-206 is up regulated by more than one thousand fold in CStrCs. Therefore, the expression levels of miR-206 in CStrCs and BM-MSCs were further evaluated by quantitative PCR. The data in FIG. 12 show that there is an about 46-fold higher expression of miR-206 in CStrC when compared to BM MSCs.

[00195] Based on the down regulation of miR-206 in T BC cells, effects of CStrCs on

T BC cell lines were evaluated in vitro. FIG. 18 compares the total cell numbers of the TNBC cell line co-cultured on a CStrC culture for 7 days in alpha MEM plus 20% FCS and on a control culture (without the addition of CStrCs). HFM3 is a human TNBC cell line which is either p53 expressing (WT) cell line or p-53 knockout (KO) cell line. The conditions for the co-culture experiments can be similar to those described in Experiment 4. The results shown in FIG. 18 displays reduction of TNBC cell proliferation in the presence of CStrCs. FIG. 19 shows culture of TNBC cells (HIM3 WT and HIM3 KO) for 7 days in alpha MEM plus 20% FCS with co-cultured on CStrCs (bottom panels) and controls (no stromal layer - top panels). As shown in FIG. 19, growth of TNBC cell lines can be inhibited by co-cultured cardiac derived stromal cells when compared with those co-cultured in the absence of the cardiac derived stromal cells. In some cases, co-culture of the wild type TNBC cell line HIM3 or the p53 KO TNBC cell line HFM3 with cardiac derived stromal cells can result in the death of the TNBC cells.

[00196] While preferred embodiments of the present invention have been shown and

described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.