AU2015252071B2 - Systemic, allogenic stem cell therapies for treatment of diseases in animals - Google Patents

Abstract

A method for treating a patient suffering from a disease or diseased state includes administering a therapeutic dose of a mesenchymal stem cell conditioned media composition to the patient. The composition comprises the media in which mesenchymal stem cells from at least one tissue selected from the group consisting of placental tissue, bone marrow, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue were cultured. The composition is further suspended in saline. The patient may be human, canine, feline, equine, or lagomorph. Typically the disease or diseased state is selected from the group consisting of degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis (beagle pain syndrome), degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, muscular dystrophy, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.

Description

SYSTEMIC, ALLOGENIC STEM CELL THERAPIES FOR TREATMENT OF DISEASES IN ANIMALS

The present application is a divisional application from Australian Patent Application No. 2011295954, the entire disclosure of which is incorporated herein by reference.

FIELD OF INVENTION

The present disclosure relates to therapies using allogeneic, mesenchymal stem cells for the treatment of diseases in animals, with particular emphasis on canine, feline, equine, human, and lagomorph species.

BACKGROUND

Mesenchymal stem cells, multipotent mesenchymal stromal cells, and/or mesenchymal-like stem cells (all referred to herein as "MSCs") derived from human origin are known to possess the potential for multiple differentiation abilities in vitro and in vivo, although the source of those cells, e.g., the tissue and species from which those cells are harvested, have proven to significantly affect the pluripotent capability of those cells. Thus, while MSCs offer great therapeutic promise for a diverse range of medical applications, the range of differentiation, and therefore, therapeutic utility, appears to be related to the tissue and species origin of the MSC.

For instance, in humans, MSCs have been shown to express a broad spectrum of differentiation potential from cell types of mesodermal origin, like osteoblasts, adipocytes, chondrocytes to ectodermal (neuronal) and endodermal (hepatocytes) origins in response to chemical, hormonal or structural stimuli. However, to date, the most common source of MSC for therapeutic treatment is bone marrow, despite the fact that isolation of MSCs from bone marrow results in: smaller number of available MSCs as compared to other sources, severe discomfort to the patient during isolation, and a drastic decrease in the available numbers of MSCs with age. Further, while some insight into the use of stem cells for therapeutic treatment has been gained in recent years, there is still a great deal to be learned about the efficacy and dosage of particular MSCs in treating specific diseases and diseased states.

Further, in order for a therapeutic treatment utilizing MSCs to be practical, there must be an available supply of the MSCs necessary for the treatment at the time the individual requires the therapy. As noted above, MSCs originating from certain tissues may be in short supply from the donor, or may result in severe discomfort or negative side effects to the donor.

As such, it would be greatly appreciated to identify sources of MSCs that are readily available for harvest during routine procedures, and which may effective in treating specific diseases or disease states in a given species, and particularly in humans and companion animals.

The discussion of the background to the invention included herein including reference to documents, acts, materials, devices, articles and the like is included to explain the context of the present invention. This is not to be taken as an admission or a suggestion that any of the material referred to was published, known or part of the common general knowledge in Australia or in any other country as at the priority date of any of the claims.

SUMMARY

Viewed from one aspect, the present invention provides a method for treating a patient suffering from a preselected disease or diseased state, comprising the step of administering a therapeutic dose of a mesenchymal stem cell conditioned media composition to a patient suffering from a preselected disease or diseased state, wherein the mesenchymal stem cell conditioned media composition comprises the media in which mesenchymal stem cells from at least one tissue selected from the group consisting of placental tissue, bone marrow, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue were cultured; wherein the mesenchymal stem cell conditioned media composition is further suspended in saline; wherein the patient is selected from the group consisting of human, canine, feline, equine, or lagomorph; and wherein the preselected disease or diseased state is selected from the group consisting of degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis (beagle pain syndrome), degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy. cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, muscular dystrophy, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.

Viewed from another aspect, the present invention provides a mesenchymal stem cell conditioned media composition comprising: a conditioned media comprising the media in which mesenchymal stem cells from at least one tissue selected from the group consisting of placental tissue, bone marrow, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue were cultured.

Viewed from another aspect, there is provided a method for treating a patient suffering from a preselected disease or diseased state, the method comprising the step of systemically administering a therapeutic dose of a mesenchymal stem cell composition to a patient suffering from a preselected disease or diseased state through an intravenous injection, the mesenchymal stem cell composition comprising mesenchymal stem cells harvested from at least one tissue selected from a group including placental tissue, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue, wherein the mesenchymal stem cell composition includes mesenchymal stem cells at a concentration of no more than 2 million cells/ml, wherein the patient is selected from the group consisting of human, canine, feline, equine, or lagomorph, and wherein the preselected disease or diseased state is selected from the group consisting of: degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis, beagle pain syndrome, degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, spinal injury, hepatitis, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.

Also described herein is a method of manufacturing compositions including MSCs, the compositions being useful for treating preselected diseases. Various embodiments, advantages, and features of the invention will be apparent from the description which follows.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments described in the following written specification. It is understood that no limitation to the scope of the invention is thereby intended. It is further understood that the present invention includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the invention as would normally occur to one skilled in the art to which this invention pertains.

According to one embodiment, a method for treating a specified disease or diseased state comprises the intravenous (IV) injection of a therapeutic dose of preselected MSCs to a patient suffering from an identified disease or diseased state. While site-specific injection of preselected MSCs to a patient suffering from an identified disease or diseased state is within the scope of the present application, systemic treatment through IV injection has proven to reduce invasiveness, improve the speed and cost of the procedure, and decrease morbidity and recovery time when compared to site-specific application.

It will be appreciated that a method for treating a specified disease or diseased state, according to certain embodiments, comprises one or more doses of the preselected MSCs at preselected time intervals. By way of nonlimiting example the preselected MSCs are delivered at approximately weekly intervals, at approximately two week intervals, at approximately three week intervals, at approximately monthly intervals, at approximately two month intervals, at approximately three month intervals, at approximately four month intervals, at approximately five month intervals, or at approximately six month intervals.

In at least one embodiment, the therapeutic dose of each IV injection of preselected MSCs comprises about six million MSCs per kg of the patient’s body weight up to a maximum of fifty million MSCs regardless of body weight. In order to obtain the necessary number oi MSCs, preselected MSCs are collected and expanded utilizing cell culture techniques described in further detail below, and those expanded MSCs are harvested and segregated into cell counts of approximately six million to about fifty million cells, as needed. As used herein, a therapeutic dose means the number of MSCs of sufficient quantity to decrease the physiological symptoms of the specified disease or diseased state in the patient.

Thereafter, according to one embodiment, those segregated cells are diluted into £ balanced saline solution or other suitable dilutive solution such that each diluted population o: cells has a concentration of approximately 2,000,000 cells or less per mL of solution. According to yet another embodiment, each cell count is diluted to a concentration of approximate!) 1,000,000 cells or less per mL of solution; approximately 500,000 cells or less per mL o: solution; approximately 250,000 cells or less per mL of solution; and approximately 100,00( cells or less per mL of solution. It will be appreciated that the diluted population of cells may br harvested such that they are free of substantially all of the culture medium upon which the MSC: were expanded, or the cells may be harvested to include at least a portion of the cell conditioner medium upon which the cells were expanded. Likewise, the diluted population of cells maj comprise additional physiological electrolyte additives. Alternatively, the medium may also b( delivered free of cells.

In certain embodiments, preselected MSCs are autologous or allogeneic to the patient, and those preselected MSCs may be isolated from a donor during health procedures that are unrelated to the purpose of harvesting MSCs. As such, MSCs may be harvested in a manner tha1 does not adversely affect the donor or result in unnecessary medical treatments to the donor. Foi instance, placental, uterine, umbilical cord, and testicle MSCs may be harvested during routine birthing procedures or during spay/neuter treatments for dogs and cats, with those tissues being banked for later expansion or later use. Further, it will be appreciated that donors are preferably screened to ensure that the donor is in good general health, and may be screened for the presence of diseases, current status of vaccinations, or presence of antibiotics in the donor’s system as necessary.

Preselected MSCs include dental derived stem cells such as stem cells harvested iron: dental pulp, periodontal ligaments, and other dental tissues; stem cells harvested from testicle tissue; stem cells harvested from bone marrow; stem cells harvested from placental tissue; sten cells harvested from uterine tissue (including endometrial regenerative cells), stem cells harvested from umbilical cord tissue, and stem cells harvested from full thickness skin biopsies.

An identified disease or diseased state includes degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease atopy, hepatitis, chronic steroid responsive meningitis-arteritis, beagle pain syndrome degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated hemolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease muscle fibrosis secondary to disease or trauma, refractory corneal ulcer, diabetes mellitus, spina trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spina injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, comeal ulcer, eczema multiple sclerosis, muscular dystrophy, spinal injury, diabetes mellitus, hepatitis, myocardia infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.

Further, according to at least one embodiment, a method for treating a specified disease or diseased state comprises the topical administration of a therapeutic dose of preselected MSCs or MSC conditioned media to a patient suffering from an identified disease or diseased state and/or in need of treatment for the identified disease or diseased state. According to at least one example, a suspension of MSCs in a saline solution is applied topically to a patient suffering from atopy or eczema. According to certain embodiments, the suspension of MSCs further contains portions of the cell conditioned media upon which the MSCs were cultured, and may contain an agent for making the suspension thicker, such as a colloid or hydrogel. According to certain embodiments, a method for treating a specified disease comprises the topical administration of a suspension of cell conditioned media (sometimes referred to as “spent media”) without the MSCs.

EXAMPLES A. Methods for Extracting and Processing MSCs from Selected Tissue.

The present application contemplates the collection and delivery of a naturally occurring population of cells derived from placental/umbilical cord, bone marrow, skin, or tooth pulp tissue. The designated name for this population is mesenchymal stem cells, or “MSCs”. MSCs are a population of adherent multipotent mesenchymal stromal cells originating from the mesoderm with some populations (such as those from teeth) having potential ectoderm origin as well (e.g. ecto-mesenchymal). In accordance with the invention, the MSCs are generally ar adherent cell population expressing markers CD90 and CD 105 (>90%) and lacking expression oJ CD34 and CD45 and MHC class II (<5%) as detected by flow cytometry. It will be appreciated that each of the MSCs were extracted and processed from the preselected tissues as noted below. 1. Placental, Testicle, and Uterine Tissue.

Placenta was collected from delivery procedures, and testicle and uterine tissue was collected from spay or neuter procedures. Regardless of which tissue was collected, the tissue was placed in sterile containers with phosphate buffered saline (“PBS”), penicillin/streptomycir and amphotericin B. Specifically, harvested tissue was first surface sterilized by multiple washes with sterile PBS, followed by immersion in 1% povidoneiodine (“PVP-1”) for 2 minutes, immersion in 0.1% sodium thiosulfate in PBS for 1 minute, and another wash in sterile PBS, Next the tissue is dissected into 5g pieces for digestion. Enzymatic digestion was performed using a mixture of collagenase type I and type II along with thermolysin as a neutral protease.

The digestion occurred in a 50cc sterile chamber for 20-45 minutes until the tissue was disaggregated and the suspending solution was turbid with cells. Next the solution was extractec leaving behind the matrix, and cold (4°C) balanced salt solution with fetal bovine serum (“FBS”^ at 5% concentration was added to quench the enzymes. This resulting suspension was centrifuged at 600 x g, supernatant is aspirated and MESENCULT® complete medium (basa medium containing MSC stimulatory supplements available from StemCell Technologies Vancouver, British Columbia) was added to a final volume of approximately 1.5 times the digestion volume to neutralize the digestion enzymes. This mixture was centrifuged at 500 g foi 5 minutes, and the supernatant aspirated. The cell pellet was be re-suspended in ffesl MESENCULT® complete medium plus 0.25 mg/mL amphotericin B, 100 IU/mL penicillin-G and 100 mg/mL streptomycin (JR Scientific, Woodland, CA). Cells were plated at an initia concentration of one starting 5g tissue digest per 225 cm2 flask. Culture flasks were monitorec daily and any contaminated flasks removed immediately and recorded. Non-contaminated flask; were monitored for cell growth, with medium changes taking place three times per week. Aftei 14 days of growth, MSC were detached using 0.25% trypsin/lmM EDTA (available fron Invitrogen, Carlsbad, CA). Cell counts and viability were assessed using flow cytometrj techniques and cells were banked by controlled rate freezing in sealed vials. 2. Bone Marrow.

Bone maiTow was collected and placed within a “washing tube”. Before the collectior procedure a “washing tube” is prepared in the class 100 Biological Safety Cabinet in a Clas: 10,000 GMP Clean Room. To prepare the washing tube, 0.2 mL amphotericin B (Sigma· Aldrich, St Louis, MO), 0.2 mL penicillin/streptomycin (Sigma 50 ug/nl) and 0.1 mL EDTA· Na2 (Sigma) were added to a 50 mL conical tube (Nunc) containing 40 mL of GMP-grad( phosphate buffered saline (PBS). Specifically, the washing tube containing the collected bom marrow was topped up to 50 mL with PBS in a class 100 Biological Safety Cabinet and cells wa; washed by centrifugation at 500 g for 10 minutes at room temperature, which produced a cel pellet at the bottom of the conical tube. Under sterile conditions supernatant was decanted anc the cell pellet was gently dissociated by tapping until the pellet appeared liquid. The pellet wa: re-suspended in 25 mL of PBS and gently mixed so as to produce a uniform mixture of cells ii PBS. In order to purify mononuclear cells, 15 mL of Ficoll-Paque (Fisher Scientific, Portsmoutl NH) density gradient was added underneath the cell-PBS mixture using a 15 mL pipette. The mixture was subsequently centrifuged for 20 minutes at 900 g. Thereafter, the huffy coat was collected and placed into another 50 mL conical tube together with 40 mL of PBS. Cells were then centrifuged at 400g for 10 minutes, after which the supernatant was decanted and the cel] pellet re-suspended in 40 mL of PBS and centrifuged again for 10 minutes at 400g. The cel pellet was subsequently re-suspended in 5 mL complete DMEM-low glucose media (GibcoBRL Grand Island, NY) supplemented with 20% Fetal Bovine Serum specified to have Endotoxir level less than or equal to 100 EU/mL (with levels routinely less than or equal to 10 EU/mL) anc hemoglobin level less than or equal to 30 mg/dl (levels routinely less than or equal to 25 mg/dl) The serum lot used was sequestered and one lot is used for all experiments. Additionally, the media was supplemented with 1% penicillin/streptomycin, 1% amphotericin B, and 1 °A glutamine. The re-suspended cells were mononuclear cells substantially free of erythrocytes anc polymorphonuclear leukocytes as assessed by visual morphology microscopically. Viability o: the cells was assessed with trypan blue. Only samples with >90% viability were selected foi cryopreservation in sealed vials. 3. Tooth Pulp.

Teeth were extracted under sterile conditions and placed into sterile chilled vial: containing 20mL of phosphate buffered saline with penicillin/streptomycin and amphotericin I (Sigma-Aldrich, St. Louis, MO). Teeth were thereafter externally sterilized and processed firs by washing several times in sterile PBS, followed by immersion in 1% povidoneiodine (PVP-1 for 2 minutes, immersion in 0.1% sodium thiosulfate in PBS for 1 minute, followed by anothe: wash in sterile PBS. The roots of cleaned teeth were separated from the crown using pliers anc forceps to reveal the dental pulp, and the pulp was placed into an enzymatic bath consisting o type I and type II collagenase (Vitacyte, Indianapolis, USA) with thermolysin as the neutra protease. Pulp tissue was allowed to incubate at 37°C for 20-40 min to digest the tissue anc liberate the cells. Once digestion was complete, MESENCULT® complete medium was addec to a final volume of 1.5x the digestion volume to neutralize the digestion enzymes. This mixtun was centrifuged at 500 g for 5 min, and the supernatant aspirated. The cell pellet were re suspended in fresh MESENCULT® complete medium plus 0.25 mg/mL amphotericin B, 10( IU/mL penicillin-G, and 100 mg/mL streptomycin (JR Scientific, Woodland, CA). Cells wen plated at an initial concentration of one tooth digest per 25 cm2 flask. Culture flasks wert monitored daily and any contaminated flasks removed immediately and recorded. Non contaminated flasks were monitored for cell growth, with medium changes taking place thre< times per week. After 14 days of growth, MSC were detached using 0.25% trypsin/lmM EDT/ (Invitrogen, Carlsbad, CA), cell counts and viability were assessed using a standard trypan blu< dye exclusion assay (Sigma) and hemacytometer, and bAU3 the DPSC divided equally betweei two 75 cm2 flasks. After the first passage, DPSC cultures were harvested once they reach 70 80% confluence. These cells were cryopreserved in sealed vials. 4. Skin Tissue. MSCs from the skin, including epidermal, dermal, and subcutaneous tissue of health] adult patients undergoing cosmetic plastic surgery were isolated by collagenase digestioi procedure. Once received, the tissue was cleaned of any unwanted adipose tissue and hair. Th< tissue was then sterilized using IX PYP-iodine solution and IX sodium thiosulfate followed b] washing twice in sterile PBS. The dermis was then minced into 1 mm3 pieces following collagenase enzymatic digestion for 30-40 minutes at 37°C. Afterwards, tissue pieces wer< dissociated by pipetting into 5 mL pipette and centrifuged at 300g for 5 min. The pellet wa; suspended in cell growth media Dulbecco's Modified Eagle Medium: Nutrient Mixture F-E (“DMEM/F12”) (available from Invitrogen, Carlsbad, CA) (1:1) containing amphoterecin penicillin and streptomycin supplemented with 10% fetal bovine serum. Cell suspensions wen transferred into T-25 tissue culture flask and grown until 80-90% confluence. The cells wen placed in a T-75 flask before being used for flow analysis and differentiation. 5. Umbilical Cord Tissue. MSCs from the umbilical cord were harvested during delivery. Once received, the tissu< was washed two to three times in sterile PBS and then divided into pieces of approximately i grams each. Thereafter, the tissue was decontaminated, and each 5 gram aliquot of tissue wa; placed in a sterile 100 mm tissue culture dish, and covered with a lid to prevent drying. Th< tissue was dissociated via enzymatic digestion in 50 cc tubes, and was minced into fragment less than 1 mm using a sterile scalpel. Then, the chopped tissue was placed in an enzyme bath and the tube was capped and transferred to an incubator. The tubes were swirled for fifteen seconds every ten minutes for forty minutes. Thereafter, the digesting enzyme was diluted by adding 45 mL of cold DME/F12 complete media (FBS, Pen/Strep and Amphotericin B), with the tubes being capped and inverted to mix the contents. Next, the tubes were centrifuged at 400 x g for fifteen minutes on low break. The top media was aspirated using a 25 mF pipette by leaving approximately 5 mL at the bottom of the tube, with special care being taken to aspirate the entire medium in the tube. The bottom 5 mF medium (containing tissue fragments and cells including MSCs) was resuspended in fresh 20 mF DME-1712 complete medium mixed well and placed into a t-75 flask, and transferred to an incubator. The tissue was washed off during the first media change after 48 hours post-digestion, and the media was changed three times per week. Cells were grown to 70%-80% confluence and then either passaged, frozen down as passage zero cells, or differentiated. Cells were not allowed to reach confluence or to remain at confluence for extended periods of time. B. Methods for Expanding Cell Populations.

Cell expansion for cells originating from any of the abovementioned tissues above took place in clean room facilities purpose built for cell therapy manufacture and meeting GMP clean room classification. In a sterile class II biologic safety cabinet located in a class 10,000 clean production suite, cells were thawed under controlled conditions and washed in a 15 ml, conical tube with 10 MF of complete DMEM-low glucose media (cDMEM) (GibcoBRF, Grand Island, NY) supplemented with 20% Fetal Bovine Serum (Atlas) from dairy cattle confirmed to have no BSE % Fetal Bovine Serum specified to have Endotoxin level less than or equal to 100 EU/mF ( with levels routinely less than or equal to 10 EU/mF) and hemoglobin level less than or equal to 30 mg/dl (levels routinely less than or equal to 25 mg/dl). The serum lot used was sequestered and one lot was used for all experiments.

Cells were subsequently placed in a T-225 flask containing 45 mF of cDMEM and cultured for 24 hours at 37°C at 5% C02 in a fully humidified atmosphere. This allowed the MSC to adhere. Non-adherent cells were washed off using cDMEM by gentle rinsing of the flask. Adherent cells were subsequently detached by washing the cells with PBS and addition of 0.05% trypsin containing EDTA (Gibco, Grand Island, NY, USA) for 2 minutes at 37°C at 5% C02 in a fully humidified atmosphere. Cells were centrifuged, washed and plated in T-225 flasl in 45 mL of cDMEM.

This resulted in approximately 6 million cells per initiating T-225 flask. The cells of the first flask were then split into 4 flasks. Cells were grown for 4 days after which approximately ( million cells per flask were present (24 million cells total). This scheme was repeated but celli were not expanded beyond 10 passages, and were then banked in 6 million cell aliquots in sealec vials for delivery.

All processes in the generation, expansion, and product production were performed undei conditions and testing that was compliant with current Good Manufacturing Processes anc appropriate controls, as well as Guidances issued by the FDA in 1998 Guidance for Industry Guidance for Human Somatic Cell Therapy and Gene Therapy; the 2008 Guidance for FDA Reviewers and Sponsors Content and Review of Chemistry, Manufacturing, and Control (CMC Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs): and the 1993 FDA points-to-consider document for master cell banks were all followed for the generation of the cell products described.

Donor cells were collected in sterile conditions, shipped to a contract manufacturing facility, assessed for lack of contamination and expanded. The expanded cells were stored ir cryovials of approximately 6 million cells/vial, with approximately 100 vials per donor. At eact step of the expansion quality control procedures were in place to ensure lack of contamination oi abnormal cell growth.

In another aspect, cells are grown in media and the cells, along with the media, are recovered after about 5-10 days. The cells are prepared in this "conditioned" media foi transfusion at concentrations of less than about 100,000 cells per mL. Physiological electrolyte additives may be added. The cell solution is administered intravenously.

In a further method, cells are grown in media for about 5-10 days. This media is ther transfused intravenously without cells or given locally to the site of the injury. Further methods involve isolation and/or concentration of stem cell produced factors and/or further refinements ol these chemicals and/or compounds.

It is contemplated in one embodiment that the above-described treatments may be administered to treat horses with systemic doses following rigorous training, particularly to address exercise induced pulmonary edema, EIPH, recurrent airway obstruction (RAO), pleuritis and other respiratory issues, as well as exertional rhabdomyolysis. Thus, the treatment is preventative in nature. In other embodiments, the treatments can be directed doses to treat dorsal displacement (DDSP). C. Description of Diseases and Diseased States.

In certain embodiments, MSC’s can be administered to an human/animal in need of treatment for one, or one or more, of the following diseases or diseased states, or others mentioned herein, potentially with or without need for treatment for any other diseases or disease states that could be treated with the MSC’s. 1. Arthritis

Arthritis is classified as non-inflammatory or inflammatory based on joint fluid analysis. Degenerative joint disease/osteoarthritis (“DJD/OA”) is considered noninflammatory, and displays degenerative changes in the joint with lack of fever, leukocytosis or other systemic signs. Osteoarthritis (“OA”) is a breakdown of articular cartilage causing increased edema in the joint, osteophyte formation and fibrosis of the periarticular soft tissues. The end result is loss of elasticity, joint degeneration and instability. A series of changes in the articular cartilage tha1 cause inflammation can eventually lead to complete loss of cartilage. The remodeling and inflammatory changes create pain that decrease the mobility of the affected joint therefore muscle atrophy results.

Osteoarthritis is further classified as primary or secondary depending on the etiology. Primary OA is due to cartilage degeneration in aging pets and occurs for unknown reasons. Secondary OA, which is more common than primary, occurs in response to an injury, abnormality or disease that causes joint instability. Either form of the disease is always considered progressive regardless of cause or treatment. Arthritis and OA are found in all breeds and ages of dogs and cats. 2. Rheumatoid Arthritis.

Rheumatoid arthritis (“RA”) is considered an inflammatory joint disease due to inflammatory changes that occur in the synovium along with systemic clinical signs. Inflammatory joint disease is further classified as infectious or immune mediated, and immune mediated disease is considered erosive or non-erosive. RA is an inflammatory, noninfectious. erosive, immune mediated polyarthritis of dogs where the synovial membrane proliferates. While the pathogenesis is not fully understood, it is characterized by the prostaglandins causing erosion of the subchondral bone beginning at the joint margins in turn causing granulation tissue to invade the bone. Antibodies called rheumatoid factors (IgG, IgM and IgA) are produced against an antigen (IgG) which produce joint inflammation. Thereafter, the synovial membrane thickens, fibrosis occurs, pannus (vascular tissue) invades the joint and releases proteolytic enzymes causing erosion of articular cartilage and subchondral bone. The articular surface then collapses which destabilizes the joint leading to subluxation or luxation that appears as a deformed joint on physical exam and radiographs.

Clinical signs vary depending on the stage of the disease. In early stages the patienl shows shifting leg lameness, possible low grade fever, inappetance, and mild lymphadenopathy Lameness later becomes more severe along with the other clinical signs. Radiographic changes usually are not visible for first few weeks until detailed radiographs show cyst like lucent lesions in the subchondral bone. Later in the disease degenerative radiographic changes are obvious as are worsening clinical signs of joint swelling subcutaneous nodules, and significant joint pain.

Along with clinical signs and radiographic findings, rheumatoid factor (“RF”) may be found in serum or joint fluid although up to 30% of dogs with RA will test seronegative Therefore a negative RF does not rule out the disease and a positive RF does not definitive diagnose RA due to false positives in non RA animals with other inflammatory diseases elsewhere in the body. The age of onset of RA is 1-9 years with an average of 4-5 years. Smal breed dogs are most commonly affected, with poodles and shelties over represented. 3. Degenerative Radiculomyelopathy.

Degenerative radiculomyelopathy and German Shepherd degenerative myelopathy are slowly progressive neurologic disorders of unknown etiology affecting most commonly middle age to older large breed dogs. A gradual loss of white matter of the spinal cord and myelir causing ataxia/weakness and eventually paraparesis of the pelvic limbs is observed. This disease most often occurs in middle-age to older large breed dogs but has been found all breeds/mixec breeds, no sex predilection, in some young animals and in cats. German Shepherd breeds appeal to be over represented.

Early in the disease the pet has difficulty getting up from a lying or sitting position. Tht back end will sway or the gait will appear ataxic. The pelvic limbs may begin to criss-cross ovei one another. As the disease progresses, the pet will drag her toenails on the ground wher walking. One characteristic of affected patients includes nails wearing down from dragging th« hind feet. Generalized weakness of the hind end of the animal becomes more obvious, mors difficulty rising and more difficulty getting stable on slick flooring. However, the disease doe: not generally result in pain to the patient. 4. Chronic Renal Failure.

Chronic renal failure (“CRF”) is defined as a progressive, irreversible renal dysfunctioi that occurs over months to years. Chronic disease is differentiated from acute based on patien history, physical exam and/or laboratory findings to suggest the disease has been present for ai extended period. CRF is characterized by azotemia along with a low urine specific gravib (dogs<1.030, cats<1.035) for a prolonged period of time. These animals also have a 75^ reduction in their functional renal mass. CRF will continue to progress negatively even after an] possible inciting cause is removed. Clinical signs include poor appetite, poor hair coat, polyuria polydipsia, weight loss, usually small kidney size on palpation and radiographs unless polycystii disease or neoplastic disease, osteodystophy may be present most often in the jaw (rubber jaw) pale mucus membranes from non-regenerative anemia, oral ulcers, acute blindness, cervica ventroflexion, hypothermic, hypertensive. 5. Dilated Cardiomyopathy.

Dilated cardiomyopathy (“DCM”) is characterized by diminished contractile dysfunctior and cardiac chamber dilation. It is the second most common form of heart disease in the dog. DCM is idiopathic, and possibly genetic due to it being found as an inherited autosomal recessive trait in the Portuguese water dog who is the only canine breed to show a juvenile form of DCM. Other canine breeds that are overrepresented include the Boxer, Doberman Pinscher. Great Dane, Newfoundland and Irish Wolfhound. These breeds are middle age when clinical signs, usually congestive heart failure, occur. Contractile dysfunction leads to congestive hearl failure (“CHF”). CHF is not purely a consequence of impaired pump function, but is also z neuroendocrine syndrome in which activation of the adrenergic nervous system and specific endocrine pathways such as the rennin-angiotensin-aldosterone system play an integral role When cardiac function declines, compensatory mechanisms activate to maintain systemic perfusion, pressures and cardiac output. 6. Chronic Hepatitis.

Chronic hepatitis (“CH”) morphology is characterized by hepatocellular apoptosis oi necrosis, mixed inflammatory cells and/or variable mononuclear cells and fibrosis. Many causes of hepatitis exist but the underlying cause of chronic hepatitis is often undetermined. Causes oi hepatitis can include copper storage disease which allows an animal to accumulate abnormal levels of copper in the liver until toxicity occurs. It is an inherited disease found in, amongsl others, mixed breed and pure breed dogs including Bedlington Terriers, West Highland White terriers, Doberman Pinchers, Skye Terriers, Dalmatians and Labrador Retrievers. Infectious diseases have been associated with hepatitis such as leptospirosis and canine viral hepatitis. CH usually occurs between 4-10 years of age, with chronic hepatitis being more common in female dogs. 7. Atopy, Eczema.

Atopy (inhalant dermatitis) is a hypersensitivity reaction to inhaled or cutaneouslj absorbed environmental antigens in individuals who are genetically predisposed. Age of onsei can be from 6 months to 6 years with the average being between 1-3 years. Clinical signs include skin erythema, generalized or local pruitis that can be seasonal or non-seasonal, areas oj moist dermatitis, papules, pustules, ulcerative eruptions, scales, hyperpigmentation, lichenification or alopecia. Self-trauma can result in secondary skin lesions including excoriations, saliva staining, lick granuloma or even open wounds. Atopy can also manifest as chronic otitis externa, conjunctivitis, epiphora, allergic bronchitis, rhinitis, secondary pyoderma Malassezia dermatitis, chronic acral lick dermatitis or rarely hyperhidrosis.

Similarly, eczema is a general term for any type of dermatitis or inflammation of the skin Eczema will cause pruritus (itching) and redness of the skin with blistering, weeping and/oi peeling being possible. There are several skin diseases that are considered eczemas with Atopic Dermatitis (AD) or Atopy being the most severe and chronic disease on the list. AD is presern worldwide but seems more common in developed countries affecting men and women equally oJ all races. AD is not contagious but is inherited; the clinical signs often develop during infancy 01 early childhood the majority of the time. Some children are fortunate enough to “outgrow’ eczema but most are affected for a lifetime. The clinical signs of AD are dry, scaly, itchy skin, cracks in the skin, and rashes on the cheeks, arms and legs. The symptoms are episodic, during £ flare up it is not uncommon to develop open weeping or crusted sores from infection or selfexcoriation. Eczema usually affects the insides of the elbows, the back of the knees and the face but can cover most of the body. People who have atopic dermatitis often either have family members or they themselves suffer from asthma, hay fever or both; these three diseases togethe] are referred to as the “Atopy triad.”

There are trigger factors that people are exposed to which worsen or cause their AD tc flare-up. Trigger factors are substances or conditions such as dry skin, allergens including fooc or environmental, stress, extreme climate changes, exercise (heat/sweat) and numerous irritant? such as smoke, fumes, fragrances, detergents, etc. When people with AD are exposed to a triggei factor which they are sensitive to, an over production of inflammatory cells migrate to the skir causing a pruritic and painful reaction, which in turns causes the person to scratch worsening the reaction. The underlying pathophysiology of AD is an over response of the immune system to ar allergen or irritant.

Traditional treatment of Eczema or AD has been topical creams that modify the skin’? immune response and thereby inflammatory response (hydrocortisone can be used only shon term, tacrolimus or pimecrolimus), skin moisturizers (petroleum based), behavior modification tc avoid irritants and in more advanced cases short term injections or oral use of steroids are used People with AD are prone to skin infections, namely staph and herpes, therefore they are taugh to watch for clinical signs of skin infection and if suspected, consult with their doctoi immediately to avoid aggravating the disease. Recent studies have identified direct links between canine atopy and the human form of atopic dermatitis making the naturally occurring disease in the canine an ideal model for analogous human studies. 8. Keratoconjunctivitis Sicca.

Keratoconjunctivitis sicca (“KCS”) is a common ocular disease in the dog characterizec by decreased aqueous tear production that can result in comeal and conjunctival pain anc disease. KCS can affect the vision, especially if left untreated, blindness or loss of the globe car occur. KCS has many underlying etiologies but the most common cause is immune mediatec lacrimoadenitis. Based on a positive response to immunomodulation therapy, greater than 75°/« of canine KCS cases are due to this immune mediated inflammation of the lacrimal gland. This immune mediated etiology also appears to be highly breed related and is most common in those dogs with atopic skin disease such as the Golden Retriever. Other causes of KCS include: a congenital lacrimal gland atresia that do not respond to immune modulating drugs and breeds commonly affected include the Yorkies, Beagles, Miniature Pinschers and Miniature Dachshunds; b) neurogenic KCS can be seen in animals that have had severe otitis extema/media/intema that now have lack of parasympathetic innervation to the lacrimal anc third eyelids; c) ocular surface infections (primary or secondary to systemic disease, distemper: d) iatrogenic KCS from amputation of third eyelid for removal of cherry eye; and f) certain drug therapy can cause decreased tear production. Breeds that are over represented include Englisl· Bulldogs, American Cocker Spaniels, West Highland White Terriers, Lhasa Apsos, Shih Tsus Pugs, Pekingese, Boston Terriers, Cavalier King Charles Spaniels, Yorkshire Terriers anc Miniature Poodles. 9. Systemic Lupus Erythematosus.

Systemic Lupus erythematosus (SLE) is a multi-systemic immune-mediated disease ir which antibodies are directed against the body’s tissues and circulating immune complexes are deposited affecting multiple systems. The most common affected areas are the joints, kidney; and skin. SLE is the result of a Type III hypersensitivity reaction although it may also be associated with type II and IV reactions. SLE is an immune complex deposition disease and car also cause heightened antibody responsiveness with a tendency to produce autoantibodies Usually either the immune complex or the autoantibody aspect of the disease predominates in the animal.

Clinical signs can be severe and variable but considering the most common systems affected the most common clinical signs of the immune complex SLE are: lameness, fever, pah (polyarthritis), polyuria/polydipsia, anorexia, nausea dehydration (renal disease) anc dermatological (mucocutaneous) disorders. The dermatological signs are one of the mos common and extremely diverse clinical signs in animals with SLE. Skin lesions tend to be symmetrical and most commonly affect the mucocutaneous junctions of the body, the feet anc the ears. The skin lesions will often present as erythematous, crusty dermatitis. The mos common disorders associated with the autoimmune aspect of SLE are hemolytic anemia anc thrombocytopenia. With either variety of SLE multiple organ systems may become involved including the cardiovascular system and central nervous system. Psychosis has even beer reported in animals with SLE similarly to human patients with SLE. Females are at slightb greater risk than males. German Shepherds, Collies and Shetland Sheepdogs are thought to be a greater risk. More than 40% of dogs diagnosed with SLE succumb to the disease within on< year of diagnosis. SLE is rare but has been documented in cats and large animals. 10. Immune-mediated Thrombocytopenia and Immune-mediated Hemolytic Anemia.

Immune-mediated thrombocytopenia (“IMT”) and Immune-mediated hemolytic anemi; (“IMHA”) can occur in the cat and dog as primary or secondary disease. When both arise simultaneously the disorder is referred to as Evans syndrome. IMT is a common cause of non traumatic bleeding in small animals and IMHA is a common cause of anemia. Primary IMT anc IMHA is the result of an autoimmune disorder, while secondary IMT or IMHA can result ii response to a variety of infectious, inflammatory or neoplastic diseases or can be attributed t( drag insult. IMT is most commonly secondary to infectious or neoplastic disease in cats, unlike IMT in dogs. The mechanism of destruction of red blood cells in IMHA is antibody mediatec cytotoxic (Type II). IMHA affects young to middle aged animals with Cocker Spaniels, Englisl Springer Spaniels, Poodles and Old English Sheepdogs being overrepresented in the dog population. IMT is most often seen in middle aged female dogs with average age of onset being 6 years. IMHA clinical signs can range from non-clinical signs of anemia such as pale mucus membranes to more significant disease including lethargy, weakness, a hemic heart murmur often compensatory tachycardia, tachypnea and bounding pulses are noted. Some patients wil have ongoing immunological or inflammatory disease clinical findings such as fever or anorexic or less commonly lymphadenopathy. Jaundice (icterus) is a common finding in IMHA patients due to extravascular hemolysis. Pulmonary thromboembolism is a common complication o: IMHA with severe anemia, especially if on aggressive steroid treatment. IMT appears as spontaneous hemorrhage in otherwise healthy appearing dogs. Cats may have other clinica symptoms of a primary disorder. Questioning of the owner may uncover previous minoi episodes of bleeding. The hallmark lesion of IMT in any species is petechial hemorrhage tha may merge into ecchymosis. 11. Steroid Responsive Meningitis-arteritis.

Steroid responsive meningitis-arteritis (“SRMA”) is of unknown etiology but is though to be immune mediated in origin. SRMA is also known as juvenile polyarteritis, necrotizing vasculitis, canine juvenile polyarteritis syndrome, and Beagle Pain Syndrome. SRMA arises it juveniles, with no sex predilection, and most notably found in the Beagle. SRMA may also be found in other breeds including Bernese Mountain dogs, German Shorthaired Pointers, Boxers Toller Retrievers and mixed breeds. Two forms of the disease are described: acute/fulminatin^ and chronic. The acute form is characterized by neutrophilic pleocytosis of the cerebrospina fluid and the chronic form by mononuclear or mixed cell pleocytosis accompanied witl neurological deficits. Either form may have systemic necrotizing vasculitis with severs subarachnoid hemorrhages throughout the length of the spinal cord and brain stem. Thrombosi: and vascular occlusion may lead to neural ischemia. Affected vessels may contain cells witl

IgG and hemosiderin filled macrophages. Amyloidosis and systemic vasculitis may occur in some dogs. SRMA is known to affect medium to large breed dogs usually less than 2 years oi age but as old as 7 years. With early aggressive immunosuppressive treatment therapy approximately 60-80% of dogs are cured with 20-40% having relapse within the treatment phase, Animals who relapse appear to have a more protracted course of signs and treatment duration There is no current way to predict who will relapse. 12. Inflammatory Bowel Disease.

Inflammatory bowel disease (“IBD”) in the canine, feline, and other species can be defined clinically as a spectrum of gastrointestinal disorders associated with chronic inflammation of the stomach, intestine and/or colon. A diagnosis of IBD is suspected only if the clinical signs have persisted chronically, usually at least 3 weeks. Often clinicians will make e presumptive diagnosis of IBD based on chronicity, clinical signs, failure to respond tc symptomatic treatment and failure to continue with diagnostics for various causes. For thii reason, many cases of IBD have an unknown etiology; however, certain forms of IBD (i.e. histiocytic in the Boxer breed of canines) is thought to have a genetic influence and there i: strong evidence to support an immune mediated etiology.

The pathology of IBD has been directed at the immune system. The exact immune mechanism responsible is still unclear but IBD is thought to be the loss of immunologic tolerance to the normal bacterial flora or food antigens, leading to abnormal T cell immune reactivity in the gut microenvironment. Genetically engineered animal models that develop IBE involve alterations of T cell function suggesting that T cell populations are responsible fo: intestinal mucosal homeostatic regulation of immune responses. Immunohistochemical studie: have shown an increase in the T cell population of the lamina propria, including CD3+ cells CD4+ cells, as well as macrophages, neutrophils and Ig-A containing plasma cells. Many of the immunologic features of canine IBD can be explained by mucosal T cell activation. Enterocyte! also play a role in the immunopathogenesis by acting like antigen presenting cells. Enterocyte: also produce interleukins (IL-7, IL-15) during inflammation and activate mucosal lymphocytes Therefore, a subset of CD4+ T cells within the intestinal epithelium that overproduc: inflammatory cytokines with a concurrent loss of another subset of CD4+ T cells, and thei: associated cytokines, which normally regulate the inflammatory response and protect the gut from injury; as well as enterocytes acting as antigen presenting cells, all contribute to the pathogenesis of IBD. 13. Feline Cholangitis.

Feline cholangitis is the second most common liver disease in cats after hepatic lipidosis. Three forms of cholangitis have been recognized in cats: neutrophilic (bacterial or rarely protozoal), lymphocytic (immune mediated) and chronic (associated with liver fluke infection). 14. Feline Eosinophilic Disease.

Feline eosinophilic disease is a broad term that encompasses several eosinophilic reactions/granulomatous. Synonyms include feline eosinophilic granuloma complex, feline indolent ulcer, rodent ulcer, eosinophilic ulcer, eosinophilic plaque, feline linear granuloma and feline collagenolitic granuloma. The underlying allergic disease appears to provoke an episode although it is thought to have a genetic predisposition as well. Clinical signs include raised, well-demarcated erythematous yellow-to-pink colored linear to circular plaques are found. These lesions may be located on the ventrum, thighs, footpads, lip margins or chin. The lesions may also become ulcerated, are usually very pruritic and extremely painful. 15. Heart Disease.

Feline heart disease occurs in different forms with the most common being hypertrophic cardiomyopathy (“HCM”) although processes such as myocarditis and/or infarction can occui with any type of heart disease. Secondary heart disease can also occur due to hyperthyroidism oi hypertension. HCM occurs due to the combination of impaired ventricular relaxation anc increased ventricular stiffness that leads to diastolic dysfunction. Most cats will also have dynamic outflow obstruction which causes the mitral valve to prolapse during systole anc regurgitation occurs. This in turn eventually leads to increased atrial pressures then tc congestive heart failure (“CHF”). HCM is most likely genetic in origin. Mutations in myosir binding protein C have been identified in Maine coons and Ragdolls with HCM, but gene mutation testing (“MBPC”) is only available for the Maine coon. 16. Exercise-induced Pulmonary Hemorrhage. (“EIPH”) is most often seen in race horses and other horses used for sport that undergc strenuous exercise for short periods of time. Epistaxis is observed only in approximately 5% o: horses who are known to have exercise-induced pulmonary hemorrhage. Although it is though all performance horses experience EIPH to some extent when exposed to strenuous activity, i rarely results in death, but does cause decrease lung function over time. Bleeding is caused b] rupture of the pulmonary capillaries with subsequent pulmonary inflammation, fibrosis anc angiogenesis which leads to further bleeding. Proposed mechanisms of the initial cause of EIPP include high pulmonary vascular pressures during maximum exercise, intrathoracic shear force: generated during exercise, failure of the pulmonary system to compensate or keep up with th< extreme increase in cardiac output to meet the demand of high intensity exercise, coagulatioi dysfunction and/or neovascularization secondary to pulmonary inflammation. Currently, it i: thought that with chronicity of disease, scarring of the lung will occur that will cause reduced ga: exchange and therefore reduced athletic potential. Clinical signs include epistaxis, blood in th( trachea after exercise, coughing, increased swallowing and/or prolonged recovery after exercise. 17. Exertional Rhabdomyolysis.

Exertional rhabdomyolysis (“ER”, also known as tying up) can affect any horse but is : common disease of performance animals and can be a recurrent problem. There are differen degrees of the disease to which a horse can be affected from subclinical to life threatening an< commonly the time of onset after exercise correlates with severity of disease, with the earlie onset relating to more significant disease. ER occurs in response to an inadequate blood flow t< the skeletal muscles of an exercising horse, with the lack of oxygenated, blood, the muscle cell begin to function anaerobically to produce the needed ATP. The longer the horse exercise and/or the more predisposing factors present, the more muscle fibers that become damaged thei the more severe the disease becomes. The more muscle cells/fibers involved then the mor clinical signs that are seen. Eventually the muscle cell membrane breaks down, enzymes an< myoglobin leak out which is then filtered by the renal system, hence the myoglobinuria and rena tubular damage that occurs with the life threatening form of the disease

An inherited and acquired form of the disease can occur. An animal with the inherited form is most likely to continue having recurrent episodes of ER. Inherited causes of ER originate from defective calcium regulation which is common in Thoroughbreds and causes 0 recurrent form of the disease. Polysaccharide storage myopathy (“PSSM”) is a comparable inheritable myopathy that is often associated with ER and occurs commonly in quarter horses as well as other breeds. Acquired causes of the disease are many and usually occur as ε combination etiology involving a horse undergoing unaccustomed exercise in addition to another predisposing factor such as: overfeeding carbohydrates (grain, pellets), sudden increase in worl; load in an animal with poor body condition, existing electrolyte or mineral imbalances (especially potassium), a deficiency in selenium or vitamin E (selenium levels should be measured before supplementation), hormone imbalance especially in frllies/mares, hypothyroidism, weather conditions being wet or cold. Females are more predisposed that males 18. Spinal Disease.

Spinal disease in rabbits, particularly in the aged rabbit, is not well defined or understooc at this time. It is known that older rabbits can suffer from spinal disease that can be progressive and leave them in a paraplegic or ataxic state in severe cases. Clinical signs can be difficult tc diagnose since rabbits do not readily show signs of pain. Veterinarians and owners must be astute in looking for the secondary signs of spinal disease and/or pain. Abnormalities of gait o: unwillingness to move about are often overlooked if the rabbit spends most of its time confmec to a hutch, rabbits may become more aggressive to owners or cage mates, they may have problems grooming themselves and therefore develop a soiled perineum area, perineal urine scald and associated dermatitis, inability to groom also leads to the buildup of scale therefore development of Cheyletiella is common, the rabbit usually can no longer reach the anus to inges cecotrophs therefore secondary digestive disorders or hypomotility may occur, the rabbit may be less interactive than usual, ataxia, loss of conscious propriorception, urinary or feca incontinence. Vertebral spondylosis, kyphosis or lordosis is a common finding in pet rabbits or radiographs and can cause pain, stiffness and degenerative disease. These spinal deformities car have many causes including congenital, a low calcium diet, metabolic bone disease, vitamin E deficiency, inactivity and/or a small cage size. Degenerative disc disease is disc protrusion anc nuclear extrusion has been confirmed post-mortem as a cause of hind limb paralysis. This can b< from forceful movement producing hyperflexion of the spine that may not result in a dislocatioi or fracture but may result in a disc lesion. Spontaneous degenerative spinal disease has also beei studied in laboratory rabbits. These consist of chondroid metaplasia of the nucleus pulposus calcification of the nucleus pulposus and spondylosis. 19. Myocardial Infarction.

Over 1.1 million Americans have a heart attack (myocardial infarction or “MI”) eacl year. Although 80% survive the initial heart attack, nearly half become disabled with hear failure over the next six years. A heart attack occurs when a coronary artery become: completely blocked so starving a section of heart muscle (myocardium) of oxygen and nutrients If the blockage remains the section of heart muscle will die. The major complication ii survivors is that in the days after the attack, tissues surrounding the dead zone are inadequatel; irrigated by collateral blood vessels, and also die off. The loss of heart muscle subsequently lead to the onset of heart failure. Enhanced blood flow to surviving heart muscle and, ultimatel; cardiac regeneration, is the essential goal in ensuring that risk of heart failure after heart attack i minimized. 20. Congestive Heart Failure.

Congestive heart failure (“CHF”) typically occurs when an injured heart muscle is unabl to pump strongly enough to maintain sufficient blood circulation to meet the needs of the body' other organs. Patients are constantly tired, short of breath, and in and out of hospital. One-thin of patients with CHF require repeat hospitalization within three months after discharge. 21. Spinal Cord Injury

Spinal Cord Injury (SCI) is damage to the spinal cord that results in a loss of functio: such as mobility or feeling. Frequent causes of damage are trauma (car accident, gunshot, falk etc.) or disease (polio, spina bifida, Friedreich's Ataxia, etc.). The spinal cord does not have to b severed in order for a loss of fimctioning to occur. In fact, in most people with SCI, the spina cord is intact, but the damage to it results in loss of functioning.

The effects of SCI depend on the type of injury and the level of the injury. SCI can be divided into two types of injury - complete and incomplete. A complete injury means that there is no function below the level of the injury; no sensation and no voluntary movement. Both sides of the body are equally affected. An incomplete injury means that there is some functioning below the primary level of the injury. A patient with an incomplete injury may be able to move one limb more than another, may be able to feel parts of the body that cannot be moved, or may have more functioning on one side of the body than the other. With the advances in acute treatment of SCI, incomplete injuries are becoming more common.

Besides a loss of sensation or motor functioning, individuals with SCI also experience other changes. For example, they may experience dysfunction of the bowel and bladder. Very high injuries (C-l, C-2) can result in a loss of many involuntary functions including the ability to breathe, necessitating breathing aids such as mechanical ventilators or diaphragmatic pacemakers. Other effects of SCI may include low blood pressure, inability to regulate blood pressure effectively, reduced control of body temperature, inability to sweat below the level oi injury, and chronic pain. 22. Skeletal Muscle Fibrosis.

Skeletal muscle fibrosis is a life changing problem in individuals who suffer from disorders that target these muscles (Muscular Dystrophy, Multiple Sclerosis) or denervation atrophy induced by trauma or neuromuscular disease. Skeletal muscle fibrosis affects individuals of all race and ages including those with specific disease that suffer denervatior fibrosis and those healthy athletes who over train or suffer a severe muscle injury. Microscopic tears that occur in musculature over time during exertion can cause muscle stiffness and fibrosis later in life that can become painful and even crippling. 23. Muscular Dystrophy.

Muscular dystrophy (“MD”) refers to a group of hereditary muscle diseases that weakens the muscles that move the human body. Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue Nine diseases including Duchene, Becker, limb girdle, congenital, scioscapulohumeral. myotonic, oculopharyngeal, distal, and Emery-Dreifuss are classified as muscular dystrophy, although there are more than 100 diseases in total with similarities to muscular dystrophy. Mosi types of MD are multi-system disorders with manifestations in body systems including the heart gastrointestinal and nervous systems, endocrine glands, skin, eyes and even brain. The conditior may also lead to mood swings and learning difficulties. 24. Multiple Sclerosis.

Multiple sclerosis (“MS”) is an autoimmune disease that affects the central nervoui system. MS is caused by damage to the myelin sheath, the protective covering that surrounds nerve cells, it is a demyelinating disease. When this nerve covering is damaged, nerve impulses are slowed down or stopped. The nerve damage is caused by inflammation. Inflammation occurs when the body's own immune cells attack the nervous system. Repeated episodes o1 inflammation can occur along any area of the brain, optic nerve, and spinal cord. Because nerves in any part of the brain or spinal cord may be damaged, patients with multiple sclerosis can have symptoms in many different organ systems. Clinical symptoms can be generalized but an usually multiple. Symptoms of MS may mimic those of many other nervous system disorders. D. Clinical Results of Treatment. 1. Intervertebral Disc Disease, Spinal Cord Injury

According to one exemplary embodiment, a canine spinal injury patient exhibiting IVDE from an acute spinal injury incurred one month pre-treatment was treated with a therapeutic dost of MSCs derived from dental tissue and testicle tissue. Prior to the treatment regime, the patien displayed hind limb ataxia with a grade 3.5/5, and the patient could not support weight to wall up and down stairs. Further, the patient could not support weight long enough to walk i significant distance, had severe crepitus in distal thoracic and cranial lumbar spine, and could no run at all. Prior steroid and non-steroidal therapy was not helping at the time of treatment although the patient was displaying normal bladder and bowel control.

The patient received three MSC intravenous injections at two week intervals, with on< dose comprising MSCs derived from testicle tissue, and the last two injections derived firon dental tissue. After receiving the treatment, the patient had a much improved hind limb ataxia t< grade 1/5, was easily moving up and down stairways, was able to walk more than one mile daily and was able to run with no administration of steroid or non-steroidal therapy. In fact, the ver mild (grade 1) ataxia post-treatment was noticed only when the patient was very tired or ove worked.

Yet another canine patient exhibiting chronic IVDD from to genetic hemi-vertebrae wa; treated with a therapeutic dose of dental tissue derived MSCs at one month intervals. Prior t< treatment, this patient was utilizing a pull-cart for mobility, as no hind leg movement had beei exhibited for approximately 1 year prior to treatment. Likewise, the patient had has develope< arthritis in the front left elbow for the prior 6 months, making it difficult to use the car effectively. The patient received three therapeutic doses of MSCs via intravenous injection a one month intervals. After the therapy, the patient developed some movement in the hind limbs and mimics a “walking action” while utilizing the pull-cart, but is not yet able to support fill body weight without use of the cart. The arthritis in the front elbow improved considerably allowing greatly improved mobility with the cart.

According to another exemplary embodiment, a lagomorph patient exhibiting chronii spinal disease (IVDD) leading to paresis of hind limbs was treated with a single therapeutic dosi of bone marrow stem cells administered intravenously. Prior to treatment, the patient ha< become non-ambulatory for several months, despite being bright and alert with a good appetifc prior to the onset of the disease. Prior to treatment with a therapeutic dose of bone marrov derived MSCs, the patient was being treated daily through physical therapy, but the patient begai to not be able to move her front legs or lift her head/neck region and showed signs of depressioj one month prior to presentation. After intravenous treatment with a therapeutic dose of MSCs the patient became bright and alert again, began eating better and began trying to move her fron limbs and head. The patient was able to start lifting her head/neck region and front arms simila to the extent shown prior to the onset of the disease. 2. Chronic Osteoarthritis.

Yet another canine patient was treated for chronic OA with a single therapeutic dose o dental tissue derived MSCs. Prior to treatment, this patient was taking long term (approximately 1.5 years) non-steroidal anti-inflammatory medications which required frequent blood work tc monitor liver and kidney function. After treatment, the patient’s energy and hair coat improved and the patient was able to cease all OA medications. The patient’s activity greatly improved and the beneficial results were maintained until another therapeutic dose of MSCs was requiret approximately 6 months after the treatment.

According to another exemplary embodiment, a geriatric feline was treated for chronii osteoarthritis with a single therapeutic dose of dental tissue derived MSCs. Prior to treatment the patient’s chronic osteoarthritis had developed to a level that significantly affected activity level, and had significantly affected the gross anatomic structure of the patient’s front limbs a the elbow level. However, the patient was on no medications prior to the treatment. Afte treatment, the patient displayed increased activity, reduced lethargy and sleep time, and showed: marked improvement in appetite.

According to another exemplary embodiment, a geriatric lagomorph patient with sever generalized arthritis, primarily of the spine and all limbs, was treated with a single therapeuti dose of bone marrow derived MSCs. Prior to treatment, the patient spend the majority of its da; with little movement, was on pain medication and non-steroidal anti-inflammatory medication t< treat the osteoarthritis, and was prone to self-soiling. After the single treatment, the patien became extremely active, moving around on his own. Further, the patient was again able ti posture to urinate and defecate, decreasing the prior problem of self-soiling. The patient’ overall health, appetite, and body weight increased, allowing the anti-inflammatory medication to be discontinued.

According to another exemplary embodiment, a geriatric lagomorph patient with sever generalized arthritis, especially of the front limbs due to a previous front limb fracture an subsequent surgery of the area, was treated with a single therapeutic dose of bone marro\ derived MSCs. Prior to the treatment, the patient’s previously fractured front limb wa significantly deformed, causing the patient to fall often. Further, the patient was quite sedentary was taking a pain medication, a glucosamine-chondroitin supplement and a non-steroidal anti inflammatory. After the single treatment, the patient was able to discontinue the paii medication, and was more ambulatory and interactive with an increased appetite. 3. Inflammatory Bowel Disease (IBD).

Another canine patient was treated for chronic OA with a two therapeutic doses of denta tissue derived MSCs spaced approximately three months apart. Prior to the treatment, the patien was on a prescription diet, and was administered several different medications twice daily to hel] control severe lymphocytic-plasmacytic colitis. As the disease progressed it was more difficult t< control with medication/diet and the patient began losing significant amounts of weight, with thi patient’s hair becoming dry and brittle, signifying poor uptake of nutrients.

After the treatment, the patient remained on the pre-treatment medication, but after tb first therapeutic dose of MSCs was administered, the patient’s feces changed from a watery bloody stool to a semi-formed, non-hemorrhagic stool. The patient regained 3A of the weight los prior to the treatment, and the patient’s hair improved dramatically. Thereafter, two of th medications for treatment of lymphocytic-plasmacytic colitis were discontinued altogether, witi all other treatments remaining unchanged. The patient became more active and displayed bette overall health. 4. Early stage Degenerative Myelopathy (DM).

Another canine patient was treated for degenerative myelopathy with a single therapeuti dose of dental derived MSCs given intravenously. Prior to treatment, the patient was becomin ataxic in the hind limbs with conscious proprioception deficits. The patient would “sway” whe: walking, and had difficulty negotiating stairs and moving around the house. Post therapy, th patient displayed increased strength and vigor overall, and had notable ataxia improvement wit no noticeable digression for approximately four months after treatment. 5. Cardiomyopathy.

According to another exemplary embodiment, another feline patient was treated for generalized inflammatory disorder of unknown etiology and feline cardiomyopathy diagnose via echocardiography with a single therapeutic dose of dental derived MSCs given intravenousl) Prior to treatment, the patient’s condition was not stabilized the patient was displaying marke lethargy. After treatment, echocardiography measurements showed noticeable improvement o the cardiomyopathy symptoms. Further, the patient showed dramatic improvement of the genera lethargy noted pre-treatment, and showed an activity and appetite associated with a norma individual. 6. Muscle Fibrosis.

According to another exemplary embodiment, a lagomorph patient was treated for IVDI and muscle fibrosis of the hind limbs with a single dose of bone marrow derived MSC intravenously. Prior to treatment, the patient displayed chronic spinal disease leading to paresi of hind limbs and eventually fibrosis of musculature of affected limbs. While the front limbs di< not show signs of muscular fibrosis, deformation of the front limbs was noted due to overuse The patient was utilizing a mobility cart for 6 months prior to treatment.

After treatment, the patient’s hind limb fibrosis greatly improved to the point that the lej could be flexed and manipulated for physical therapy to occur, and the patient’s hind limbs wen no longer frozen in one position. As physical therapy continued, the patient began to regain thi ability to bend her legs underneath her under her own power, and was able to balance hersel without her cart and take some steps on her own. The patient displayed improved overall genera health.

According to another exemplary embodiment, an equine patient was treated for long tern muscle fibrosis and OA from previous right scapular fracture with one therapeutic dose o placental derived MSCs administered three times at monthly intervals. Prior to treatment, th< patient’s front right shoulder showed severe supraspinatus and infraspinatus muscle fibrosis, lacl of blood supply to the site according to a thermography study, decreased range of motion in th< affected limb as well as atrophy of all shoulder and proximal arm musculature. Osteoarthritis o the shoulder became a problem for the arm as a sequential disease to the trauma as well. Afte: the treatments, the patient’s muscle fibrosis greatly improved, and blood supply to the are; greatly improved as shown by thermography comparison studies. Further, the patient’s range o: motion in the affected limb improved dramatically, the shoulder muscle mass as measured by th< circumference of the leg measurably improved from previous records, and the patient was able t( discontinue prior OA medication. 7. Exercise Induced Pulmonary Hemorrhage (EIPH).

According to one exemplary embodiment, an equine patient diagnosed with EIPH vi; endoscopy and bronchoalveolar lavage (“BAL”) was treated with four therapeutic doses o: placental derived MSCs administered intravenously, with each dose administered at monthl· intervals. Prior to treatment, the patient would tire easily and failed to finish well in races Further, the patient displayed a thin overall body condition score, measuring a 2/5. Afte: treatment, a post-race endoscopy and BAL testing showed marked improvement in th< pulmonary hemorrhage (EIPH), the patient was not winded after racing, and consistently placet in finishing positions in races. Further, the patient’s body condition score improved to 3.5/5. 8. Rhabdomyolysis.

An equine patient diagnosed with exercised induced rhabdomyolysis, severe generalizet muscle soreness with associated electrolyte abnormalities, was treated with one therapeutic dos< of dental derived MSCs intravenously. Prior to treatment, clinical signs of exercised inducec rhabdomyolysis persisted for days after and episode of extreme exercise. After treatment, th< patient was able to move around normally, eat normally and begin exercise regimen. Further, th< patient showed diminished signs of pain in normal stance and gait. 9. Spinal Cord Injury.

An equine patient displaying a caudal spinal cord injury from an impact with a wal during a race was treated with four therapeutic doses of dental derived MSC, with each dosi administered at monthly intervals. Prior to the treatment, the patient was ataxic in the hind limb; and without control of urinary, rectal, or penile function. Initial emergency treatment improve< the hind limb ataxia to a mild ataxia, allowing the patient to walk/trot. However the patien required catheterization in order to relieve his bladder and avoid bladder rupture, which occurrec in one instance. After treatment, the patient regained control of his rectal function and furthe regained enough control of penile function to allow at least partial re-sheathing of the member t< prevent damage from drying out. Further, the patient regained the ability to urinate withou being catheterized.

Claims (20)

1. The claims defining the invention are as follows:

   1. A method for treating a patient suffering from a preselected disease or diseased state, comprising the step of administering a therapeutic dose of a mesenchymal stem cell conditioned media composition to a patient suffering from a preselected disease or diseased state, wherein the mesenchymal stem cell conditioned media composition comprises the media in which mesenchymal stem cells from at least one tissue selected from the group consisting of placental tissue, bone marrow, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue were cultured; wherein the mesenchymal stem cell conditioned media composition is further suspended in saline; wherein the patient is selected from the group consisting of human, canine, feline, equine, or lagomorph; and wherein the preselected disease or diseased state is selected from the group consisting of degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis (beagle pain syndrome), degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, muscular dystrophy, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.
2. 2. The method of claim 1, wherein said administering comprises systemic administration of the mesenchymal stem cell conditioned media composition.
3. 3. The method of claim 1 or claim 2, wherein said systemic administration comprises intravenous administration of the mesenchymal stem cell conditioned media composition.
4. 4. The method of claim 1, wherein said administering comprises local administration of the mesenchymal stem cell conditioned media composition.
5. 5. The method of claim 4, wherein said local administration comprises a local injection of the mesenchymal stem cell conditioned media composition.
6. 6. The method according to any one of the preceding claims, wherein the mesenchymal stem cell conditioned media composition is administered without cells.
7. 7. A mesenchymal stem cell conditioned media composition comprising: a conditioned media comprising the media in which mesenchymal stem cells from at least one tissue selected from the group consisting of placental tissue, bone marrow, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue were cultured.
8. 8. The mesenchymal stem cell conditioned media composition of claim 7, wherein the composition is operable to reduce or eliminate the symptoms of one or more diseases or diseased states in a target patient, wherein the diseases or diseased states are selected from the group consisting of degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis, beagle pain syndrome, degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, comeal ulcer, eczema, multiple sclerosis, spinal injury, hepatitis, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.
9. 9. The mesenchymal stem cell conditioned media composition of claim 7 or claim 8, wherein the mesenchymal stem cells are autologous or allogeneic to a target patient.
10. 10. The mesenchymal stem cell conditioned media composition according to any one of claims 7 to 9, wherein the media is essentially cell-free.
11. 11. The mesenchymal stem cell conditioned media composition according to any one of claims 7 to 10, wherein the mesenchymal stem cells were cultured in said media for a period comprising at least 5 days.
12. 12. A method for treating a patient suffering from a preselected disease or diseased state, the method substantially as hereinbefore described with reference to any one of the embodiments as described in the Examples.
13. 13. A mesenchymal stem cell conditioned media composition substantially as hereinbefore described with reference to any one of the embodiments as described in the Examples.
14. 14. A method for treating a patient suffering from a preselected disease or diseased state, the method comprising the step of systemically administering a therapeutic dose of a mesenchymal stem cell composition to a patient suffering from a preselected disease or diseased state through an intravenous injection, the mesenchymal stem cell composition comprising mesenchymal stem cells harvested from at least one tissue selected from a group including placental tissue, dental tissue, testicle tissue, uterine tissue, umbilical cord tissue, and skin tissue, wherein the mesenchymal stem cell composition includes mesenchymal stem cells at a concentration of no more than 2 million cells/ml, wherein the patient is selected from the group consisting of human, canine, feline, equine, or lagomorph, and wherein the preselected disease or diseased state is selected from the group consisting of: degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis, beagle pain syndrome, degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated memolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, spinal injury, hepatitis, myocardial infarction, congestive heart failure, and muscle fibrosis secondary to disease or trauma.
15. 15. The method of claim 14, wherein the therapeutically effective dose is about 6 million mesenchymal stem cells per kg of a patient's body weight.
16. 16. The method of claim 14 or 15, wherein the therapeutically effective dose does not exceed about 50 million mesenchymal stem cells regardless of the patient's body weight.
17. 17. The method of any one of claims 14 to 16, wherein the mesenchymal stem cell composition consists essentially of mesenchymal stem cells and saline.
18. 18. The method of any one of claims 14 to 17, wherein the mesenchymal stem cell composition further comprises factors from a stem cell conditioned media.
19. 19. The method of any one of claims 14 to 18, wherein the mesenchymal stem cells are harvested from a human tissue source.
20. 20. The method of any one of claims 14 to 19, wherein the mesenchymal stem cells autologous or allogeneic to the patient.