Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0085—Brain, e.g. brain implants; Spinal cord
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2523/00—Culture process characterised by temperature

Definitions

• the present invention relates to treatment regimens for multiple sclerosis comprising administration of adipose-derived stem cells.
• the invention relates to treatment of progressive forms of multiple sclerosis using intra-ventricular or intrathecal administration of adipose-derived stem cells.
• MS Multiple sclerosis
• CNS central nervous system
• RRMS relapsing-remitting
• PMS period of partial or complete recovery
• SPMS secondary progressive MS
• PPMS primary progressive MS
• Patients with relapsing-remitting MS are typically treated with corticosteroids during acute attacks (relapses), and with immunomodulatory- or immunosuppressive drugs to prevent new relapses and progression of disability.
• immunomodulatory- or immunosuppressive drugs include interferon beta (Avonex®, Rebif®, Betaseron®), glatiramer acetate (Copaxone®), dimethyl fumarate (Tecfidera®), fmgolimod (Gilenya®) natalizumab (Tysabri®) and the chemotherapeutic agent mitoxantrone in more severe cases.
• Ocrelizumab is a humanized anti-CD20 monoclonal antibody, which was granted Breakthrough Therapy Designation for PPMS by the Food and Drug Administration (FDA) in 2016 (Investor Update by Roche, Basel, February 17, 2016).
• MSCs Mesenchymal stem cells
• MSCs are a source of multipotent self-renewing cells, originally identified in adult bone marrow. Naturally, they differentiate to produce osteoblasts, chondrocytes and adipocytes. MSCs provide an accessible source of multipotent stem cells alternative to embryonic stem (ES) cells. MSCs potentially circumvent the need for immunosuppression in cellular therapies since they can be derived from an autologous source and also because they are characterized by an immuno-privileged nature advantageous for allogeneic use.
• ES embryonic stem
• MSCs based therapies have shown to be effective in preclinical studies for a number of indications including graft versus host disease, stroke, myocardial infarction, pulmonary fibrosis and autoimmune disorders. MSCs are also being extensively researched as a therapeutic tool against neurodegenerative diseases. MSCs have been discussed with regard to two aspects in the context of neurodegenerative diseases: their ability to transdifferentiate into neural cells under specific conditions and their neuroprotective and immunomodulatory effects. When transplanted into the brain, MSCs produce neurotrophic and growth factors that protect and induce regeneration of damaged tissue. Additionally, MSCs have also been explored as gene delivery vehicles, for example being genetically engineered to over express glial-derived or brain-derived neurotrophic factor in the brain. Clinical trials involving MSCs are currently underway for MS, ALS, traumatic brain injuries, spinal cord injuries and stroke.
• ADSCs Adipose-derived stem cells
• adipose tissue is in addition to its main function as an energy reservoir also an abundant resource for multipotent stromal cells (Zuk et al, Mol Biol Cell 2002; 13: 4279-4295).
• WO 2010/045645 discloses methods of recovering adipose stem cells from adipose tissue.
• US 8,021,882 discloses a method for producing stem cell conditioned media for the treatment of neurological insults, by providing a culture of adipose stem cells and collecting the supernatants thereof.
• WO 2006/057003 discloses, inter alia, methods of stem cell therapy using bone marrow-derived stem cells in combination with glatiramer.
• the present invention provides according to some aspects therapies for multiple sclerosis (MS), including progressive MS, particularly primary progressive MS, using adipose-derived stem cells (ADSCs) administered into the central nervous system (CNS).
• MS multiple sclerosis
• ADSCs adipose-derived stem cells
• CNS central nervous system
• the present invention further provides improved methods for obtaining ADSCs, which are more cost effective and which provide higher yields compared to currently used methods.
• the present invention is based, in part, on the surprising effect of human ADSCs administered into the CNS on the clinical score in an animal model of chronic multiple sclerosis.
• the cells were found to be particularly effective in reducing the maximal disease score and, importantly, in slowing the progression of the disease.
• the cells were isolated using an improved method as exemplified herein below.
• the improved method disclosed herein for the first time, involves freezing and thawing a sample of adipose tissue obtained by lipoaspiration, i.e. a lipoaspirate, before further processing.
• the improved method also avoids the need for applying a buffer to destroy red blood cells found in the lipoaspirate, as required by currently used methods.
• the improved method thus also eliminates subsequently separating the ADSCs from cell debris. It was surprisingly found that even though a red blood cell lysis buffer is not applied, the resulting ADSCs sample grows as effectively as a sample isolated according to standard methods.
• the present invention provides a method of treating primary progressive multiple sclerosis comprising administering non- genetically modified human adipose-derived stem cells (hADSCs) into the central nervous system (CNS) of a subject in need thereof.
• hADSCs non- genetically modified human adipose-derived stem cells
• the hADSCs are hADSCs obtained from human subcutaneous fat by:
• step (d) resuspending the pellet obtained in step (c) in a suspension medium capable of supporting cell viability and selecting hADSCs from the population of cells in the resuspended pellet;
• the hADSCs are hADSCs obtained from human subcutaneous fat by: (i) freezing a lipoaspirate; (ii) thawing the lipoaspirate and dissociating with a tissue-dissociation enzyme or by mechanical disruption; (iii) pelleting a cellular fraction comprising the hADSCs by centrifugation; (iv) washing the pellet with an isotonic buffer or culture medium and subjecting to a further centrifugation; (v) resuspending the pellet obtained in step (iv) in an isotonic buffer or culture medium and conducting at least one filtration; (vi) selecting hADSCs from the population of cells in the resuspended pellet; and (vii) culturing the hADSCs for at least 3 passages.
• the method of obtaining ADSCs avoids the need for applying a buffer to cause lysis of red blood cells originally found in the lipoaspirate.
• the lipoaspirate is processed to isolate adipose-derived stem cells without applying a red blood cells lysis buffer.
• the cellular fraction comprising the hADSCs obtained following centrifugation is not exposed to a red blood cell lysis buffer.
• the present invention provides a method of injecting human adipose-derived stem cells (hADSCs) into the central nervous system (CNS) of a subject in need thereof, the method comprising obtaining hADSCs from human subcutaneous fat by:
• step (d) resuspending the pellet obtained in step (c) in a buffer or culture medium and conducting at least one filtration;
• the present invention provides a method for obtaining human adipose-derived stem cells (hADSCs) from human subcutaneous fat for administration to a subject in need thereof, the method consisting of:
• step (d) resuspending the pellet obtained in step (c) in a suspension medium capable of supporting cell viability and conducting at least one filtration;
• hADSCs from the population of cells in the resuspended pellet; and (f) optionally culturing the hADSCs for at least 3 passages prior to their administration to the subject.
• the cellular fraction comprising the hADSCs obtained following centrifugation is not exposed to a red blood cell lysis buffer.
• the hADSCs are selected by adherence to a cell culture vessel.
• the cells adhere to a plastic tissue culture vessel.
• freezing is carried out at -80°C followed by vapor phase liquid nitrogen.
• the tissue-dissociation enzyme is selected from a collagenase, a dispase or a combination thereof. In some specific embodiments, the tissue- dissociation enzyme is a collagenase.
• the hADSCs are characterized by positive expression of CD44, CD73 and CD90 by at least 95% of the cells, positive expression of CD 105 by at least 90% of the cells, and negative expression of CD45, CD19, CDl lb and HLA-DR by at least 95% of the cells. According to some embodiments, the hADSCs are further characterized by positive expression of CD34 by 1-10% of the cells.
• the hADSCs are characterized by positive expression of CD44, CD73 and CD90 by at least 98% of the cells, positive expression of CD 105 by at least 90% of the cells, and negative expression of CD45, CD19, CDl lb and HLA-DR by at least 98% of the cells.
• At least 50% of the cells are positive for CD105, CD73, CD44 and CD90, and negative for CD45, CD19, CDl lb and HLA-DR.
• the hADSCs are hADSCs cultured to a passage number between 3-10 prior to their administration. In additional embodiments, the hADSCs are hADSCs cultured to a passage number between 3-5 prior to their administration.
• the hADSCs are hADSCs cultured in a xeno-free medium prior to their administration.
• the hADSCs are administered once.
• the hADSCs are administered more than once, for example twice, three times, four times, etc. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the hADSCs are administered once every 2-8 months. According to additional embodiments, the hADSCs are administered once every 3-12 months.
• administering the hADSCs is by intrathecal administration.
• administering the hADSCs is by intraventricular or intracerebroventricular (ICV) administration, namely into the brain ventricles.
• ICV intracerebroventricular
• the hADSCs are derived from human subcutaneous fat obtained by liposuction aspiration.
• the hADSCs are autologous.
• the hADSCs are allogeneic.
• administering the hADSCs comprises
• the subject to be treated as described herein is typically a human.
• the methods and compositions of the present invention are useful for the treatment of multiple sclerosis, particularly progressive forms of multiple sclerosis (MS).
• MS progressive forms of multiple sclerosis
• the subject is suffering from MS.
• the subject is a subject suffering from a progressive form of MS.
• the progressive MS is secondary progressive MS.
• the progressive MS is primary progressive MS.
• Figure 7 Cell surface marker expression of ADSCs as a function of passage number. The cells were isolated from adipose tissue and cultured as described in the Examples section, and analyzed for the expression of the indicated markers. The results are the average +/- standard deviation of nine (9) samples.
• Figure 8 SVF cell counting data following isolation with or without red blood cell lysis. (A) cells/ml; (B) % viability.
• Figure 9 Cell counting data at passage 0 following isolation with or without red blood cell lysis. (A) cells/ml; (B) % viability.
• FIG. 10 SVF and ADSC yields as a function of initial volume of lipoaspirated tissue sample - 1 st batch. Total number of viable cells immediately after SVF isolation (A), at P#0 (B), and at P#l (C).
• FIG. 11 SVF and ADSC yields as a function of initial volume of lipoaspirated tissue sample - 2 nd batch. Total number of viable cells immediately after SVF isolation (A), at P#0 (B), and at P#l (C).
• tissue sample - 3 batch Total number of viable cells immediately after SVF isolation (A), at P#0 (B), and at P#l (C).
• the present invention provides according to some aspects therapies for multiple sclerosis (MS) including progressive MS using adipose -derived stem cells (ADSCs).
• MS multiple sclerosis
• ADSCs adipose -derived stem cells
• a method of treating primary progressive multiple sclerosis comprising administering human adipose-derived stem cells (hADSCs) into the central nervous system (CNS) of a subject in need thereof is provided.
• hADSCs human adipose-derived stem cells
• MOG is a glycoprotein believed to be important in the process of myelinization of nerves in the central nervous system (CNS).
• CNS central nervous system
• Immunization with MOG 35-55 peptide is generally used for induction of a chronic EAE in C57BL/6 mice.
• ADSCs were injected into the brain ventricles (ICV) of mice in which EAE has been induced using MOG. Mice were treated with various doses of stem cells.
• the surprising observations disclosed herein for the first time demonstrate a clear effect of human ADSCs on chronic EAE, including clinical score, maximum mean disease score and disease progression.
• the present invention thus provides treatment methods and regimens comprising administration of human mesenchymal adipose-derived stem cells to subjects suffering from progressive forms of multiple sclerosis. These treatments afford superior therapeutic efficacy for multiple sclerosis, compared to intravenous treatment with stem cells. The current treatment resulted in improved and prolonged effects as determined by various clinical scores.
• treating refers to prevention, suppression or alleviation of a symptom or of a plurality of symptoms after the onset of primary progressive or secondary progressive multiple sclerosis.
• the symptom is selected from the group consisting of impaired coordination, impaired walking capability, impaired balance, weakness of the leg, stiffness of the leg, impaired memory, impaired cognitive function, a difficulty to swallow, impaired vision, general fatigue, pain, impaired bladder function, impaired bowel function, and any combination thereof.
• treating multiple sclerosis according to the present invention comprises slowing disease progression, i.e., slowing the progression of disability.
• the "subject" to which the cells are administered is a mammal, typically a human.
• the subject according to some embodiments is suffering from a progressive form of multiple sclerosis, namely, diagnosed with a progressive form of multiple sclerosis.
• multiple sclerosis refers to an auto-immune disease of the central nervous system which is accompanied by one or more of the symptoms described hereinabove.
• the progressive MS is secondary progressive MS.
• the progressive MS is primary progressive MS.
• the progressive MS is progressive relapsing MS.
• the ADSCs are administered once.
• the ADSCs are administered multiple times, for example every 2-8 months, every 3-12 months, or less frequent.
• the ADSCs are administered once every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months. Each possibility represents a separate embodiment of the present invention.
• the present invention utilizes adipose-derived mesenchymal stem cells.
• adipose -derived mesenchymal stem cells or "adipose-derived stem cells”, abbreviated “ADSCs” or “hADSCs” (i.e., human adipose-derived stem cells)
• ADSCs adipose-derived mesenchymal stem cells
• hADSCs human adipose-derived stem cells
• the cell population is characterized by positive expression of CD44, CD73 and CD90 by at least 95% of the cells, positive expression of CD105 by at least 90% of the cells, and negative expression of CD45, CD19, CD1 lb and HLA-DR by at least 95% of the cells.
• the cell population is characterized by positive expression of CD44, CD73 and CD90 by at least 98% of the cells, positive expression of CD105 by at least 90% of the cells, and negative expression of CD45, CD19, CDl lb and HLA-DR by at least 98% of the cells.
• the cell population is further characterized by positive expression of CD34 by up to 10%-20% of the cells. In some embodiments, the cell population is characterized by positive expression of CD34 by up to 5% of the cells.
• At least 50% of the cells are positive for CD105, CD73, CD44 and CD90, and negative for CD45, CD19, CDl lb and HLA-DR.
• 90-100% of the human ADSCs are positive for the markers: CD44, CD73 and CD90. According to additional embodiments, at least 95% of the human ADSCs are positive for the markers: CD44, CD73 and CD90. According to yet additional embodiments, at least 98% of the human ADSCs are positive for the markers: CD44, CD73 and CD90.
• 65-100% of the human ADSCs are positive for
• CD105 CD105.
• 80-100% of the hADSCs are positive for CD105.
• 90-100% of the hADSCs are positive for CD105.
• 80-95% of the hADSCs are positive for CD105.
• 0.1-20% of the human ADSCs express the marker CD34. According to additional embodiments, 0.1-10% of the human ADSCs express the marker CD34.
• 1-10% of the hADSCs are positive for the marker CD34. According to other embodiments, 1-5% of the hADSCs are positive for the marker CD34. According to some embodiments, at least 90% of the cells, for example at least 95% of the cells, are negative for the marker CD34.
• At least 90% of the administered human ADSCs are negative for the markers: CD45, CD19, CDl lb and HLA-DR.
• at least 95% of the administered human ADSCs are negative for the markers: CD45, CD19, CDl lb and HLA-DR.
• at least 98% of the administered human ADSCs are negative for the markers: CD45, CD19, CDl lb and HLA-DR.
• At least 50% of the injected human ADSCs are positive for CD105, CD73, CD44 and CD90, and negative for CD45, CD19, CDl lb, and HLA-DR.
• at least 60%, 70%, 80% or 90% of the injected human ADSCs are positive for CD105, CD73, CD44 and CD90, and negative for CD45, CD19, CDl lb and HLA-DR.
• Characterization of cell surface marker expression can be performed by methods known in the art, for example using fluorescence-activated cell sorting (FACS).
• FACS protocols for FACS are reviewed, for example, in: Flow Cytometry Protocols, Methods in Molecular Biology Volume 699 2011, Editors: Maria S. Hawley, Robert G. Hawley Humana Press. Exemplary procedures are described below.
• Adipose tissue as a source for multipotent stromal/stem cells has several advantages over other sources (Baer PC, Geiger H. Stem Cells Int 2012; 2012: 812693).
• subcutaneous fat is omnipresent in humans and is easily accessible in large quantities by liposuction aspiration.
• Liposuction is a well-tolerated procedure yielding large quantities of tissue aspirate.
• the lipoaspirate is typically discarded as medical waste, qualifying it as a good starting material for adipose-derived stromal/stem cell (ASC) isolation.
• ASC adipose-derived stromal/stem cell isolation.
• the tissue contains a large number of multipotent cells which can be isolated and proliferated in culture.
• the ADSCs are derived from human subcutaneous fat.
• the cells are derived from human subcutaneous fat obtained by liposuction aspiration.
• the ADSCs may be obtained by liposuction procedures in various areas of the body including stomach, hips, thighs, arms, neck and buttocks. Any procedure of liposuction may be used according to the present invention for obtaining ADSCs, including but not limited to laser, ultrasound and fat removal by abdominoplasty, as known in the art.
• the adipose tissue is processed to isolate the adipose-derived stem cells.
• Preparation methods typically include steps of washing the tissue with buffers such as PBS and saline, and/or with growth media (typically without any additives such as external cytokines or growth factors) e.g. DMEM, StemMACSTM or Plasma-Lyte, and treating the tissue with a tissue-dissociation enzyme such as collagenase and/or subjecting the tissue to non-enzymatic mechanical disruption, for example, using devices such as Tulip Nano TransferTM. Enzymatic digestion of the sample can also be performed using a combination of dispase and collagenase.
• buffers such as PBS and saline
• growth media typically without any additives such as external cytokines or growth factors
• a tissue-dissociation enzyme such as collagenase and/or subjecting the tissue to non-enzymatic mechanical disruption, for example, using devices such as Tulip Nano TransferTM.
• Enzymatic digestion of the sample can also be performed using a combination of dispase and collagenase.
• Liposomes which are generally aggregated, can be separated from free stromal cells which include the stem cells and other cells such as red blood cells, endothelial cells, and fibroblast cells, by centrifugation. Erythrocytes may be lysed from the suspended pellet using a suitable lysis buffer and the remaining cells can be filtered or centrifuged.
• an improved method for isolating the adipose-derived stem cells comprises freezing and thawing the adipose tissue prior to further processing.
• the tissue can be processed to isolate the stem cells without the need to apply a lysis buffer to destroy the red blood cells.
• a standard lysis buffer is typically a solution of ammonium chloride (NH 4 CI), potassium bicarbonate (KHCO 3 ) and EDTA at pH 7.3.
• NH 4 CI ammonium chloride
• KHCO 3 potassium bicarbonate
• EDTA EDTA
• Freezing according to some embodiments is performed as follows: a lipoaspirate is placed in a freezing bag and DMSO is added at a final concentration of 10%. The freezing bag is placed in a freezing canister and the sample is kept at -80°C overnight ( ⁇ 24 hours). The frozen sample is then transferred to a vapor phase liquid nitrogen tank. Stability tests can be performed during the storage period.
• the sample is taken out and left at room temperature for a few minutes, typically 5-10 minutes.
• the sample is then thawed in a water bath at 37°C for a few minutes, typically between 5-10 minutes, or until most of the sample is thawed.
• the sample is then washed with a suspension medium capable of supporting cell viability, defined herein as an isotonic buffer or culture medium suitable for mesenchymal stem cells, for example, a buffer, such as PBS, at 37°C.
• a suspension medium capable of supporting cell viability defined herein as an isotonic buffer or culture medium suitable for mesenchymal stem cells, for example, a buffer, such as PBS, at 37°C.
• a buffer such as PBS
• the process comprises conducting at least one filtration.
• Filtration is performed, in some embodiments, through a 100 micron mesh and subsequently through a 40 micron mesh in order to further dissociate the tissue and facilitate collection of the SVF fraction.
• ADSCs In order to isolate the ADSCs from within the population of cells in the SVF fraction, processes such as selection by adherence to a cell culture vessel (e.g. by plastic adherence) and/or via beads/antibodies are typically applied. Optionally, cells may be separated by cell sorting or separated immunohistochemically. Bunnell et al. (2008) Methods., 45(2): 115-120, review methods for isolation of ADSCs.
• Selection by adherence to the culture vessel is typically carried out by: seeding the SVF cells in a culture flask (e.g. plastic flask) with a suitable culture medium; incubating the seeded cells overnight (in some embodiments for at least 12 hours, or between 24-48 hours); washing the flask to remove non-adherent cells and tissue debris; and adding fresh culture medium to the flask.
• the adherent cells may be cultured to a desired level of confluency, following which they may be collected and stored or sub-cultured to further passages.
• the hADSCs are hADSCs obtained from human subcutaneous fat by: (a) freezing a lipoaspirate; (b) thawing the lipoaspirate and dissociating with a tissue-dissociation enzyme or by mechanical disruption; (c) pelleting a cellular fraction comprising the hADSCs by centrifugation, and optionally washing the pellet with a suspension medium capable of supporting cell viability and subjecting the suspension to at least one additional centrifugation; (d) resuspending the pellet obtained in step (c) in a suspension medium capable of supporting cell viability and selecting plastic adherent hADSCs by overnight incubation in plastic flasks; (e) optionally conducting at least one filtration to isolate the hADSCs prior to the plastic adherence selection; and (f) optionally culturing the hADSCs for at least 3 passages.
• the hADSCs are hADSCs obtained from human subcutaneous fat by: (a) freezing a lipoaspirate; (b) thawing the lipoaspirate and digesting with a collagenase; (c) pelleting a cellular fraction comprising the hADSCs by centrifugation and optionally washing the pellet with a buffer or culture medium and subjecting to a further centrifugation; (d) resuspending the pellet obtained in step (c) in a suitable culture medium; and selecting hADSCs by adherence to the cell culture vessel.
• the method for obtaining hADSCs from human subcutaneous fat for administration to a subject in need thereof consists essentially of:
• step (d) resuspending the pellet obtained in step (c) in a suspension medium capable of supporting cell viability and conducting at least one filtration;
• the ADSCs are cultured before being provided to a subject in need thereof (and/or before being stored for later use).
• the cells are cultured in a xeno-free medium.
• the ADSCs are grown to about 80 -100 , confiuency for example to about 80% confiuency, and sub-cultured to a passage number between 3-10, preferably between 3-5 or 3-4, before administration to the subject.
• the administered cells are at a passage between 3 to 10.
• the administered cells are at passage number between 3 to 5.
• the ADSCs are sub-cultured to passage number 3.
• the ADSCs are sub-cultured to passage number 4. In some embodiments, the ADSCs are sub-cultured to passage number 5.
• cells are counted and prepared for injection in a pharmaceutically acceptable diluent/carrier. Typically, the cells are concentrated before administration to the subject. The concentration typically ranges from 1.5xl0 4 /ml to 100xl0 6 /ml.
• a stem cell composition for single administration according to the methods of the invention
• present invention comprises, in some embodiments, 10 - 3x10 human ADSCs.
• the composition comprises 10 -10 human ADSCs.
• 10 6 -10 7 human ADSCs are injected in one administration.
• 200xl0 6 -300xl0 6 human ADSCs are injected in one administration.
• 2x10 human ADSCs are injected in one administration.
• the ADSC composition of the present invention is for use by systemic administration.
• the administration is into the central nervous system (CNS) of a subject.
• CNS central nervous system
• Such administration may be aimed at bypassing the blood brain barrier.
• the ADSCs are administered directly to a specific region of the brain.
• the composition is administered to the CNS, for example, by intraspinal administration.
• the composition is administered intrathecally.
• the composition is administered by intraventricular or intracerebroventricular (ICV) route, namely into the brain ventricles.
• ICV intracerebroventricular
• Intraventricular drug delivery is the delivery of medication within the cerebrospinal fluid of the cistern (Cl-2 vertebra) and intracranial ventricles. By administering medication directly, less medication is needed, and fewer side effects are seen than with orally administered drugs.
• the medicine is typically delivered through an implanted catheter connected to a pump, as known in the art.
• the pump may be programmable, and either implanted or external.
• Intrathecal administration is a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) and is useful in spinal anesthesia, chemotherapy, or pain management applications. This route is also used to introduce drugs that fight certain infections, particularly post-neurosurgical. The drug needs to be given this way to avoid the blood brain barrier.
• Intrathecal and epidural drug delivery comprise the intraspinal routes of drug administration. Each route delivers drug to the cerebrospinal fluid (CSF).
• Intrathecal delivery involves the direct injection of the drug into the CSF within the intrathecal space of the spinal column, whereas drugs injected in the epidural space have to cross the dura membrane in order to reach the CSF. As such, epidurally administered drugs can also reach the systemic circulation whereas intrathecally administered drugs are confined within the CSF circulating in the spinal column and the brain ventricles.
• Also encompassed by the present invention is a combination therapy of adipose- derived stem cells and optionally at least one other active agent.
• Active agents within the scope of the present invention include, but are not limited to interferons, e.g. pegylated or non-pegylated a-interferons, or ⁇ -interferons, e.g. interferon ⁇ -la or interferon ⁇ -lb, or ⁇ -interferons; immunosuppressants with optionally antiproliferative/antineoplastic activity, e.g. mitoxantrone, methotrexate, azathioprine, cyclophosphamide, or steroids, e.g. methylprednisolone, prednisone or dexamethasone, or steroid-secreting agents, e.g.
• interferons e.g. pegylated or non-pegylated a-interferons, or ⁇ -interferons, e.g. interferon ⁇ -la or interferon ⁇ -lb, or ⁇ -interfer
• ACTH ACTH
• adenosine deaminase inhibitors e.g. cladribine
• IV immunoglobulin G e.g. as disclosed in Neurology, 1998, May 50(5):1273-81
• monoclonal antibodies to various T-cell surface markers e.g. natalizumab (ANTEGREN®) or alemtuzumab
• TH2 promoting cytokines e.g. IL-4, IL-10
• compounds which inhibit expression of TH1 promoting cytokines e.g. phosphodiesterase inhibitors, e.g.
• antispasticity agents including baclofen, diazepam, piracetam, dantrolene, lamotrigine, rifluzole, tizanidine, clonidine, beta blockers, cyproheptadine, orphenadrine or cannabinoids; AMPA glutamate receptor antagonists, e.g.
• ⁇ 4 ⁇ 1 integrin VLA-4 and/or ⁇ -4- ⁇ -7 integrins e.g. natalizumab (ANTEGREN®); anti-macrophage migration inhibitory factor (Anti-MIF); xii) Cathepsin S inhibitors; xiii) mTor inhibitors.
• natalizumab ANTEGREN®
• Anti-MIF anti-macrophage migration inhibitory factor
• xii) Cathepsin S inhibitors xiii) mTor inhibitors.
• Currently preferred one other active agent is FTY720 (2-amino-2-[2-(4- octylphenyl) ethyl] propane- 1, 3-diol; fingolimod) belonging to the class of immunosuppressants.
• the invention encompasses the combination of ADSCs, with at least one additional drug, preferably, an immunosuppressant, particularly fmgolimod.
• the present invention also provides use in the treatment of multiple sclerosis of human ADSCs, optionally together with an additional drug being suitable for the treatment of multiple sclerosis.
• Example 1 Effect of human adipose derived stem cells (hADSCs)
• Lipoaspirate obtained from human healthy donors was frozen at -80°C and then in vapor phase liquid nitrogen (for over a week). The lipoaspirate was then thawed and washed twice with an equal volume of PBS at 37°C. The lipoaspirate was then digested with collagenase (NB4, Serva) at 37°C for 35 minutes with shaking. An equivalent volume of DMEM was added and the sample span down once at RT, 300-500g, for 10 min. The precipitated fraction was washed with DMEM, span down again and the pellet resuspended in culture medium (StemMACSTM, Miltentyi), without applying red blood cells lysis buffer.
• stemMACSTM pellet resuspended in culture medium
• the amount of culture medium was calculated as follows: 50ml for every 150ml of initial lipoaspirated tissue. The sample was then filtered through a ⁇ filter, followed by the addition of additional 5ml medium and centrifugation at 500g for 10 minutes at RT. The procedure was repeated with a 40 ⁇ filter and the cells were counted. The resulting cells are referred as stromal vascular fraction (SVF).
• SVF stromal vascular fraction
• the SVF fraction was plated at a density of ⁇ 2x10 6 cells per 75 cm 2 without any coating and incubated overnight to select for plastic adherent cells (ADSCs). The next day the flask was washed to remove non-adherent cells and tissue debris, fresh medium was added, and the cells were grown to 80-100% confluency, trypsinized and sub-cultured up to passage 3-5.
• ADSCs plastic adherent cells
• Cells were then collected and analyzed for the markers: CD 105, CD73, CD90, CD45, CD44, CD19, CDl lb, HLA-DR and CD34.
• the cells were stored frozen in liquid nitrogen until use. For use, frozen cells were thawed and plated at a concentration of 5000cells/cm and incubated overnight.
• ICV injection Prior to cells ICV injection the cells were trypsinized, counted and prepared for injection at the selected concentration detailed in Table 2 below and kept on ice until use for a period no longer than 30min. ICV injection was performed using a stereotactic system and care was taken not to injure the mice brain.
• EAE Experimental Autoimmune Encephalomyelitis
• EAE had been induced by injection of this emulsion subcutaneously (SC) on the shaved back of the mouse at one site, followed by an intraperitoneal injection of Bordetella pertussis toxin (Sigma) in PBS on Day 0, and 48 hours post MOG immunization.
• SC subcutaneously
• Bordetella pertussis toxin Sigma
• AUC Area under the curve
• the area under the curve (AUC) of Mean Clinical Score calculated as a function of cell dose at day 28 post immunization was significantly lower for the 2xl0 5 ADSCs and 4x10 ADSCs animal groups compared to the vehicle and to 1x10 ADSC ( Figure 5). Furthermore, the Mean Maximal Disease Score in the 2x10 5 ADSCs group at day 28 post immunization was significantly lower than that of the lxlO 5 ADSCs and the control group ( Figure 6).
• the experimental data show a clear advantage of the ICV transplantation of ADSC with a maximum effect at 2x10 5 cells per mice.
• This ADSC dose yielded a long term effect on the attenuation of EAE symptoms that led to a significant reduction of disease burden.
• Example 2- Analyses of cell surface markers of ADSCs Tables 4-12 summarize FACS analyses of nine (9) samples of ADSCs prepared as described in Example 1 above, following 1-5 passages. Tables 13-14 and Figure 7 show the average and standard deviation ("StDev") values. As can be seen in the tables and figure, the markers are stabilized after P3. Passage numbers P3-P4 are equivalent to no more than approximately 14 population doublings.
• CD44 99.9 100 99.8 99.7 99.8 99.9
• CD44 99.8 99.8 99.8 99.9 99.8 99.9
• Example 3- SVF isolation with vs. without red blood cell lysis
• coUagenase solution 142ml PBS, 7.5ml of 5% coUagenase stock solution, 0.6ml CaCi 2 1M
• DMEM LG DMEM Low Glucose
• the pellet was resuspended in 10ml of ACK red blood cell lysis buffer and incubated for approximately 10 min at 15-25°C.
• 20ml of PBS (15-25°C) were added to the tube and the tube was centrifuged for lOmin at 500x g, 21 ⁇ 2°C. Following centrifugation, the supernatant was removed and the pellet was resuspended in 5ml of DMEM LG.
• Both tubes designated A and tubes designated B were then subjected to two filtration steps in a similar manner: a ⁇ mesh filter was placed on top of new pre- labeled 50ml tubes (labeled as "A” or “B”) and the suspensions were filtered. Each filter was washed with additional 5ml DMEM LG and the filtrate was then centrifuged for lOmin at 500 x g, 21 ⁇ 2°C. The pellets were resuspended in 5ml DMEM LG. Next, a 40 ⁇ filter was placed on top of another set of new pre-labeled 50ml tubes (labeled as "A” or "B”) and the suspensions were filtered.
• NC-200 Nucleo-Counter was used to count the number of cells in each tube. The following parameters were recorded: viable cells/ml , dead cells /ml, viability ( ), average cell size ⁇ m) and total number of viable cells. Each tube was counted twice and an average of the two counts was calculated.
• Cells from each tube were analyzed by FACS for the following markers: CD73, CD90, CD45, CD31 , and IgGl as a control.
• Tables 15 and 17 below and Figures 8A-8B, 9A-9B show cell counting data (average of two counts for each sample) immediately after SVF isolation and at passage 0 (P0).
• the standard deviation (SD) data are summarized in Tables 16, 18.
• samples that were not subjected to the red blood cell lysis reached 70-80% confiuency faster than samples that were subjected to the lysis step.
• Table 19 summarizes the immunophenotype data (marker expression) at passage 0.
• marker expression was essentially the same for samples not subjected to the red blood cell lysis and for samples that were subjected to the lysis step, indicating that omission of this step does not negatively affect the phenotype of the obtained cells.
• Example 4- Effect of initial tissue volume on the yield of stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs)
• the amount of ADSCs that can be obtained from a lipoaspirated tissue sample depends, among others, on the initial volume of the lipoaspirated tissue sample. For therapeutic applications such as treating multiple sclerosis as described herein, it is suggested that at least 10 5 ADSCs be obtained, or even at least 10 6 ADSCs.
• the following study aimed at defining a minimum volume of fat tissue (lipoaspirated tissue) that can be used in order to obtain an amount of ADSCs that is sufficient for therapeutic purposes such as treating multiple sclerosis according to the present invention, and at evaluating possible changes to the production process that may improve the yield in cases where volumes lower than 100ml are available for processing. Study design
• the SVF fractions obtained from the 20, 50 and 150ml samples were seeded at a concentration of 35xl0 3 /cm 2 .
• the SVF fractions obtained from the 5 and 10ml samples were seeded at a concentration of ⁇ 4xl0 3 cells/cm 2 .
• ADSCs plastic adherent cells
• the flasks were washed, fresh medium was added, and the cells were grown in StemMACSTM Complete medium to -80% confluency. At this stage the cell passage was defined as P#0.
• Cells were then trypsinized, harvested, seeded in fresh flasks at a concentration of 5xl0 3 cells/cm 2 , and grown again to -80% confluency to the next passage, defined as P#l. Cells were then trypsinized, harvested, and kept frozen at a concentration of ⁇ 5xl0 6 /ml in 1ml cryotubes until use.
• Tables 20-22 below and Figures lOA-C, 11A-C, 12A-C show cell counting data for each sample immediately after SVF isolation and at passages 0 and 1 (P#0, P#l).
• the P#l samples of the three batches were harvested and cryopreserved at a concentration of ⁇ 5xl0 6 cells/ml. The cells were then thawed, seeded overnight with StemMACSTM Complete, harvested and analyzed by FACS. The markers that were analyzed are listed in Table 23. The results of the analysis are specified in Tables 24-27.
• the positive markers showed a high percentage of mesenchymal stem cells (as defined by the ISCT guidelines) in all of the processed volumes from all of the three tested batches (Positive CD73, CD105, CD90, CD44).
• the negative markers showed higher percentages than what is normally seen for adipose-derived mesenchymal stem cells, indicating the presence of hematopoietic progenitors as impurities.
• cells were cultured for five (5) passages, and it was demonstrated that the negative markers are decreased as a function of passage.
• tissue aggregates may trap more hematopoietic progenitors, which result in higher expression of CD34 as well as higher CD45 and CD 19 expressing cells. This assumption is supported by 50ml-aggregates cultures, which displayed the highest CD34 expression.
• ADSCs for therapeutic applications such as treatment of MS as disclosed herein can be obtained even from very low initial tissue volumes.
• certain adaptations to the production process may be applied, such as omission of filtration and culturing for at least 4 passages, in order to reduce loss of cells and obtain an ADSC population with sufficient purity.
• initial tissue volumes of at least 50ml. It is further suggested to apply a production process which includes filtration through a set of filters (but is devoid of red blood cell lysis, as disclosed herein), inter alia, to improve the purity of the ADSCs at earlier passages.
• the adapted process may be applied.

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