EP2035093A1 - Procédés pour traiter une lésion de la moelle épinière et pour minimiser l'étendue de la cicatrisation - Google Patents

Procédés pour traiter une lésion de la moelle épinière et pour minimiser l'étendue de la cicatrisation

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Publication number
EP2035093A1
EP2035093A1 EP07777388A EP07777388A EP2035093A1 EP 2035093 A1 EP2035093 A1 EP 2035093A1 EP 07777388 A EP07777388 A EP 07777388A EP 07777388 A EP07777388 A EP 07777388A EP 2035093 A1 EP2035093 A1 EP 2035093A1
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EP
European Patent Office
Prior art keywords
cells
spinal cord
derived therefrom
cell
cord injury
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07777388A
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German (de)
English (en)
Other versions
EP2035093A4 (fr
Inventor
Vivienne S. Marshall
Diana L. Clarke
George L. Sing
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Stemnion LLC
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Stemnion LLC
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Publication date
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Publication of EP2035093A1 publication Critical patent/EP2035093A1/fr
Publication of EP2035093A4 publication Critical patent/EP2035093A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • A61K35/48—Reproductive organs
    • 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the field of the invention is directed to methods of promoting the healing of spinal cord injury.
  • the field of the invention is further directed to methods of minimizing the extent of scarring following spinal cord injury.
  • Such methods utilize novel compositions, including but not limited to extraembryonic cytokine secreting cells (herein referred to as ECS cells), including, but not limited to, amnion-derived multipotent progenitor cells (herein referred to as AMP cells) and conditioned media derived therefrom (herein referred to as amnion-derived cellular cytokine suspension or ACCS), each alone or in combination with each other and/or other agents.
  • ECS cells extraembryonic cytokine secreting cells
  • AMP cells amnion-derived multipotent progenitor cells
  • ACCS amnion-derived cellular cytokine suspension
  • Sankar, V. et al. (Neuroscience 2003, Letter to Neuroscience, 118(1):11-7) studied the role of human amniotic epithelial cell transplantation in transected spinal cord injury repair and reported that the human amniotic epithelial cells survived in monkey transected spinal cord, the graft was penetrated by host axons and there was no glial scar at the transection lesion site.
  • the fibrous scar that develops after spinal cord injury is considered a major impediment for axonal regeneration.
  • Spinal cord scarring generally means the recruitment and proliferation of glial cells to the site of injury.
  • the densely packed cells primarily reactive astrocytes
  • their secretions form a dense cellular plaque known as the glial scar.
  • This scar prevents axons from projecting through.
  • reactive astrocytes appear and accumulate in the wounded area, leading to glial scar formation.
  • Glial scar is the physical barrier to axonal regeneration of injured neurons. Chondroitin sulfate proteoglycans are inhibitory to axon outgrowth and are upregulated in reactive astrocytes upon CNS injury.
  • keratin sulfate proteoglycans are also augmented after CNS injury and act as inhibitory cues.
  • Therapeutic treatments aimed at suppression of fibrous scarring have been shown to promote axon regeneration in various lesion paradigms of the mammalian spinal cord.
  • ECS cells extraembryonic cytokine secreting cells
  • AMP cells amnion-derived multipotent progenitor cells
  • ACCS amnion-derived cellular cytokine suspension or ACCS
  • ECS cells extraembryonic cytokine secreting cells
  • AMP cells amnion-derived multipotent progenitor cells
  • ACCS amnion-derived cellular cytokine suspension or ACCS
  • a first aspect of the invention is a method for treating spinal cord injury in a patient in need thereof comprising administering to the patient a therapeutically effective amount of one or more compositions comprising ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom.
  • ECS cells are AMP cells.
  • conditioned media is ACCS.
  • AMP cells are pooled AMP cells and in another particular embodiment, the ACCS is pooled ACCS.
  • a second aspect of the invention is method for promoting the healing of a spinal cord injury in a patient in need thereof comprising administering to the patient a therapeutically effective amount of one or more compositions comprising ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom.
  • ECS cells are AMP cells.
  • conditioned media is ACCS.
  • AMP cells are pooled AMP cells and in another particular embodiment, the ACCS is pooled ACCS.
  • a third aspect of the invention is a method for stimulating growth or regeneration of spinal cord cells following spinal cord injury in a patient in need thereof comprising administering to the patient a therapeutically effective amount of one or more compositions comprising ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom.
  • ECS cells are AMP cells.
  • the conditioned media is ACCS.
  • the AMP cells are pooled AMP cells and in another particular embodiment, the ACCS is pooled ACCS.
  • the spinal cord cells are neurons or oligodendrocytes.
  • the neurons are motor neurons, interneurons or sensory neurons.
  • a fourth aspect of the invention is a method for preventing or ameliorating scarring following spinal cord injury in a patient in need thereof comprising administering to the patient a therapeutically effective amount of one or more compositions comprising ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom.
  • ECS cells are AMP cells.
  • conditioned media is ACCS.
  • AMP cells are pooled AMP cells and in another particular embodiment, the ACCS is pooled ACCS.
  • a fifth aspect of the invention is a method for neuroprotecting spinal cord neurons following spinal cord injury in a patient in need thereof comprising administering to the patient a therapeutically effective amount of one or more compositions comprising ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom.
  • ECS cells are AMP cells.
  • conditioned media is ACCS.
  • AMP cells are pooled AMP cells and in another particular embodiment, the ACCS is pooled ACCS.
  • the spinal cord injury is complete spinal cord injury or incomplete spinal cord injury.
  • the spinal cord injury is caused by contusion of the spinal cord, bruising of the spinal cord, loss of blood to the spinal cord, pressure on the spinal cord, cut spinal cord or severed spinal cord and the incomplete spinal cord injury is anterior cord syndrome, central cord syndrome, Brown- Sequard syndrome, injuries to individual nerve cells or spinal contusion.
  • the patient is a human.
  • the ECS cells, conditioned media derived therefrom, cell lysate derived therefrom or cell products derived therefrom are administered in combination with other agents.
  • the other agents are active agents.
  • the active agents are neuroregenerative agents, neuroprotective agents, neurotrophic factors, growth factors, cytokines, chemokines, antibodies, inhibitors, antibiotics, immunosuppressive agents, steroids, anti-fungals, anti-virals or other cell types.
  • the neuroprotective agent is for example dopamine D3 receptor agonists
  • the neurotrophic factors are for example BDNF, NT-3, NT-4, CNTF, NGF, or GDNF
  • the antibodies are for example IN-I anti-NOGO antibodies
  • the inhibitor is for example the PDE4 inhibitor rollipram
  • the immunosuppressive agents are for example corticosteroids (i.e.
  • glucocorticoids cyclosporine, tacrolimus, sirolimus, methotrexate, azathiopine, mercatopurine, cytotoxic antibiotics, polyclonal and monoclonal antibodies such as anti-T-cell receptor (CD23) and anti-IL2 receptor (CD25) antibodies, interferon, opioids, TNF binding proteins, mycophenolate, and small biological agents such as FTY720;
  • the antibiotics are pikromycin, narbomycin, methymycin, neomethymycin;
  • the steroid is methylprednisolone; and the other cell types are for example differentiated AMP cells, or a mixture of differentiated and undifferentiated AMP cells, or a mixture of AMP cells (differentiated and/or undifferentiated) and other cells such as neural stem cells or any other progenitor cell or cells that are treated in such a way as to augment the AMP cells or AMP cell activity.
  • isolated refers to material removed from its original environment and is thus altered “by the hand of man” from its natural state.
  • a “gene” is the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region, as well as intervening sequences (introns) between individual coding segments (exons).
  • the term "protein marker” means any protein molecule characteristic of the plasma membrane of a cell or in some cases of a specific cell type.
  • enriched means to selectively concentrate or to increase the amount of one or more materials by elimination of the unwanted materials or selection and separation of desirable materials from a mixture (i.e. separate cells with specific cell markers from a heterogeneous cell population in which not all cells in the population express the marker).
  • substantially purified means a population of cells substantially homogeneous for a particular marker or combination of markers.
  • substantially homogeneous is meant at least 90%, and preferably 95% homogeneous for a particular marker or combination of markers.
  • placenta means both preterm and term placenta.
  • totipotent cells shall have the following meaning. In mammals, totipotent cells have the potential to become any cell type in the adult body; any cell type(s) of the extraembryonic membranes (e.g., placenta). Totipotent cells are the fertilized egg and approximately the first 4 cells produced by its cleavage.
  • pluripotent stem cells are true stem cells with the potential to make any differentiated cell in the body, but cannot contribute to making the components of the extraembryonic membranes which are derived from the trophoblast. The amnion develops from the epiblast, not the trophoblast.
  • Three types of pluripotent stem cells have been confirmed to date: Embryonic Stem (ES) Cells (may also be totipotent in primates), Embryonic Germ (EG) Cells, and Embryonic Carcinoma (EC) Cells. These EC cells can be isolated from teratocarcinomas, a tumor that occasionally occurs in the gonad of a fetus. Unlike the other two, they are usually aneuploid.
  • multipotent stem cells are true stem cells but can only differentiate into a limited number of types.
  • the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but may not be able to differentiate into other cells types.
  • extraembryonic tissue means tissue located outside the embryonic body which is involved with the embryo's protection, nutrition, waste removal, etc. Extraembryonic tissue is discarded at birth. Extraembryonic tissue includes but is not limited to the amnion, chorion (trophoblast and extraembryonic mesoderm including umbilical cord and vessels), yolk sac, allantois and amniotic fluid (including all components contained therein). Extraembryonic tissue and cells derived therefrom have the same genotype as the developing embryo.
  • extraembryonic cytokine secreting cells or "ECS cells” means a population of cells derived from the extraembryonic tissue which have the characteristic of secreting a unique combination of physiologically relevant cytokines in a physiologically relevant temporal manner into the extracellular space or into surrounding culture media.
  • the ECS cells secrete at least one cytokine selected from VEGF, Angiogenin, PDGF and TGF ⁇ 2 and at least one MMP inhibitor selected from TIMP-I and TIMP-2.
  • the ECS cells secrete more than one cytokine selected from VEGF, Angiogenin, PDGF and TGF ⁇ 2 and more than one MMP inhibitor selected from TIMP-I and TIMP-2.
  • the ECS cells secrete the cytokines VEGF, Angiogenin, PDGF and TGF ⁇ 2 and the MMP inhibitors TIMP-I and TIMP-2.
  • the physiological range of the cytokine or cytokines in the unique combination is as follows: ⁇ 5-16ng/ml for VEGF, -3.5-4.5 ng/ml for Angiogenin, ⁇ 100-165pg/ml for PDGF, -2.5- 2.7ng/ml for TGF ⁇ 2, ⁇ 0.68 ⁇ g ml for TIMP-I and -1.04 ⁇ g/ml for TIMP-2.
  • ECS cells may be selected from populations of cells and compositions described in this application and in US2003/0235563, US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.
  • amnion-derived multipotent progenitor cell or "AMP cell” means a specific population of ECS cells that are epithelial cells derived from the amnion.
  • AMP cells have the following characteristics. They grow without feeder layers, do not express the protein telomerase and are non-tumorigenic. AMP cells do not express the hematopoietic stem cell marker CD34 protein. The absence of CD34 positive cells in this population indicates the isolates are not contaminated with hematopoietic stem cells such as umbilical cord blood or embryonic fibroblasts.
  • AMP cells have previously been described as "amnion- derived cells" (see U.S. Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067, U.S. Provisional Application Nos. 60/813,759, U.S. Application No. 11/333,849, U.S. Application No. 11/392,892, and PCTUS06/011392, each of which is incorporated herein in its entirety).
  • animal-free when referring to certain compositions, growth conditions, culture media, etc. described herein, is meant that no animal-derived materials, such as animal-derived serum, other than human materials, such as native or recombinantly produced human proteins, are used in the preparation, growth, culturing, expansion, or formulation of the certain composition or process.
  • the term “expanded”, in reference to cell compositions, means that the cell population constitutes a significantly higher concentration of cells than is obtained using previous methods.
  • the level of cells per gram of amniotic tissue in expanded compositions of AMP cells is at least 50 and up to 150 fold higher than the number of cells in the primary culture after 5 passages, as compared to about a 20 fold increase in such cells using previous methods.
  • the level of cells per gram of amniotic tissue in expanded compositions of AMP cells is at least 30 and up to 100 fold higher than the number of cells in the primary culture after 3 passages.
  • an “expanded” population has at least a 2 fold, and up to a 10 fold, improvement in cell numbers per gram of amniotic tissue over previous methods.
  • the term “expanded” is meant to cover only those situations in which a person has intervened to elevate the number of the cells.
  • the term "passage” means a cell culture technique in which cells growing in culture that have attained confluence or are close to confluence in a tissue culture vessel are removed from the vessel, diluted with fresh culture media (i.e. diluted 1 :5) and placed into a new tissue culture vessel to allow for their continued growth and viability.
  • cells isolated from the amnion are referred to as primary cells.
  • Such cells are expanded in culture by being grown in the growth medium described herein. When such primary cells are subcultured, each round of subculturing is referred to as a passage.
  • "primary culture” means the freshly isolated cell population.
  • conditioned medium is a medium in which a specific cell or population of cells has been cultured, and then removed. When cells are cultured in a medium, they may secrete cellular factors that can provide support to or affect the behavior of other cells. Such factors include, but are not limited to hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors and granules.
  • the medium containing the cellular factors is the conditioned medium. Examples of methods of preparing conditioned media are described in U.S. Patent 6,372,494 which is incorporated by reference in its entirety herein.
  • conditioned medium also refers to components, such as proteins, that are recovered and/or purified from conditioned medium or from ECS cells, including AMP cells.
  • amnion-derived cellular cytokine suspension or "ACCS” means conditioned medium that has been derived from AMP cells or expanded AMP cells.
  • lysate refers to the composition obtained when cells, for example, AMP cells, are lysed and optionally the cellular debris (e.g., cellular membranes) is removed. This may be achieved by mechanical means, by freezing and thawing, by sonication, by use of detergents, such as EDTA, or by enzymatic digestion using, for example, hyaluronidase, dispase, proteases, and nucleases.
  • physiological level means the level that a substance in a living system is found and that is relevant to the proper functioning of a biochemical and/or biological process.
  • the term “pooled” means a plurality of compositions that have been combined to create a new composition having more constant or consistent characteristics as compared to the non-pooled compositions. For example, pooled AMP cells have more constant or consistent characteristics compared to non-pooled AMP cells.
  • the term “substrate” means a defined coating on a surface that cells attach to, grown on, and/or migrate on.
  • the term “matrix” means a substance that cells grow in or on that may or may not be defined in its components.
  • the matrix includes both biological and non-biological substances,
  • the term "scaffold” means a three-dimensional (3D) structure (substrate and/or matrix) that cells grow in or on. It may be composed of biological components, synthetic components or a combination of both. Further, it may be naturally constructed by cells or artificially constructed. In addition, the scaffold may contain components that have biological activity under appropriate conditions.
  • cell product refers to any and all substances made by and secreted from a cell, including but not limited to, protein factors (i.e. growth factors, differentiation factors, engraftment factors, cytokines, morphogens, proteases (i.e. to promote endogenous cell delamination, protease inhibitors), extracellular matrix components (i.e. fibronectin, etc.).
  • protein factors i.e. growth factors, differentiation factors, engraftment factors, cytokines, morphogens, proteases (i.e. to promote endogenous cell delamination, protease inhibitors), extracellular matrix components (i.e. fibronectin, etc.).
  • terapéuticaally effective amount means that amount of a therapeutic agent necessary to achieve a desired physiological effect (i.e. promote functional recovery following spinal cord injury).
  • the term "pharmaceutically acceptable” means that the components, in addition to the therapeutic agent, comprising the formulation, are suitable for administration to the patient being treated in accordance with the present invention.
  • therapeutic protein includes a wide range of biologically active proteins including, but not limited to, growth factors, enzymes, hormones, cytokines, inhibitors of cytokines, blood clotting factors, peptide growth and differentiation factors.
  • transplantation refers to the administration of a composition comprising cells that are either in an undifferentiated, partially differentiated, or fully differentiated form, or a combination thereof, into a human or other animal.
  • the term “differentiation” means the process by which cells become progressively more specialized.
  • the term “differentiation efficiency” means the percentage of cells in a population that are differentiating or are able to differentiate.
  • tissue refers to an aggregation of similarly specialized cells united in the performance of a particular function.
  • co-administer can include simultaneous or sequential administration of two or more agents.
  • central nervous system or “CNS” means the brain and/or spinal cord.
  • central nervous system disorder or “CNS disorder” means any condition or disease that causes or results in a functional and/or physical deficit in the brain and/or spinal cord.
  • neurodegeneration means the progressive loss of neurons in the central nervous system. This includes but is not limited to immediate loss of neurons followed by subsequent loss of connecting or adjacent neurons.
  • neurodegeneration or “neuroprotecting” means the arrest and/or reverse progression of neurodegeneration following a central nervous system injury.
  • neurological disease refers to a disease or condition associated with any defects in the entire integrated system of nervous tissue in the body: the cerebral cortex, cerebellum, thalamus, hypothalamus, midbrain, pons, medulla, brainstem, spinal cord, basal ganglia and peripheral nervous system.
  • Neuron neuronal cell
  • neural cell including neural progenitor cells and neural stem cells
  • nerve cells i.e., cells that are responsible for conducting nerve impulses from one part of the body to another.
  • telodendria Most neurons consist of three distinct portions: a cell body which contains the nucleus, and two different types of cytoplasmic processes: dendrites and axons.
  • Dendrites which are the receiving portion of the neuron, are usually highly branched, thick extensions of the cell body.
  • the axon is typically a single long, thin process that is specialized to conducts nerve impulses away from the cell body to another neuron or muscular or glandular tissue. Axons may have side branches called "axon collaterals.” Axon collaterals and axons may terminate by branching into many fine filaments called telodendria.
  • telodendria The distal ends of telodendria are called synaptic end bulbs or axonal terminals, which contain synaptic vesicles that store neurotransmitters.
  • Axons may be surrounded by a multilayered, white, phospholipid, segmented covering called the myelin sheath, which is formed by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. Axons containing such a covering are "myelinated.”
  • Neurons include sensory (afferent) neurons, which transmit impulses from receptors in the periphery to the brain and spinal cord and from lower to higher centers of the central nervous system.
  • a neuron can also be motor (efferent) neurons which convey impulses from the brain and spinal cord to effectors in the periphery and from higher to lower centers of the central nervous system.
  • Other neurons are association (connecting or interneuron) neurons which carry impulses from sensory neurons to motor neurons and are located within the central nervous system.
  • the processes of afferent and efferent neurons arranged into bundles are called “nerves" when located outside the CNS or fiber tracts if inside the CNS.
  • Neuronal tissue includes any tissue that comprises a neural cell or a nerve.
  • astrocytes and "astroglial cells” refer to a type of glial cell that become reactive and up-regulates intermediate filament (IF) proteins, such as glial fibrillary acid protein (GFAP) and vimentin (Vim), under pathological conditions or after transplantation in the brain and retina.
  • IF intermediate filament
  • GFAP glial fibrillary acid protein
  • Vim vimentin
  • GFAP refers to "glial fibrillary acid protein" which is one of the major intermediate filament proteins of mature astrocytes. It is used as a marker to distinguish astrocytes from other glial cells during development.
  • axonal growth or axon growth refers to the elongation or extension of an axon of a neural cell. An axon can elongate for distances of microns to meters. Extension or elongation of an axon is also referred to as "regeneration" of the axon of a neural cell and may result in the reestablishment of nerve cell connectivity.
  • spinal cord injury means an injury in which the axons or nerve fibers of the spinal cord are interrupted, generally by mechanical forces.
  • spinal cord scarring refers to the recruitment and proliferation of glial cells to the site of spinal cord injury.
  • the densely packed cells primarily reactive astrocytes
  • their secretions form a dense cellular plaque known as the glial scar. This scar prevents axons from projecting through thus interfering with axonal regeneration and functional recovery.
  • parenteral administration and “administered parenterally” are art-recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, epidural, intracerebral and intrasternal injection or infusion.
  • Treatment covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; (c) relieving and or ameliorating the disease or condition, i.e., causing regression of the disease or condition; or (d) curing the disease or condition, i.e., stopping its development or progression.
  • the population of subjects treated by the methods of the invention includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.
  • a "wound” is any disruption, from whatever cause, of normal anatomy (internal and/or external anatomy) including but not limited to traumatic injuries such as mechanical (i.e. contusion, penetrating), thermal, chemical, electrical, concussive and incisional injuries; elective injuries such as operative surgery and resultant incisional hernias, fistulas, etc.; acute wounds, chronic wounds, infected wounds, and sterile wounds, as well as wounds associated with disease states (i.e. ulcers caused by diabetic neuropathy or ulcers of the gastrointestinal or genitourinary tract).
  • wound healing refers to improving, by some form of intervention, the natural cellular processes and humoral substances of tissue repair such that healing is faster, and/or the resulting healed area has less scaring and/or the wounded area possesses tissue strength that is closer to that of uninjured tissue and/or the wounded tissue attains some degree of functional recovery.
  • spinal Cord Injury Common causes of spinal cord injury include fractures of the vertebrae, which can damage the spinal cord from the concussive effect of injury due to displaced bony fragments, or damaged blood vessels, or contusion of emerging nerve roots. Dislocation of vertebrae can also cause spinal cord damage; dislocation is often the result of the rupture of an intervertebral disk, and may result in partial or complete severance of the spinal cord. Penetrating wounds can also cause severance or partial severance of the cord. Epidural hemorrhage and spinal subdural hematoma can result in progressive paraparesis due to pressure on the spinal cord. Examples of indirect injury to the spinal cord include damage induced by a blow to the head or a fall on the feet.
  • Intramedullary injury can be the result of direct pressure on the cord or the passage of a pressure wave through the cord, laceration of the cord by bone, or the rupture of a blood vessel during the passage of a pressure wave through the cord with a hemorrhage into the cord.
  • Intramedullary bleeding and hematoma formation can also be caused by rupture of a weakened blood vessel. Ischemic damage can occur following compression of the anterior spinal artery, pressure on the anastomotic arteries, or damage to major vessels (Gilroy, in Basic Neurology, McGraw-Hill, Inc. New York, N. Y. (1990).
  • Incomplete spinal cord injuries generally do not result in complete loss of movement and sensation below the injury site.
  • a variety of patterns exist to classify such injuries including 1) anterior cord syndrome which results from damage to the motor and sensory pathways in the anterior areas of the spinal cord. Effects include loss of movement and overall sensation, although some sensations that travel by way of the still intact pathways can be felt; 2) central cord syndrome which results from injury to the center of the cervical area of the spinal cord.
  • the damage affects the corticospinal tract, which is responsible for carrying signals between the brain and spinal cord to control movement.
  • Patients suffering from central cord syndrome experience weakness or paralysis in the arms and some loss of sensory reception. The loss of strength and sensation is much less in the legs than in the arms.
  • Brown-Sequard syndrome results from injury to the right or left side of the spinal cord.
  • movement and sensation are lost below the level of the injury.
  • temperature and pain sensation are lost due to the crossing of these pathways in the spinal cord;
  • injuries to individual nerve cells results in loss of sensory and motor function in the area of the body to which the injured nerve root corresponds.
  • symptoms from these injuries vary depending on the location and function of the particular nerve root affected;
  • spinal contusions which are the most common type of spinal cord injury.
  • the spinal cord is bruised, not severed, but the consequence is inflammation and bleeding from blood vessels near the injury.
  • a spinal contusion may result in temporary (usually one to two days) incomplete or complete debilitation of the spinal cord or the incomplete or complete debilitation of the spinal cord may be longer term, including a permanent incomplete or complete debilitation of the spinal cord.
  • a goal of treating spinal cord injury includes promoting motor recovery. Another goal is promoting sensory recovery. Various modalities have been attempted to achieve such motor and sensory recovery, most with only limited success. These studies include application of various growth factors and cytokines to the site of injury as well as transplantation of brain-derived stem cells or healthy spinal cord tissue. Stem cell transplantation therapies are among the most promising therapies currently being studied (see, for example, Cummings, B.J., et al., Neurol Res 2006 28(5):474-81; Pallini, R., et al., Neurosurgery 2005 57(5): 1014-25; Cummings, BJ., et al., PNAS USA 2005 102(39): 14069-74; Sankar, V. et al., Neuroscience 2003 118(1):11-7).
  • compositions and methods described herein to promote spinal cord injury repair and functional recovery.
  • Such methods utilize novel compositions, including but not limited to extraembryonic cytokine secreting cells (herein referred to as ECS cells), conditioned media and cell products derived therefrom, each alone and/or in combination with each other and/or with other agents including active and/or inactive agents.
  • ECS cells extraembryonic cytokine secreting cells
  • conditioned media and cell products derived therefrom each alone and/or in combination with each other and/or with other agents including active and/or inactive agents.
  • the methods utilize novel compositions including, but not limited to, amnion-derived multipotent progenitor cells (herein referred to as AMP cells), conditioned media derived therefrom (herein referred to as amnion-derived cellular cytokine suspension or ACCS), and cell products derived therefrom, each alone and/or in combination with each other and/or with other agents including active and/or inactive agents.
  • AMP cells amnion-derived multipotent progenitor cells
  • ACCS amnion-derived cellular cytokine suspension
  • cell products derived therefrom each alone and/or in combination with each other and/or with other agents including active and/or inactive agents.
  • the AMP cells are pooled AMP cells and the ACCS is pooled ACCS.
  • the instant invention is based upon the discovery that ECS cells, conditioned medium therefrom, cell lysates therefrom, extracellular matrices therefrom, alone or in combination, as well as compositions of AMP cells can promote such spinal cord injury repair and functional recovery.
  • ECS cells including AMP cells, and/or conditioned medium and/or cell lysates from such cells, alone or in combination with each other and/or other agents.
  • all types of spinal cord injury will repair and heal and regain functionality more rapidly than similar spinal cord injuries left to heal naturally or which are treated with currently available methods.
  • the compositions described herein will prevent or reduce swelling and/or inflammation of the injured spinal cord.
  • ECS cells including AMP cells and/or conditioned medium therefrom (i.e. ACCS), and/or cell lysates thereof are administered to the injury to promote spinal cord injury healing and functional recovery in the patient.
  • This direct administration can be as a single dose or repeated doses given at multiple designated intervals. It will be readily appreciated by those skilled in the art that the preferred dosage regimen will vary with the type and severity of the injury being treated.
  • ACCS conditioned medium therefrom
  • ECS cells Various methods for isolating cells from the extraembryonic tissue, which may then be used to produce the ECS cells of the instant invention are described in the art (see, for example, US2003/0235563, US2004/0161419, US2005/0124003, U.S. Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. Application No. 11/333,849, U.S. Application No. 11/392,892, PCTUS06/011392, US2006/0078993, PCT/USOO/40052, U.S. Patent No. 7,045,148, US2004/0048372, and US2003/0032179).
  • ECS cells Once extraembryonic tissue is isolated, it is necessary to identify which cells in the tissue have the characteristics associated with ECS cells (see definition above). For example, cells are assayed for their ability to secrete a unique combination of cytokines into the extracellular space or into surrounding culture media. Suitable cells are those in which the cytokine or cytokines occurs in the physiological range of ⁇ 5.0-16ng/ml for VEGF, ⁇ 3.5-4.5 ng/ml for Angiogenic ⁇ 100-165pg/ml for PDGF, ⁇ 2.5-2.7ng/ml for TGF ⁇ 2, ⁇ 0.68 ⁇ g ml for TIMP-I and ⁇ 1.04 ⁇ g/ml for TIMP-2.
  • AMP cells are prepared using the steps of a) recovery of the amnion from the placenta, b) dissociation of the cells from the amniotic membrane, c) culturing of the cells in a basal medium with the addition of a naturally derived or recombinantly produced human protein; and optionally d) further proliferation of the cells using additional additives and/or growth factors. Details are contained in US Publication No. 2006- 0222634-A1, which is incorporated herein by reference.
  • AMP cells are cultured as follows: The AMP cells are cultured in a basal medium.
  • a basal medium includes, but is not limited to, Epilife (Cascade Biologicals), Opti-pro, VP-SFM, IMDM, Advanced DMEM, K/O DMEM, 293 SFM II (all made by Gibco; Invitrogen), HPGM, Pro 293S- CDM, Pro 293A-CDM, UltraMDCK, UltraCulture (all made by Cambrex), Stemline I and Stemline II (both made by Sigma-Aldrich), DMEM, DMEM/F-12, Ham's F12, M199, and other comparable basal media.
  • Such media should either contain human protein or be supplemented with human protein.
  • human protein is one that is produced naturally or one that is produced using recombinant technology.
  • Human protein also is meant to include a human fluid or derivative or preparation thereof, such as human serum or amniotic fluid, which contains human protein. Details on this procedure are contained in US Publication No. 2006-0222634-A1, which is incorporated herein by reference.
  • the cells are cultured using a system that is free of animal products to avoid xeno-contamination.
  • the culture medium is Stemline I or II, Opti-pro, or DMEM, with human albumin added up to concentrations of 10%.
  • the invention further contemplates the use of any of the above basal media wherein animal-derived proteins are replaced with recombinant human proteins and animal-derived serum, such as BSA, is replaced with human albumin.
  • the media is serum-free in addition to being animal-free. Details on this procedure are contained in US Publication No. 2006-0222634- Al, which is incorporated herein by reference.
  • the culture medium may be supplemented with serum derived from mammals other than humans, in ranges of up to 40%.
  • EGF epidermal growth factor
  • TGF ⁇ or TGF ⁇ 5ng/ml; range 0.1 -100ng/ml
  • activin A cholera toxin (preferably at a level of about O.l ⁇ g/ml; range 0-10 ⁇ g/ml), transferrin (5 ⁇ g/ml; range 0.1- lOO ⁇ g/ml)
  • fibroblast growth factors bFGF 40ng/ml (range 0-200ng/ml)
  • aFGF, FGF-4, FGF-8 all in range 0-200ng/ml
  • bone morphogenic proteins i.e. BMP-4
  • ECS conditioned medium- is obtained as described below for ACCS, except that ECS cells are used.
  • the AMP cells of the invention can be used to generate ACCS.
  • the AMP cells are isolated as described herein and 10 xlO 6 cells are seeded into T75 flasks containing between 5-30ml culture medium, preferably between 10-25ml culture medium, and most preferably about 10ml culture medium.
  • the cells are cultured until confluent, the medium is changed and in one embodiment the ACCS is collected 1 day post-confluence. In another embodiment the medium is changed and ACCS is collected 2 days post-confluence. In another embodiment the medium is changed and ACCS is collected 4 days post-confluence. In another embodiment the medium is changed and ACCS is collected 5 days post-confluence.
  • the medium is changed and ACCS is collected 3 days post-confluence. In another preferred embodiment the medium is changed and ACCS is collected 3, 4, 5, 6 or more days post- confluence.
  • Skilled artisans will recognize that other embodiments for collecting ACCS from AMP cell cultures, such as using other tissue culture vessels, including but not limited to cell factories, flasks, hollow fibers, or suspension culture apparatus, or collecting ACCS from sub-confluent and/or actively proliferating cultures, are also contemplated by the methods of the invention. It is also contemplated by the instant invention that the ACCS be cryopreserved following collection. It is also contemplated by the invention that ACCS be lyophilized following collection. Skilled artisans are familiar with cryopreservation and lyophilization methodologies.
  • compositions of the invention can be prepared in a variety of ways depending on the intended use of the compositions.
  • a composition useful in practicing the invention may be a liquid comprising an agent of the invention, i.e. ECS cells, including AMP cells and/or ACCS, in solution, in suspension, or both (solution/suspension).
  • ECS cells i.e. ECS cells
  • ACCS AMP cells and/or ACCS
  • solution/suspension refers to a liquid composition where a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form, in suspension in a liquid matrix.
  • a liquid composition also includes a gel.
  • the liquid composition may be aqueous or in the form of an ointment, salve, cream, or the like.
  • An aqueous suspension or solution/suspension useful for practicing the methods of the invention may contain one or more polymers as suspending agents.
  • Useful polymers include water- soluble polymers such as cellulosic polymers and water-insoluble polymers such as cross-linked carboxyl-containing polymers.
  • An aqueous suspension or solution/suspension of the present invention is preferably viscous or muco-adhesive, or even more preferably, both viscous and rnuco- adhesive.
  • compositions of ECS cells including AMP cells and/or ACCS and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, and still others are familiar to skilled artisans.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the invention also provides for an article of manufacture comprising packaging material and a pharmaceutical composition of the invention contained within the packaging material, wherein the pharmaceutical composition comprises compositions of ECS cells, including AMP cells and/or ACCS.
  • the packaging material comprises a label or package insert which indicates that the ECS cells, including AMP cells and/or ACCS can be used for treating spinal cord injury.
  • compositions comprising ECS cells, including AMP cells and/or ACCS may be administered to a subject to provide various cellular or tissue functions, for example, to treat spinal cord injury due to trauma, surgery, etc.
  • subject may mean either a human or non-human animal.
  • compositions may be formulated in any conventional manner using one or more physiologically acceptable carriers optionally comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.
  • the compositions may be packaged with written instructions for their use in treating spinal cord injury.
  • the compositions may also be administered to the recipient in one or more physiologically acceptable carriers.
  • Carriers for the ECS cells including AMP cells, may include but are not limited to solutions of phosphate buffered saline (PBS) or lactated Ringer's solution containing a mixture of salts in physiologic concentrations.
  • ECS cells including AMP cells and/or ACCS
  • concentration, or dose of the ECS cells, including AMP cells and/or ACCS
  • a preferred dose is one which produces a therapeutic effect, such as functional recovery following spinal cord injury, in a patient in need thereof.
  • proper doses of the ECS cells, including AMP cells and/or ACCS will require empirical determination at time of use based on several variables including but not limited to the severity and type of injury, disorder or condition being treated; patient age, weight, sex, health; other medications and treatments being administered to the patient; and the like.
  • doses dosing regimen
  • number of doses (dosing regimen) to be administered needs also to be empirically determined based on, for example, severity and type of injury, disorder or condition being treated. In a preferred embodiment, one dose is sufficient. Other preferred embodiments contemplate 2, 3, 4, or more doses.
  • the present invention provides a method of treating a spinal cord injury by administering to a subject ECS cells, including AMP cells and/or ACCS in a therapeutically effective amount.
  • therapeutically effective amount is meant the dose of ECS cells, including AMP cells and/or ACCS that is sufficient to elicit a therapeutic effect.
  • concentration of ECS cells, including AMP cells and/or ACCS in an administered dose unit in accordance with the present invention is effective in the treatment spinal cord injury.
  • at least one additional agent may be combined with the ECS cells, including AMP cells and/or ACCS to enhance healing and functional recovery following spinal cord injury.
  • Such agents include but are not limited to neuroregenerative agents, neuroprotective agents, neurotrophic factors, growth factors, cytokines, chemokines, antibodies, inhibitors, antibiotics, immunosuppressive agents, steroids, anti-fungals, anti-virals or other cell types.
  • the neuroprotective agents is for example a dopamine D3 receptor agonist
  • the neurotrophic factors are BDNF, NT-3, NT-4, CNTF, NGF, or GDNF
  • the antibodies are for example IN-I anti-NOGO antibodies
  • the inhibitor is for example the PDE4 inhibitor rollipram
  • the immunosuppressive agents are for example corticosteroids (i.e.
  • glucocorticoids cyclosporine, tacrolimus, sirolimus, methotrexate, azathiopine, mercatopurine, cytotoxic antibiotics, polyclonal and monoclonal antibodies such as anti-T-cell receptor (CD23) and anti-IL2 receptor (CD25) antibodies, interferon, opioids, TNF binding proteins, mycophenolate, and small biological agents such as FTY720;
  • the antibiotics are for example pikromycin, narbomycin, methymycin, neomethymycin;
  • the steroid is for example methylprednisolone;
  • the other cell types are for example differentiated AMP cells, or a mixture of differentiated and undifferentiated AMP cells, or a mixture of AMP cells (differentiated and/or undifferentiated) and other cells such as neural stem cells or any other progenitor cell or cells that are treated in such a way as to augment the AMP cells or AMP cell activity.
  • Inactive agents include carriers, diluents, stabilizers, gelling agents, delivery vehicles, ECMs (natural and synthetic), scaffolds, and the like.
  • ECS cells including AMP cells and/or ACCS are administered conjointly with other pharmaceutically active agents, even less of the ECS cells, including AMP cells and/or ACCS may be needed to be therapeutically effective.
  • ECS cells including AMP cells and/or ACCS
  • a delivery device such as a tube, e.g., catheter.
  • the tube additionally contains a needle, e.g., a syringe, through which the cells (i.e. ECS cells, including AMP cells) and/or conditioned media (i.e. ACCS) can be introduced into the subject at a desired location.
  • the cells i.e. ECS cells, including AMP cells
  • conditioned media i.e. ACCS
  • administering cells to subjects may also include administration by subcutaneous injection, intramuscular injection, intravenous injection, intraarterial injection, intracardiac injection, intradermal injection, intrathecal injection, epidural injection, intraperitoneal injection, intracerebral injection, direct injection into spinal cord tissue, or any injection that results in introduction into CNS tissues or spaces.
  • Infusions are also contemplated by the methods of the invention (i.e. subdural, intrathecal or intracerebral infusion, or direct infusion into spinal cord tissue, or any injection that results in introduction into CNS tissues or spaces).
  • an injectable liquid suspension of cells can be prepared and administered by a continuous drip or as a bolus.
  • ECS cells including AMP cells and/or ACCS
  • the ECS cells, including AMP cells and/or ACCS are administered as soon as possible after the injury.
  • the ECS cells, including AMP cells and/or ACCS are administered more than one time following injury.
  • the ECS cells, including AMP cells and/or ACCS are administered at the time of stabilizing surgery.
  • the ECS cells, including AMP cells and/or ACCS are administered at the time of stabilizing surgery as well as after surgery.
  • Such post-surgical administration may take the form of a single administration or multiple administrations.
  • ECS cells including AMP cells
  • a delivery device e.g., a syringe
  • the cells can be suspended in a solution contained in such a delivery device.
  • solution includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Solutions of the invention can be prepared by incorporating ECS cells, including AMP cells or partially or fully differentiated cells as described herein, in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above.
  • ECS cells, including AMP cells may be administered systemically (for example intravenously) or locally (for example by direct application under visualization during surgery).
  • the cells may be in an injectable liquid suspension preparation or in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged tissue.
  • a controllable endoscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter (e.g. 30 gauge or larger) that shear forces will not damage the cells being delivered.
  • ECS cells may be administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.
  • ECS cells including AMP cells
  • ECS cells can be used in therapy by direct administration, or as part of a bioassist device that provides temporary or permanent organ function.
  • ECS cells, including AMP cells may be grown in a bioreactor to provide extracorporeal organ support for organ relief, such as in the case of a liver assist device, to provide a plentiful source of cells for transplantation to restore organ function, or provide a source of conditioned medium that may be used to stimulate tissue regeneration and/or healing.
  • ECS cells including AMP cells
  • Tissues are an aggregation of similarly specialized cells united in the performance of a particular function. Tissue is intended to encompass all types of biological tissue including both hard and soft tissue. Also included in the definition of tissues are neural tissues, including the functional cells of the nervous system, neurons, and various supporting cells such as glial cells, oligodendrocytes, and the like.
  • Support matrices into which the ECS cells, including AMP cells, can be incorporated or embedded include matrices which are recipient-compatible and which degrade into products which are not harmful to the recipient. These matrices provide support and protection for ECS cells, including AMP cells, in vivo and are, therefore, the preferred form in which such cells are transplanted into the recipient subjects.
  • Natural and/or synthetic biodegradable matrices are examples of such matrices. Natural biodegradable matrices include plasma clots, e.g., derived from a mammal, collagen, fibronectin, and laminin matrices.
  • Suitable synthetic material for a cell transplantation matrix must be biocompatible to preclude migration and immunological complications, and should be able to support extensive cell growth and differentiated cell function. It must also be resorbable, allowing for a completely natural tissue replacement.
  • the matrix should be configurable into a variety of shapes and should have sufficient strength to prevent collapse upon implantation. Recent studies indicate that the biodegradable polyester polymers made of polyglycolic acid fulfill all of these criteria (Vacanti, et al. J. Ped. Surg. 23:3-9 (1988); Cima, et al. Biotechnol. Bioeng. 38:145 (1991); Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 (1991)).
  • synthetic biodegradable support matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. Further examples of synthetic polymers and methods of incorporating or embedding cells into these matrices are also known in the art. See e.g., U.S. Pat. Nos. 4,298,002 and 5,308,701.
  • Attachment of the cells to the polymer may be enhanced by coating the polymers with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof, and other materials known to those skilled in the art of cell culture. All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation.
  • the polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release and degradation studies.
  • GPC gel permeation chromatography
  • DSC differential scanning calorimetry
  • IR infrared
  • biodegradable polymeric matrix angiogenic and other bioactive compounds (i.e. neurotrophic factors) can be incorporated directly into the support matrix so that they are slowly released as the support matrix degrades in vivo. As the cell-polymer structure is vascularized and the structure degrades, ECS cells, including AMP cells, may differentiate according to their inherent characteristics.
  • bioactive compounds i.e. neurotrophic factors
  • vascular growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and heparin binding epidermal growth factor like growth factor (HB-EGF), could be incorporated into the matrix or be provided in conjunction with the matrix.
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • HB-EGF heparin binding epidermal growth factor like growth factor
  • polymers containing peptides such as the attachment peptide RGD can be synthesized for use in forming matrices (see e.g. U.S. Pat. Nos. 4,988,621, 4,792,525, 5,965,997, 4,879,237 and 4,789,734).
  • the ECS cells may be transplanted in a gel matrix (such as Gelfoam from Upjohn Company) which polymerizes to form a substrate in which the cells can grow.
  • a gel matrix such as Gelfoam from Upjohn Company
  • encapsulation technologies have been developed (e.g. Lacy et al., Science 254:1782-84 (1991); Sullivan et al., Science 252:718-712 (1991); WO 91/10470; WO 91/10425; U.S. Patent No. 5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538).
  • ECS cell a cell that can be repeatedly transplanted at intervals until a desired therapeutic effect, i.e. functional recovery from spinal cord injury, is achieved.
  • the present invention also relates to the use of ECS cells, including AMP cells, in three dimensional cell and tissue culture systems to form structures analogous to tissue counterparts in vivo.
  • ECS cells including AMP cells
  • the resulting tissue will survive for prolonged periods of time, and perform tissue-specific functions following transplantation into the recipient host.
  • Methods for producing such structures are described in U.S. Pat. No. 5,624,840 and 6,428,802, which are incorporated herein in their entireties.
  • the three-dimensional matrices to be used are structural matrices that provide a scaffold for the cells, to guide the process of tissue formation.
  • Scaffolds can take forms ranging from fibers, gels, fabrics, sponge-like sheets, and complex 3-D structures with pores and channels fabricated using complex Solid Free Form Fabrication (SFFF) approaches.
  • SFFF Solid Free Form Fabrication
  • Cells cultured on a three-dimensional matrix will grow in multiple layers to develop organotypic structures occurring in three dimensions such as ducts, plates, and spaces between plates that resemble sinusoidal areas, thereby forming new liver tissue.
  • the present invention provides a scaffold, multi-layer cell and tissue culture system.
  • the term "scaffold” means a three-dimensional (3D) structure (substrate and/or matrix) that cells grow in or on. It may be composed of biological components, synthetic components or a combination of both. Further, it may be naturally constructed by cells or artificially constructed. In addition, the scaffold may contain components that have biological activity under appropriate conditions.
  • the structure of the scaffold can include a mesh, a sponge or can be formed from a hydrogel.
  • Examples of such scaffolds include a three-dimensional stromal tissue or living stromal matrix which has been inoculated with stromal cells that are grown on a three dimensional support.
  • the extracellular matrix proteins elaborated by the stromal cells are deposited onto the scaffold, thus forming a living stromal tissue.
  • the living stromal tissue can support the growth of ECS cells, including AMP cells, or partially or fully differentiated cells derived therefrom later inoculated to form the three-dimensional cell culture. Examples of other three dimensional scaffolds are described in U.S. Pat. No. 6,372,494.
  • the design and construction of the scaffolding to form a three-dimensional matrix is of primary importance.
  • the matrix should be a pliable, non-toxic, injectable porous template for vascular ingrowth.
  • the pores should allow vascular ingrowth. These are generally interconnected pores in the range of between approximately 100 and 300 microns, i.e., having an interstitial spacing between 100 and 300 microns, although larger openings can be used.
  • the matrix should be shaped to maximize surface area, to allow adequate diffusion of nutrients, gases and growth factors to the cells on the interior of the matrix and to allow the ingrowth of new blood vessels and connective tissue.
  • a porous structure that is relatively resistant to compression is preferred, although it has been demonstrated that even if one or two of the typically six sides of the matrix are compressed, that the matrix is still effective to yield tissue growth.
  • the polymeric matrix may be made flexible or rigid, depending on the desired final form, structure and function.
  • a flexible fibrous mat is cut to approximate the entire defect then fitted to the surgically prepared defect as necessary during implantation.
  • An advantage of using the fibrous matrices is the ease in reshaping and rearranging the structures at the time of implantation.
  • a sponge-like structure can also be used to create a three-dimensional framework.
  • the structure should be an open cell sponge, one containing voids interconnected with the surface of the structure, to allow adequate surfaces of attachment for sufficient ECS cells, including AMP cells, or differentiated cells to form a viable, functional implant.
  • the invention also provides for the delivery of ECS cells, including AMP cells, including compositions described herein, in conjunction with any of the above support matrices as well as amnion-derived membranes.
  • ECS cells including AMP cells, including compositions described herein, in conjunction with any of the above support matrices as well as amnion-derived membranes.
  • Such membranes may be obtained as a by-product of the process described herein for the recovery of AMP cells, or by other methods, such as are described, for example, in U.S. Patent No. 6,326,019 which describes a method for making, storing and using a surgical graft from human amniotic membrane, US 2003/0235580 which describes reconstituted and recombinant amniotic membranes for sustained delivery of therapeutic molecules, proteins or metabolites, to a site in a host, U.S.
  • ECS cells including AMP cells
  • conditioned media i.e. ACCS
  • cell lysates may be added to such membranes.
  • amniotic tissue in which amnion epithelial cells have not been stripped away may be used to deliver ECS cells, including AMP cells, to a particular site.
  • ECS cells, including AMP cells used in conjunction with amniotic tissue or other matrices can be used in combination with other therapeutically useful cells and/or cells expressing biologically active therapeutics such as those described in below.
  • ECS cells including AMP cells, may be genetically engineered to produce a particular therapeutic protein.
  • Therapeutic protein includes a wide range of biologically active proteins including, but not limited to, growth factors, neurotrophic factors, enzymes, hormones, cytokines, inhibitors of cytokines, blood clotting factors, peptide growth and differentiation factors.
  • Particular differentiated cells may be engineered with a protein that is normally expressed by the particular cell type.
  • neural cells can be engineered to produce chemical transmitters or neurotrophic factors or axonal guidance factors.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid encoding the protein of interest linked to appropriate transcriptional/translational control signals.
  • Suitable methods for transferring vector or plasmids into ECS cells, including AMP cells, or cells differentiated therefrom include lipid/DNA complexes, such as those described in U.S. Pat. Nos. 5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202; and 6,051,429.
  • Suitable reagents include lipofectamine, a 3:1 (w/w) liposome formulation of the poly-cationic lipid 2,3- dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-d- imethyl-1 -propanaminium trifluoroacetate (DOSPA) (Chemical Abstracts Registry name: N-[2-(2,5-bis[(3-aminopropyl)amino]-l- oxpentyl)amino)ethyl- ]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-l-propanamin-trifluoroacetate), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water.
  • DOSPA poly-cationic lipid 2,3- dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N
  • Exemplary is the formulation Lipofectamine 200 OTM (available from Gibco/Life Technologies # 11668019).
  • Other reagents include: FuGENETM 6 Transfection Reagent (a blend of lipids in non- liposomal form and other compounds in 80% ethanol, obtainable from Roche Diagnostics Corp. # 1814443); and LipoTAXITM transfection reagent (a lipid formulation from Invitrogen Corp., # 204110).
  • Transfection of ECS cells can be performed by electroporation, e.g., as described in Roach and McNeish (Methods in MoI. Biol. 185:1 (2002)).
  • Suitable viral vector systems for producing stem cells with stable genetic alterations may be based on adenoviruses, lentiviruses, retroviruses and other viruses, and may be prepared using commercially available virus components.
  • ECS cells including AMP cells, or cells that are partially differentiated or fully differentiated therefrom may be administered or transplanted to a subject to provide various cellular or tissue functions specific to the differentiated cell type.
  • the present invention provides for administration of neural cells derived from ECS cells, including AMP cells, for treatment of spinal cord injury and scarring.
  • the ECS cells may be used in in vitro priming procedures that result in neural stem cells becoming neurons when grafted into non-neurogenic or neurogenic areas of the CNS.
  • ECS cells including AMP cells
  • the ECS cells may be contacted with various growth factors (termed differentiation factors) that influence differentiation of such stem cells into particular cell types such as neural cells, hepatocytes, pancreatic cells, vascular endothelial cells, muscle cells and cardiomyocytes.
  • growth factors include neural cells, hepatocytes, pancreatic cells, vascular endothelial cells, muscle cells and cardiomyocytes.
  • Ectoderm neural differentiation. Clusters are removed from the large-scale apparatus and transferred to ultra-low adherence 6-well plates. The differentiation protocol described by Carpenter, M.K. et al. (2001) Exp Neurol 172:383-397 for human embryonic stem cells is followed for differentiation as follows. 10 mM all-trans retinoic acid (RA) will added to the culture medium (80% KO-DMEM, 1 mM glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% non-essential amino acids, and 20% FBS) containing these clusters in suspension.
  • RA all-trans retinoic acid
  • clusters are plated onto poly-L-lysine/f ⁇ bronectin-coated plates in differentiation medium (DMEM/F-12 with B27 (Gibco), 10 ng/ml human epidermal growth factor (hEGF), 10 ng/ml human basic fibroblast growth factor (hbFGF) (Gibco), 1 ng/ml human platelet-derived growth factor- AA (hPDGF-AA) (R & D Systems), and 1 ng/ml human insulin-like growth factor- 1 (hIGF-1) (R & D Systems) for 3 days. After 3 days under these conditions, the cells are harvested for RNA or fixed.
  • differentiation medium DMEM/F-12 with B27
  • hEGF human epidermal growth factor
  • hbFGF human basic fibroblast growth factor
  • hPDGF-AA human platelet-derived growth factor- AA
  • hIGF-1 R & D Systems
  • RNA is analyzed by reverse transcriptase-PCR (RT-PCR) for nestin, GFAP and MAP-2.
  • Differentiated cells derived from ECS cells may be detected and/or enriched by the detection of tissue-specific markers by immunological techniques, such as flow immunocytochemistry for cell-surface markers, immunohistochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods.
  • differentiated cells may be detected using selection markers.
  • ECS cells can be stably transfected with a marker that is under the control of a tissue-specific regulatory region as an example, such that during differentiation, the marker is selectively expressed in the specific cells, thereby allowing selection of the specific cells relative to the cells that do not express the marker.
  • the marker can be, e.g., a cell surface protein or other detectable marker, or a marker that can make cells resistant to conditions in which they die in the absence of the marker, such as an antibiotic resistance gene (see e.g., in U.S. Pat. No. 6,015,671).
  • Example 1 Preparation of AMP cell compositions
  • Method of obtaining selected AMP cells Cells were plated immediately upon isolation from the amnion. After ⁇ 2 days in culture non-adherent cells were removed and the adherent cells were kept. This attachment to plastic tissue culture vessel is the selection method used to obtain the desired population of cells. Adherent and non-adherent AMP cells appear to have a similar cell surface marker expression profile but the adherent cells have greater viability and are the desired population of cells. Adherent AMP cells were cultured until they reached -120,000-150,000 cells/cm 2 . At this point, the cultures were confluent. Suitable cell cultures will reach this number of cells between ⁇ 5-14 days.
  • Attaining this criterion is an indicator of the proliferative potential of the AMP cells and cells that do not achieve this criterion are not selected for further analysis and use.
  • the AMP cells Once the AMP cells reach ⁇ 120,000-150,000 cells/cm 2 , they were collected and cryopreserved. This collection time point is called p0 and all subsequent analyses are done with thawed pO AMP cells.
  • the AMP cells of the invention can be used to generate ACCS.
  • the AMP cells were isolated as described herein and 10 xlO 6 cells were seeded into T75 flasks containing 10ml culture medium. The cells are cultured until confluent, the medium is changed and ACCS was collected 3 days post- confluence. Skilled artisans will recognize that other embodiments for collecting ACCS from confluent cultures, such as using other tissue culture vessels, including but not limited to cell factories, flasks, hollow fibers, or suspension culture apparatus, are also contemplated by the methods of the invention. It is also contemplated by the instant invention that the ACCS be cryopreserved following collection.
  • Example 3 Use of AMP cells to treat spinal cord injury in NOD SCID mice.
  • the improvement observed for the epicenter transplanted animals reflects the gain of coordinated locomotion.
  • histological analyses need to be done to determine if there is evidence of differentiation and/or regeneration.
  • Example 4 Histology of spinal cord from AMP cell transplanted NOD SCID mice
  • mice are anesthetized and transcardially perfused with 30 ml of PBS followed by 100 ml of 4% paraformaldehyde.
  • Spinal cords are dissected, and the segments corresponding to T2-T5, T6-T12, and T13-L3 are blocked. Blocks are post-fixed overnight in 4% paraformaldehyde.
  • blocks are equilibrated in 30% sucrose/PBS for 12 h, embedded in OCT compound, and frozen at -65 0 C in isopentane for sectioning. Tissue sections are analyzed for markers indicating differentiation and/or regeneration of cells.
  • Example 5 Use of ACCS to treat spinal cord injury in NOD SCID mice
  • Example 3 An experiment is performed as described in Example 3 except that the NOD SCID mice are treated with ACCS (see Example 2 for ACCS collection). The same analyses are performed on this experimental group.

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Abstract

L'invention a pour objet des procédés favorisant la guérison d'une lésion de la moelle épinière. L'invention a également pour objet des procédés pour minimiser l'ampleur de la cicatrice résultant d'une lésion de la moelle épinière. De tels procédés utilisent de nouvelles compositions, comprenant, mais sans y être limitées, des cellules sécrétant de la cytokine extra-embryonnaire (actuellement appelées cellules ECS), comprenant, mais sans y être limitées, des cellules souches pluripotentes issues de l'amnios (actuellement appelées cellules AMP) et des milieux conditionnés issus de celles-ci (actuellement appelées suspension de cytokine cellulaire issue de l'amnios ou ACCS), ces constituants étant présents individuellement ou en combinaison entre eux et/ou avec d'autres agents.
EP07777388A 2006-06-14 2007-06-04 Procédés pour traiter une lésion de la moelle épinière et pour minimiser l'étendue de la cicatrisation Withdrawn EP2035093A4 (fr)

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US20090054339A1 (en) * 2007-08-22 2009-02-26 Marshall Vivienne S Novel cellular factor-containing solution compositions
MX2013006592A (es) * 2010-12-17 2014-02-06 Anthrogenesis Corp Tratamiento de lesion de la medula espinal y lesion traumatica de cerebro empleando celulas adherentes obtenidas del amnios.

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EP2316919B1 (fr) 2001-02-14 2015-10-07 Anthrogenesis Corporation Placenta post-gravidique de mammifère, son utilisation et céllules souches placentaires correspondantes
EP1442115B9 (fr) 2001-11-15 2009-12-16 Children's Medical Center Corporation Techniques d'isolation, de developpement et de differenciation de cellules souches provenant de villosites choriales, de liquide amniotique ainsi que de placenta et applications therapeutiques
US20040161419A1 (en) 2002-04-19 2004-08-19 Strom Stephen C. Placental stem cells and uses thereof
CA2483010A1 (fr) 2002-04-19 2003-10-30 University Of Pittsburgh Of The Commonwealth System Of Higher Education Cellules souches placentaires et utilisations
JP2005534345A (ja) 2002-07-29 2005-11-17 エス セル インターナショナル ピーティーイー リミテッド インスリン陽性、グルコース応答性細胞の分化のための多段階方法
WO2005041944A2 (fr) * 2003-06-27 2005-05-12 Schepens Eye Research Institute Procedes et compositions favorisant la regeneration des axones et le remplacement therapeutique de cellules
WO2005001080A2 (fr) * 2003-06-27 2005-01-06 Ethicon, Incorporated Cellules derivees de tissus post-partum destinees a etre utilisees pour traiter des maladies du coeur et du systeme circulatoire
PL1789534T3 (pl) 2004-08-16 2015-03-31 Cellresearch Corp Pte Ltd Wyodrębnienie komórek macierzystych/progenitorowych z błony owodniowej pępowiny
US20060222634A1 (en) 2005-03-31 2006-10-05 Clarke Diana L Amnion-derived cell compositions, methods of making and uses thereof
CN103356706B (zh) * 2005-03-31 2016-01-20 斯丹姆涅恩有限公司 促进无疤愈合或改善皮肤外观的美容制剂

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