WO2010096177A1 - Isolated monocyte populations and related therapeutic applications - Google Patents

Isolated monocyte populations and related therapeutic applications Download PDF

Info

Publication number
WO2010096177A1
WO2010096177A1 PCT/US2010/000477 US2010000477W WO2010096177A1 WO 2010096177 A1 WO2010096177 A1 WO 2010096177A1 US 2010000477 W US2010000477 W US 2010000477W WO 2010096177 A1 WO2010096177 A1 WO 2010096177A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
isolated
monocyte
population
cell
Prior art date
Application number
PCT/US2010/000477
Other languages
English (en)
French (fr)
Inventor
Martin Friedlander
Matthew R. Ritter
Stacey K. Moreno
Mohammad A. El-Kalay
Original Assignee
The Scripps Research Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Scripps Research Institute filed Critical The Scripps Research Institute
Priority to CA2752679A priority Critical patent/CA2752679A1/en
Priority to CN2010800079738A priority patent/CN102317446A/zh
Priority to EA201190109A priority patent/EA201190109A1/ru
Priority to MX2011008826A priority patent/MX2011008826A/es
Priority to JP2011551065A priority patent/JP2012518407A/ja
Priority to BRPI1008392-8A priority patent/BRPI1008392A2/pt
Priority to AU2010216374A priority patent/AU2010216374A1/en
Priority to EP10744061A priority patent/EP2398900A4/en
Publication of WO2010096177A1 publication Critical patent/WO2010096177A1/en
Priority to ZA2011/05869A priority patent/ZA201105869B/en
Priority to US13/136,940 priority patent/US20120009166A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/051—Lipid A (MPA, MPL)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G01N15/042—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1497—Particle shape

Definitions

  • Ocular vascular diseases such as age related macular degeneration (ARMD) and diabetic retinopathy (DR) are due to abnormal choroidal or retinal neovascularization respectively. They are the leading causes of visual loss in industrialized countries. Since the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small disturbances such as those seen in vascular proliferation or edema can lead to significant loss of visual function. Inherited retinal degenerations, such as Retinitis Pigmentosa (RP), are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy.
  • RP Retinitis Pigmentosa
  • the present invention provides isolated cell populations containing substantially pure monocytes that express CD33 antigen and CD14 antigen. Some of these isolated cell populations are isolated from a mammalian peripheral blood sample, a cord blood sample or a bone marrow sample. Some of the isolated cell populations are comprised of human cells or murine cells. In some of the isolated cell populations, at least 70%, 80% or 90% of the cells express surface markers CD 14 and CD33. Some of the isolated cell populations do not contain cells that express CD34. Some of isolated cell populations are substantially free of ALDH r cells. The isolated cell populations can be further activated in vitro or ex vivo. This can be accomplished with any monocyte-activating compounds, e.g., LPS, MPLA, or MCP-I .
  • monocyte-activating compounds e.g., LPS, MPLA, or MCP-I .
  • the invention provides methods for treating ocular vascular disorders.
  • the methods involve administering to a subject suffering from an ocular vascular disorder an isolated monocyte population in an amount that is sufficient to treat or ameliorate the ocular vascular disorder.
  • the monocyte population is isolated from a blood sample or a bone marrow sample from the subject.
  • the subject to be treated with the methods is a human.
  • the monocyte population comprises substantially pure CD14 + /CD33 + cells. For example, at least 80% of the cells in the isolated monocyte population are CD14 + /CD33 + .
  • the isolated monocyte population is activated in vitro or ex vivo prior to being administered to the subject.
  • the isolated monocyte cells can be activated with LPS, MPLA, or MCP-I .
  • an untreated monocyte population (or an in vitro or ex vivo activated monocyte population) is co-administered to a subject along with such a monocyte- activating compound.
  • ocular vascular disorders can be treated with methods of the invention. Examples include ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlutions, retina edema, macular degeneration and retinitis pigmentosa.
  • the isolated monocyte population is administered to the subject via a local route, e.g., via intravitreal injection.
  • the monocyte population is administered to the subject via a systemic route, e.g., via intravenous injection.
  • the invention provides other methods of treating or ameliorating an ocular disease in a subject.
  • These methods entail (i) isolating from a blood sample or a bone marrow sample of a subject having an ocular vascular disease a substantially pure monocyte population; and (ii) administering the isolated monocyte population to the subject in an amount sufficient to treat or ameliorate the ocular vascular disease.
  • Some of these methods additionally entail activating the isolated monocyte population ex vivo prior to administering the cells to the subject. Any compounds known to be able to activate monocytes can be used in these embodiments.
  • the isolated monocyte cells can be activated with LPS, MPLA, or MCP-I.
  • the isolated monocyte population is coadministered to the subject along with a monocyte-activating compound.
  • the monocyte population used in these methods contains substantially pure CD14 + /CD33 + cells.
  • at least about 80% of the cells in the isolated monocyte population express surface markers CD33 and CD14.
  • the monocyte population is isolated by (i) debulking red blood cells from the sample; and (ii) separating remaining red blood cells and other cell types in the sample from monocytes based on their size, granularity or density.
  • the remaining red blood cells and other cell types are separated from monocytes by density centrifugation or fluorescence- activated cell sorting (FACS).
  • FACS fluorescence- activated cell sorting
  • red blood cells can be debulked by Hespan differential centrifugation or Ficoll density gradient centrifugation.
  • These methods can additional include a step of assaying the isolated cell population for expression of surface marker CD14 and CD33.
  • Figures 1A-1B show properties and therapeutic activities of isolated monocyte populations.
  • A Flow cytometry plot showing population of monocytes (gated) that are distinct from lymphocytes. No labeling was used to discriminate these populations; and
  • B Data obtained from the mouse oxygen-induced retinopathy model demonstrating that human peripheral blood (HuPB) monocytes isolated in the described manner significantly reduce both neovascular tuft area (black bars) as well as vascular obliteration (white bars) compared to vehicle injection. These results were similar to mouse bone marrow-derived CD44hi cells used as a positive control.
  • Figures 2A-2B show results from flow cytometry analysis of fractions generated by density centrifugation. The data show that the sample is depleted of CD2 + /CD3 + lymphocytes (A) and enriched for CD14 + /CD33 + monocytes (B).
  • Figure 3 shows results from ALDH labeling of peripheral blood indicating negligible ALDH br /SSC population.
  • Figure 4 shows results from flow cytometry analysis indicating the presence of small number of CD34 + cells (top right) relative to the target CD14 + monocytes (top left) in the isolated cell population.
  • Figure 5 shows post-sort analysis of human peripheral blood monocytes or lymphocytes selected on the basis of light scattering properties as described above.
  • the monocyte fraction is shown to be composed of ⁇ 88% CD14 + cells while the lymphocyte population contains virtually no CD14 + cells. Also shown is analysis of CDl Ib and CD33 showing high expression of both of these myeloid markers on the monocyte fraction and few positive cells in the lymphocyte fraction.
  • Figure 6 shows results of an in vitro chemotaxis assay showing dose-dependent increase in migration of monocytes (Mono) in response to MCP-I .
  • Lymphocytes (Lympho) failed to respond to MCP-I.
  • Mouse CD44hi bone marrow cells (CD44Hi), which contains monocytes, also responded to MCP-I.
  • Figure 7 shows in vitro differential adhesion assay demonstrating the ability of increasing numbers of monocytes to adhere to untreated cell culture plastic. Lymphocytes were unable to adhere in significant number to the same substrate.
  • Figure 8 shows images from retinal whole mounts which indicate the presence of
  • GFP-expressing cells in the retina after intracardiac injection 5 days earlier. Injury was created in the retina through exposure to hyperoxia.
  • FIG. 9 shows cytometric bead array (CBA) analysis of secreted cytokines from
  • LPS-treated monocyte-enriched F5 cells (ActF5).
  • the data showed increased secretion of IL- lbeta, 11-6, IL-8 and TNF after LPS stimulation.
  • approximate ED50 is given as a reference for quantity and biological activity of protein present in media. Units are in pg/ml.
  • Figure 10 shows cytometric bead array data demonstrating increased secretion of cytokines after incubation with LPS, MPLA or mouse MCP-I for 1 hr or 4 hrs. Two concentrations of LPS and MPLA are shown. Values represent the ratio of the treated
  • Figure 11 shows cytometric bead array data following 4h and 19h stimulation with
  • LPS LPS, mouse MCP-I, human MCP-I and MPLA at different concentrations.
  • the 19h time point shows that, in addition to LPS and MPLA, mouse and human MCP-I also stimulate secretion of IL-8 and IL-6, albeit at lower levels.
  • the present invention relates to isolated and substantially pure populations of monocyte cells which are useful for treating or ameliorating ocular vascular diseases or degenerative disorders.
  • the monocyte populations isolated by the present inventors contain substantially pure CD14 + /CD33 + monocytes.
  • the isolated monocyte populations possess the activity of promoting vascular repair as examined in eye disease models.
  • the monocyte populations are also distinct from other known hematopoietic cell populations for clinical use, as evidenced by a lack of AldeFluor Bright labeling and independence on CD34 + cells for their therapeutic activities.
  • the isolated cell populations are also characterized by being CD34 " and/or containing a very low amount of cells with high level expression of aldehyde dehydrogenase (ALDH br cells).
  • ADH br cells aldehyde dehydrogenase
  • the inventors found that some of the isolated monocyte populations upon activation ex vivo have enhanced ability to promote blood vessel repair.
  • monocytes isolated from donors with retina vascular disorders can also be activated ex vivo and promote vascular repair in a mouse model of ischemic retinopathy, similar to cells isolated from normal donors.
  • the present invention provides isolated or substantially purified monocyte populations that are therapeutically effective.
  • the invention also provides novel methods for isolating such monocyte populations.
  • the invention further provides methods of treating or ameliorating diseases or disorders related to or mediated by aberrant ocular vascularization.
  • methods are provided for producing highly active monocyte cells by in vitro or ex vivo activation with compounds capable of activating monocyte (e.g., agonist compounds of CD14 or TLR4), as well as methods for identifying novel compounds that can activate monocyte cells in a similar fashion.
  • the invention also encompasses therapeutic methods using a combination of the isolated monocyte populations and a compound capable of activating and recruiting the cells (e.g., MCP-I). In these methods, the cells can be activated upon administration to the subject, and a sustained effect can be mediated by additional recruited cells.
  • Hematopoietic stem cells that do not express significant levels of these antigens are commonly referred to a lineage negative (Lin .).
  • Human hematopoietic stem cells commonly express other surface antigens such as CD31, CD34, CDl 17 (c-kit) and/or CDl 33.
  • Murine hematopoietic stem cells commonly express other surface antigens such as CD34, CDl 17 (c-kit), Thy-1, and/or Sca-1.
  • the cells that circulate in the bloodstream are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets or thrombocytes.
  • Leukocytes include granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leucocytes).
  • Granulocytes are leukocytes characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. There are three types of granulocytes: neutrophils, basophils, and eosinophils.
  • Agranulocytes are leukocytes characterized by the apparent absence of granules in their cytoplasm. Although the name implies a lack of granules, these cells do contain nonspecific azurophilic granules, which are lysosomes. Agranulocytes include lymphocytes, monocytes, and macrophages.
  • Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissues monocytes mature into different types of macrophages at different anatomical locations. Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (aprox. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.
  • Ocular neovascularization or ocular vascular disorder is a pathological condition characterized by altered or unregulated proliferation and invasion of new blood vessels into the structures of ocular tissues such as the retina or cornea.
  • ocular neovascular diseases include ischemic retinopathy, iris neovascularization, intraocular neovascularization, age-related macular degeneration, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic retinal ischemia, retinal degeneration and diabetic retinopathy.
  • corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.
  • Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, retinitis pigmentosa, retina edema (including macular edema), Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-
  • ROP Retinopathy of prematurity
  • Macular degeneration is a medical condition predominantly found in elderly adults in which the center of the inner lining of the eye, known as the macula area of the retina, suffers thinning, atrophy, and in some cases, bleeding. This can result in loss of central vision, which entails inability to see fine details, to read, or to recognize faces. According to the American Academy of Ophthalmology, it is the leading cause of central vision loss (blindness) in the United States today for those over the age of fifty years. Although some macular dystrophies that affect younger individuals are sometimes referred to as macular degeneration, the term generally refers to age-related macular degeneration (AMD or ARMD).
  • AMD age-related macular degeneration
  • Age-related macular degeneration begins with characteristic yellow deposits in the macula (central area of the retina which provides detailed central vision, called fovea) called drusen between the retinal pigment epithelium and the underlying choroid. Most people with these early changes (referred to as age-related maculopathy) have good vision. People with drusen can go on to develop advanced AMD. The risk is considerably higher when the drusen are large and numerous and associated with disturbance in the pigmented cell layer under the macula. Large and soft drusen are related to elevated cholesterol deposits and may respond to cholesterol lowering agents or the Rheo Procedure.
  • Advanced AMD which is responsible for profound vision loss, has two forms: dry and wet.
  • Central geographic atrophy the dry form of advanced AMD, results from atrophy to the retinal pigment epithelial layer below the retina, which causes vision loss through loss of photoreceptors (rods and cones) in the central part of the eye.
  • photoreceptors rods and cones
  • vitamin supplements with high doses of antioxidants, lutein and zeaxanthin have been demonstrated by the National Eye Institute and others to slow the progression of dry macular degeneration and in some patients, improve visual acuity.
  • Retinitis pigmentosa is a group of genetic eye conditions. In the progression of symptoms for RP, night blindness generally precedes tunnel vision by years or even decades. Many people with RP do not become legally blind until their 40s or 50s and retain some sight all their life. Others go completely blind from RP, in some cases as early as childhood. Progression of RP is different in each case.
  • RP is a type of hereditary retinal dystrophy, a group of inherited disorders in which abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the retina lead to progressive visual loss. Affected individuals first experience defective dark adaptation or nyctalopia (night blindness), followed by reduction of the peripheral visual field (known as tunnel vision) and, sometimes, loss of central vision late in the course of the disease.
  • Macular edema occurs when fluid and protein deposits collect on or under the macula of the eye, a yellow central area of the retina, causing it to thicken and swell. The swelling may distort a person's central vision, as the macula is near the center of the retina at the back of the eyeball. This area holds tightly packed cones that provide sharp, clear central vision to enable a person to see form, color, and detail that is directly in the line of sight. Cystoid macular edema is a type of macular edema that includes cyst formation.
  • subject and patient are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc.
  • Preferred subjects for practicing the therapeutic methods of the present invention are human.
  • Subjects in need of treatment include patients already suffering from an ocular vascular disease or disorder as well as those prone to developing the disorder.
  • substantially pure or “substantial purity” when referring to an isolated cell population means the percentage of a given cell (target cell) in the population is significantly higher than that found in a natural environment (e.g., in a tissue or a blood stream of a subject).
  • percentage of the target cell (e.g., monocyte) in a substantially pure cell population is at least about 50%, preferably at least about 60%, 70%, 75%, and more preferably at least about 80%, 85%, 90% or 95% of total cells in the cell population.
  • treating includes (i) preventing a pathologic condition (e.g., macular degeneration) from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition (e.g., macular degeneration) or arresting its development; and (iii) relieving symptoms associated with the pathologic condition (e.g., macular degeneration).
  • pathologic condition e.g., macular degeneration
  • treatment includes the administration of an isolated cell population of the invention and/or other therapeutic compositions or agents to prevent or delay the onset of the symptoms, complications, or biochemical indicia of an ocular disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Treatment further refers to any indicia of success in the treatment or amelioration or prevention of the ocular disease, condition, or disorder described herein, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • Detailed procedures for the treatment or amelioration of an ocular disorder or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.
  • the invention provides methods for isolating a population of monocytes that are useful to treat various ocular vascular disorders as described herein.
  • the monocyte populations can be isolated from suitable biological samples obtained from a mammalian subject, e.g., peripheral blood or bone marrow.
  • the methods of the present invention enable isolation of substantially pure (e.g., with at least 50%, 75% or 85% purity) monocyte populations from a bone marrow or a blood sample.
  • the blood sample can be any sample that contains the bulk of white blood cells or mononuclear leukocytes from whole blood. For example, it can be whole blood or leukapheresis product from whole blood.
  • Leukapheresis is a laboratory procedure in which white blood cells are separated from a sample of blood.
  • the monocytes present in the isolated cell populations are CD14 + /CD33 + .
  • CD33 is a transmembrane receptor expressed on cells of monocytic/myeloid lineage.
  • CD14 is a membrane-associated glycosylphosphatidylinositol- linked protein expressed at the surface of cells, especially macrophages.
  • Bone marrow, peripheral blood, and umbilical cord blood each include a sub-population of monocytes that express the CD 14 antigen and CD33.
  • these biological samples are preferred for isolating monocyte populations enriched for CDH + and CD33 + cells in accordance with the methods disclosed herein.
  • the isolated cell populations are also characterized by being CD34- and/or expressing no or low levels of aldehyde dehydrogenase (ALDH).
  • ADH aldehyde dehydrogenase
  • the monocyte populations are isolated from human bone marrow, human peripheral blood, human umbilical cord blood or other related blood samples.
  • the methods entail first removal the majority of red blood cells (RBCs) from the sample ("debulking"). This step is accompanied by separation of other blood cells (e.g., platelets, granulocytes and lymphocytes) and remaining red blood cells, if any, from monocytes.
  • a beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid.
  • a number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).
  • FSC Forward Scatter
  • SSC Segment Scatter
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source.
  • This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to derive various types of information about the physical and chemical structure of each individual particle.
  • Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
  • the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter.
  • a vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet.
  • Function and biochemical activity of the isolated cells can also be analyzed by measuring chemotaxis of the cells, e.g., using a monocyte chemotactic protein such as MCP- 1. Results from such an activity assay also provide a readout of the relative purity of the preparation and an indication of the viability and function of the isolated cells. Additional methods for examining purity and viability of the isolated monocytes include an assay that is based on differential adhesion to cell culture substrata by monocytes relative to other monoclear cells. As demonstrated in the Examples, it was found that cells generated by the isolation methods of the invention are primarily monocytes as evidenced by their ability to adhere under the described assay conditions.
  • CD34 + stem cells In addition to being CD14 + /CD33 + , some of the monocyte populations of the present invention are also characterized by a lack of CD34 + cells or a very low amount of CD34 + cells. As exemplified in the Examples below, a small amount of CD34 + cells that may be present in the initial cell preparations can be further depleted from the final isolated monocyte populations. Importantly, as disclosed herein, removal of the CD34 + cells does not result in any change of the therapeutic activities of the monocyte populations.
  • the isolated monocyte populations of the invention contain negligible amount (about 0.04%) of ALDH br cells.
  • some preferred embodiments of the invention provide isolated or purified monocyte populations that are substantially free of ALDH br cells. As measured by fluorescence-activated cell sorting, these monocyte cell populations should contain less than about 5%, 2%, or 1% of ALDH br cells. More preferably, the percentage of ALDH br cells in these cell populations should be less than 0.5%, less than 0.1%, or less than 0.05%.
  • cells from the CD14 + /CD33 + monocyte population of the invention may exert their therapeutic effect by selectively targeting astrocytes, incorporating into developing vasculature and then differentiating to become vascular endothelial cells.
  • the cells may promote neuronal rescue in the retina and promote upregulation of anti-apoptotic genes.
  • a mammalian subject e.g., a human or a mouse
  • the cells are useful for the treatment of retinal neovascular and retinal vascular degenerative diseases, and for repair of retinal vascular injury.
  • a systemic route of administration of the isolated monocyte population is employed.
  • the cells can be administered to the subject by intravenous injection that is routinely practiced in the art.
  • non-human subjects may also be administered with the cells via intracardiac injection. This can be accomplished based on procedures routinely practiced in the art. See, e.g., Iwasaki et al., Jpn. J. Cancer Res. 88:861-6, 1997; Jespersen et al., Eur. Heart J. 11:269-74, 1990; and Martens, Resuscitation 27: 177, 1994.
  • Other routes of administration may also be employed in the practice of the present invention. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20 th ed., 2000.
  • the cells Prior to administering the treated cells to a subject, the cells can also be examined in vitro to ascertain their activation. This can be typically carried out by qualitatively or quantitatively monitoring cytokine secretions by the treated monocytes. As shown in the Examples, activated monocytes have increased secretions of cytokines such as IL- l ⁇ , IL-8, IL-6 and TNF. As exemplified in the Examples, cytokine secretion profiles of monocytes can be easily assessed with routinely practiced methods such as cytometric bead array (CBD) analysis. See e.g., Elshal et al., Methods. 38:317-329, 2006; and Morgan et al., Clin. Immunol. 110:252-266, 2004.
  • CBD cytometric bead array
  • FACS fluorescence-activated cell sorting
  • a second method of isolating monocyte populations which discriminates cells based on density relies on differential mobility during centrifugation.
  • This method has certain advantages in clinical applications because disposable tubing sets can be used to ensure sterility and eliminate cross-contamination of samples.
  • human blood sample was first treated to debulk red blood cells (RBCs) by sedimentation using HESpan. Thereafter, an appropriate volume of 6% HESpan was added to anti-coagulated blood product to reach final concentration of 1.5%. The bag was gently mixed and was incubated upright, at room temperature for 45 minutes to allow the RBCs to sediment.
  • the nucleated cell fraction (NCF) was then expressed off using a manual plasma expressor and collected into a separate sterile 60OmL empty blood bag.
  • the resulting cell product was used as the starting material for further separation based on gradient density centrifugation.
  • An Elutra ® device (Gambro BCT Inc., Lakewood, Colorado) designed to enrich for Monocyte population was then utilized for processing the starting cell product.
  • the disposable tubing set was connected to the Elutra ® device.
  • the starting cell product, primary and secondary media bags containing HBSS and 0.5% HSA were then connected to the appropriate connection on the tubing set.
  • the tubing set was primed using the secondary bag.
  • the program number one (see table 1 below) was used to process the starting cell product.
  • the program automatically loaded the starting cell product into the chamber and processed it using the primary media bag.
  • the cells were then continuously centrifuged, separated and collected in multiple fractions at various flow rates.
  • the program was designed to collect 5 fractions each enriched with a particular cell population as follows. Platelets were collected in fraction one, RBC in fraction two, lymphocytes in fraction three, monocytes in fraction four and granulocytes in fraction five. Each fraction was sampled and analyzed for cell count, viability by nuclear cell counter and purity by flow cytometry. The flow rates and collection volumes for each fraction are shown in Table 1. Based on the purity and cell count, appropriate volume containing monocytes was collected and then centrifuged at 300 x g-
  • Example 2 Treating ocular vascular disorder with isolated monocyte populations [0078]
  • a murine model of oxygen-induced retinopathy was employed to examine therapeutic activities of the monocyte populations isolated with the methods described herein. Mice with oxygen-induced retinopathy were generated as described in Ritter et al., J. Clin. Invest. 116:3266-76, 2006. Specifically, oxygen-induced retinopathy was induced in C57BL/6J mice according to the protocol described by Smith et al., Invest. Ophthalmol. Vis. Sci. 35:101-11 1, 1994. For comparison, BALB/cByJ mice were also subjected to the same conditions.
  • Example 3 Other properties and activities of isolated monocyte populations [0080] To demonstrate that the cells we isolated are distinct from other known cell populations in clinical use or development, we have labeled peripheral blood samples for the expression of aldehyde dehydrogenase which, when expressed at high levels (ALDH br ), identifies CD34 + cells, CD133 + cells, kit + cells, Lineage-antigen negative (Lin ) cells. We found essentially no such labeling in peripheral blood samples ( Figure 3), fitting with the idea that stem cells are expected to be exceedingly rare in unmobilized peripheral blood. [0081] CD34 is a marker of hematopoietic stem cells and has been used to select cells for various clinical applications. We have found that such cells might comprise or adversely affect the outcome of the therapeutic applications described herein.
  • mice embryonic and human mesenchymal stem cells which, like CD34 + stem cells, are undifferentiated cells
  • OIR oxygen-induced retinopathy
  • Example 4 In vitro assays for purity and function of isolated monocytes
  • the first assay was to measure the purity of the monocyte preparation. It used an antibody against the monocyte marker CD 14 and flow cytometry ( Figure 5). As shown in Figure 5, this assay allowed us to determine the number of non-monocyte cells present in the isolated cell population and to validate the efficiency of our isolation methods.
  • the second assay was a measure of the activity of the isolated monocytes. It quantified chemotaxis of cells toward a gradient of monocyte chemotactic protein 1 (MCP-I).
  • This Example describes intracardiac administration of CD44 hl myeloid cells for therapeutic applications in mouse retinopathy model.
  • This systemic route of delivery differs from the typical local administration route (intraocular injection) used in the above Examples.
  • GFP-expressing CD44 hl myeloid cells were prepared and obtained as described in Ritter et al., J. Clin. Invest. 116:3266-76, 2006.
  • Intracardiac injection of the cells into C57BL/6J mice with oxygen-induced retinopathy typically, postnatal mice at day 7
  • Vascular targeting activity of the cells was demonstrated by analyzing GS lectin-stained retinas of the injected mice several days after the injection (e.g., 7 days or 10 days thereafter).
  • Example 6 Enhanced activities of monocyte population activated in vitro [0087] This Example describes activation of monocyte populations ex vivo and then- enhanced activities relative to non-activated cells.
  • lymphocyte-enriched population (F3) does not substantially activate after LPS, monocyte- enriched population (F5) are clearly activated with LPS.
  • cells derived from diabetic donor activate normally as measured by intracellular cytokine staining.
  • flow cytometry analysis that LPS treatment has little effect on the morphology of F5 cells as measured by forward scatter vs. side scatter.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Ophthalmology & Optometry (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/US2010/000477 2005-02-24 2010-02-19 Isolated monocyte populations and related therapeutic applications WO2010096177A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CA2752679A CA2752679A1 (en) 2009-02-20 2010-02-19 Isolated monocyte populations and related therapeutic applications
CN2010800079738A CN102317446A (zh) 2009-02-20 2010-02-19 分离的单核细胞群及相关的治疗应用
EA201190109A EA201190109A1 (ru) 2009-02-20 2010-02-19 Изолированные популяции моноцитов и связанные с ними терапевтические применения
MX2011008826A MX2011008826A (es) 2009-02-20 2010-02-19 Poblaciones de monocitos aisladas y aplicaciones terapeuticas relacionadas.
JP2011551065A JP2012518407A (ja) 2009-02-20 2010-02-19 単離された単球集団及び関連した治療応用
BRPI1008392-8A BRPI1008392A2 (pt) 2009-02-20 2010-02-19 Populações de monócito isoladas e aplicações terapêuticas relacionadas
AU2010216374A AU2010216374A1 (en) 2009-02-20 2010-02-19 Isolated monocyte populations and related therapeutic applications
EP10744061A EP2398900A4 (en) 2009-02-20 2010-02-19 INSULATED MONOCYTIC POPULATIONS AND ASSOCIATED THERAPEUTIC APPLICATIONS
ZA2011/05869A ZA201105869B (en) 2009-02-20 2011-08-11 Isolated monocyte populations and related therapeutic applications
US13/136,940 US20120009166A1 (en) 2005-02-24 2011-08-15 Isolated monocyte populations and related therapeutic applications

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US20817309P 2009-02-20 2009-02-20
US61/208,173 2009-02-20
US28324409P 2009-11-30 2009-11-30
US61/283,244 2009-11-30

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/658,440 Continuation-In-Part US20100254952A1 (en) 2005-02-24 2010-02-05 Isolated myeloid-like cell populations and methods of treatment therewith

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/136,940 Continuation US20120009166A1 (en) 2005-02-24 2011-08-15 Isolated monocyte populations and related therapeutic applications

Publications (1)

Publication Number Publication Date
WO2010096177A1 true WO2010096177A1 (en) 2010-08-26

Family

ID=42634163

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/000477 WO2010096177A1 (en) 2005-02-24 2010-02-19 Isolated monocyte populations and related therapeutic applications

Country Status (11)

Country Link
EP (1) EP2398900A4 (ja)
JP (1) JP2012518407A (ja)
KR (1) KR20110123778A (ja)
CN (1) CN102317446A (ja)
AU (1) AU2010216374A1 (ja)
BR (1) BRPI1008392A2 (ja)
CA (1) CA2752679A1 (ja)
EA (1) EA201190109A1 (ja)
MX (1) MX2011008826A (ja)
WO (1) WO2010096177A1 (ja)
ZA (1) ZA201105869B (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014188436A1 (en) * 2013-05-22 2014-11-27 Yeda Research And Development Co. Ltd Human monocyte sub-population for treatment of eye diseases and disorders

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101668462B1 (ko) * 2015-02-26 2016-10-24 충북대학교 산학협력단 혈액성분을 유효성분으로 포함하는 녹내장의 예방 또는 치료용 약제학적 조성물
FR3084463B1 (fr) * 2018-07-30 2023-04-21 Francais Du Sang Ets Procede d'analyse des plaquettes d'un echantillon sanguin
CN114441419B (zh) * 2022-01-29 2022-11-22 杭州翔宇医学检验实验室有限公司 流式圈门方法及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070231306A1 (en) * 2005-02-24 2007-10-04 The Scripps Research Institute Isolated myeloid-like cell populations and methods of treatment therewith
US20080241171A1 (en) * 2004-02-11 2008-10-02 Aldagen , Inc. Stem Cell Populations and Methods of Use

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006031467A2 (en) * 2002-07-25 2006-03-23 The Scripps Research Institute Isolated lineage negative hematopoietic stem cells and methods of treatment therewith
CA2656463C (en) * 2006-06-28 2015-12-01 Yeda Research And Development Co. Ltd. Method of treatment of age-related macular degeneration

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080241171A1 (en) * 2004-02-11 2008-10-02 Aldagen , Inc. Stem Cell Populations and Methods of Use
US20070231306A1 (en) * 2005-02-24 2007-10-04 The Scripps Research Institute Isolated myeloid-like cell populations and methods of treatment therewith

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CRAVENS ET AL.: "Human peripheral blood dendritic cells and monocyte subsets display similar chemokine receptor expression profiles with differential migratory responses.", SCAND J IMMUNOL, vol. 65, no. 6, June 2007 (2007-06-01), pages 514 - 524 *
REHMAN ET AL.: "Peripheral blood ''endothelial progenitor cells' are derived from monocyte/macrophages and secrete angiogenic growth factors.", CIRCULATION, vol. 107, no. 8, 4 March 2003 (2003-03-04), pages 1164 - 1169 *
See also references of EP2398900A4 *
VIRIYAKOSOL ET AL.: "Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide.", J. BIOL CHEM, vol. 275, no. 5, 4 February 2000 (2000-02-04), pages 3144 - 3149 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014188436A1 (en) * 2013-05-22 2014-11-27 Yeda Research And Development Co. Ltd Human monocyte sub-population for treatment of eye diseases and disorders
KR20160016874A (ko) * 2013-05-22 2016-02-15 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 안질환 및 장애의 치료를 위한 인간 단핵구 하위집단
AU2014269935B2 (en) * 2013-05-22 2019-01-24 Yeda Research And Development Co. Ltd Human monocyte sub-population for treatment of eye diseases and disorders
US10188679B2 (en) 2013-05-22 2019-01-29 Yeda Research And Development Co. Ltd Human monocyte sub-population for treatment of eye diseases and disorders
KR102257163B1 (ko) * 2013-05-22 2021-05-27 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 안질환 및 장애의 치료를 위한 인간 단핵구 하위집단

Also Published As

Publication number Publication date
BRPI1008392A2 (pt) 2015-08-25
CN102317446A (zh) 2012-01-11
KR20110123778A (ko) 2011-11-15
CA2752679A1 (en) 2010-08-26
EP2398900A1 (en) 2011-12-28
AU2010216374A1 (en) 2011-08-18
EP2398900A4 (en) 2012-08-22
ZA201105869B (en) 2012-10-31
EA201190109A1 (ru) 2012-07-30
JP2012518407A (ja) 2012-08-16
MX2011008826A (es) 2011-11-02

Similar Documents

Publication Publication Date Title
US8951793B2 (en) Method of making an isolated population of FOXP3+ regulatory T cells
US20150210982A1 (en) Isolation and Use of Human Regulatory T Cells
RU2403906C2 (ru) Способ лечения ретинопатии при недоношенности и родственных ретинопатических заболеваний
RU2331669C2 (ru) Популяция стволовых кроветворных клеток и ее применение
Friedlander et al. Progenitor cells and retinal angiogenesis
JP2009132725A (ja) 幹細胞集団および使用方法
JP2020518260A (ja) 向上した免疫抑制作用を有する間葉系前駆または幹細胞
EP1402005B1 (en) A method of purification of cells
WO2023216799A1 (zh) 一种人nkt细胞系及其应用
TW201130978A (en) Method of isolation of stem cell populations from peripheral blood using sized-based separation (elutriation)
JP2016192970A (ja) Ｔｒ１細胞を単離する新規な方法
JPH08509377A (ja) ヒトのエリスロイド前駆細胞集団
EP2398900A1 (en) Isolated monocyte populations and related therapeutic applications
US20120009166A1 (en) Isolated monocyte populations and related therapeutic applications
US20110268716A1 (en) Methods for the enrichment of viable foxp3+ cells and uses thereof
JPWO2009145238A1 (ja) 免疫調節剤及びその利用
JP3917652B2 (ja) 造血促進細胞及びその使用
Collins et al. T Cell Depletion of Allogeneic Human Bone Marrow Grafts by Soybean Lectin Agglutination and Either Sheep Red Blood Cell Rosetting or Adherence on the CD5/CD8 Cellector™
CN107050436B (zh) Mbl在制备预防或治疗效应t细胞引发疾病药物中的应用
EA035338B1 (ru) Композиция гемопоэтических стволовых клеток с пониженной воспалительной активностью, ее получение и применение
EP2527428A1 (en) Tolerogenic dendritic cells and their use in cell therapy
CN112007165B (zh) 巨噬细胞极化调节剂及其在促进血小板生成中的应用
WO2024166969A1 (ja) 天疱瘡を治療または予防するための医薬組成物
Amar Study of extracellular vesicles released by allogeneic transplants and secondary lymphoid organs in mice
Derventzi et al. An in vitro model for investigating human autologous neuronal–astrocyte and immune cell interactions underlying neurodegenerative and immunosuppressive processes in neuropathy

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201080007973.8

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10744061

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 201190109

Country of ref document: EA

WWE Wipo information: entry into national phase

Ref document number: 2752679

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2010216374

Country of ref document: AU

Date of ref document: 20100219

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2011551065

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: MX/A/2011/008826

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2010744061

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20117021989

Country of ref document: KR

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: PI1008392

Country of ref document: BR

ENP Entry into the national phase

Ref document number: PI1008392

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20110819