Ways to treat the effects of a stroke

Abstract

The present invention provides a method for improving brain function or preventing brain function loss and / or treating or preventing movement disorders and / or regenerating brain neurons in a subject suffering from stroke, the method comprising administering to the subject a STRO-1-rich + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

Description

[0001] Related Applications

[0002] This application is a divisional application of Chinese patent application No. 201280033263.1. This application claims priority to U.S. patent application No. 61 / 493,057, entitled “Methods for treating the effects of stroke”, filed on June 3, 2012. The entire contents of said application are incorporated herein by reference.

[0003] Sequence Listing

[0004] The sequence listing is submitted electronically with this application. The entire contents of the sequence listing are incorporated herein by reference.

[0005] field

[0006] The present invention is directed to methods of treating the effects of stroke in a subject.

[0007] background

[0008] In Australia, stroke is the second leading cause of death and the leading cause of disability after heart disease. In the United States, it is the third leading cause of death, with more than 140,000 people dying from stroke each year. It is also the leading cause of severe long-term disability in the United States. The projected cost of stroke in the United States between 2005 and 2050 is $2.2 trillion.

[0009] Disability affects 75% of stroke survivors and can be severe enough to reduce their ability to work. A stroke can affect a subject physically, mentally, emotionally, or a combination of all three.

[0010] Some of the physical disabilities that a stroke may cause include muscle weakness, numbness, pressure sores, pneumonia, urinary incontinence, apraxia (inability to perform learned movements), difficulty carrying out daily activities, loss of appetite, loss of speech, vision loss, and pain. If the stroke is severe enough or in a certain area, such as the brain stem, it may lead to coma or death.

[0011] Emotional problems caused by stroke may result from direct damage to the emotional centers of the brain or from frustration or difficulty adjusting to new limitations. Emotional disorders after stroke include depression, anxiety, panic attacks, flat affect (inability to express emotion), mania, apathy, and psychosis.

[0012] Cognitive deficits caused by stroke include perceptual impairments, language impairments, dementia, and problems with attention and memory. A stroke patient may be unaware of his or her disability, a condition called anosognosia. In a condition called hemispatial neglect, the patient is unable to attend to anything on the side of space opposite to the damaged hemisphere.

[0013] Up to 10% of all stroke patients develop seizures, most commonly in the weeks following the event. The severity of the stroke increases the likelihood of seizures.

[0014] A stroke is a rapidly developing loss of brain function due to a disturbance in the blood supply to the brain. This can be due to ischemia (lack of blood flow) caused by a blockage (thrombosis, arterial embolism) or hemorrhage (blood leakage). As a result, the affected area of ​​the brain cannot function, which may result in the subject being unable to move one or more limbs on one side of the body, being unable to understand or formulate language, or being unable to see one side of the visual field. A stroke often results in neuronal cell death or may result in death.

[0015] There are two common types of stroke: (i) ischemic stroke, which is caused by a temporary or permanent blockage of blood flow to the brain and accounts for 85% of stroke cases, and (ii) hemorrhagic stroke, which is caused by a ruptured blood vessel and accounts for most of the remaining cases. The most common cause of ischemic stroke is occlusion of the middle cerebral artery (intracranial artery downstream of the internal carotid artery), which damages the brain (e.g., cerebral cortex), such as the motor and sensory cortex of the brain. The damage causes hemiplegia, hemi-sensory loss, and language or visuospatial deficits depending on the damaged cerebral hemisphere.

[0016] Current treatments for ischemic stroke have focused on rescuing the ischemic penumbra, a moderately hypoperfused area of ​​the brain that maintains structural integrity but loses electrical function. Currently, there are only four clinically proven treatments to improve outcomes after stroke:

[0017] aspirin to prevent further strokes,

[0018] Acute thrombolysis with tissue plasminogen activator to reverse occlusion,

[0019] Management of the stroke unit, and

[0020] Hemicraniectomy to reduce intracranial pressure.

[0021] However, such treatments only attempt to mitigate ongoing neurological damage and do not restore lost neurons or brain function.

[0022] Several neuroprotective agents have been tested for efficacy in treating stroke and have failed, including N-methyl-D-aspartate receptor antagonists, nalmefene, lubeluzole, clomethiazole, calcium channel blockers (including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid antagonists, serotonin receptor agonists (e.g., rabinotan), and transmembrane potassium channel modulators), tirilazad, anti-ICAM-1 antibodies, human anti-leukocyte antibodies (Hu23F2G), anti-platelet antibodies (e.g., abciximab), citicoline (an exogenous form of cytidine-5'-diphosphocholine), and basic fibroblast growth factor.

[0023] From the foregoing, it is apparent that there is a need in the art for treatments for stroke. A desired treatment would repair at least some of the neural damage caused by the stroke and / or restore at least some of the brain function lost as a result of the stroke.

[0024] Overview

[0025] The present invention is based on the inventors' identification of a cell population useful for treating the effects of stroke. As exemplified herein, the inventors have shown that STRO-1 + The cells and / or their progeny and / or factors secreted thereby are capable of restoring brain function lost after a stroke. + The cells are obtained from bone marrow and / or dental pulp and may be cultured and / or stored prior to their use in therapy.The inventors used an accepted animal model of ischemic stroke (eg, affecting the brain) to illustrate the efficacy of cells and / or secreted factors to treat the effects of stroke.

[0026] For example, the inventors have shown that they can restore brain function after stroke and / or treat movement disorders. The inventors have also shown that STRO-1 + The cells secrete factors (eg, SDF-1) that induce the growth of neuronal axons. Without being bound by any theory or mode of action, the inventors propose that these factors may contribute to neuroprotection, angiogenesis, immunomodulation, and / or neuroplasticity.

[0027] The inventors also showed that STRO-1 + The cells were able to differentiate into neuron-like cells. However, after injection into the brains of subjects with stroke, few of these cells survived for more than a few weeks. These results suggest that STRO-1 + The cells and / or their progeny and / or factors secreted thereby contribute to the survival and / or regeneration of the subject's own neurons following a stroke.

[0028] The findings presented herein provide the basis for methods for treating one or more effects of stroke. For example, the methods include administering to a subject having stroke a protein rich in STRO-1. + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0029] The present invention provides a method for improving brain function or preventing brain function loss in a subject suffering from stroke, the method comprising administering to the subject a protein rich in STRO-1. + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0030] In one embodiment, improving the subject's brain function improves the subject's movement disorder. In one embodiment, the brain function is cerebral cortex function, such as motor cortex function and / or sensory cortex function.

[0031] The present invention additionally or alternatively provides a method of treating or preventing movement disorders in a subject suffering from a stroke, the method comprising administering to the subject a protein rich in STRO-1. + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0032] In one embodiment of the methods described herein, the movement disorder is paralysis, partial paralysis, slurred speech, incoordination, muscle weakness, hypotonia, hypertonia, or involuntary abnormal movements.

[0033] The present invention additionally provides a method of regenerating brain neurons in a subject suffering from a stroke, the method comprising administering to the subject a protein rich in STRO-1. + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0034] The present invention additionally provides a method for treating, reducing or preventing brain atrophy in a subject suffering from stroke, the method comprising administering to the subject a protein rich in STRO-1. + A cell population of cells and / or their progeny and / or soluble factors obtained therefrom. In one embodiment, the atrophy is in the corpus callosum.

[0035] In one embodiment, the brain neurons regenerated by performing the methods of the present invention are in the cerebral cortex, for example, in the motor cortex and / or sensory cortex.

[0036] In one embodiment, the stroke is an ischemic stroke.

[0037] An exemplary stroke contemplated by the present invention is an ischemic stroke caused by occlusion of the subject's middle cerebral artery.

[0038] In one embodiment, the method of the invention comprises administering to the subject a STRO-1-rich protein between 1 hour and 1 month, such as between 1 hour and 2 weeks, such as between 1 hour and 1 week after stroke. + For example, administering a STRO-1-rich cell population within about 72 hours after a stroke + For example, administering a STRO-1-rich cell population within about 48 hours after a stroke + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0039] In another embodiment, the method comprises administering to the subject a STRO-1-enriched + For example, administering STRO-1-rich cells approximately 24 hours after a stroke - A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0040] In one embodiment, the method comprises administering a STRO-1-enriched 亮 In one embodiment, the progeny are additionally enriched for STRO-1. 亮 cell.

[0041] Exemplary cells and / or progeny additionally express tissue non-specific alkaline phosphatase (TNAP) and / or heat shock protein 90β (HSP90β) and / or CD146.

[0042] In one embodiment, the cell population is derived from bone marrow or dental pulp.

[0043] In one embodiment, STRO-1 is enriched + The population of cells and / or their progeny and / or soluble factors derived therefrom are administered systemically.

[0044] In an alternative embodiment, STRO-1 is enriched + The population of cells and / or their progeny and / or soluble factors derived therefrom are administered locally to the brain of a subject. + The population of cells and / or progeny thereof and / or soluble factors obtained therefrom are locally administered to the brain of a subject. In one embodiment, the population and / or progeny and / or soluble factors are additionally (or alternatively) administered to the striatum of a subject.

[0045] In one embodiment, STRO-1 is enriched + The population of cells and / or progeny thereof are administered to a site distal to the stroke site and migrate to the stroke site, for example, to an ischemic infarct or a border zone surrounding an ischemic infarct.

[0046] An exemplary method described herein according to any embodiment comprises administering a dose of the population and / or the progeny and / or the soluble factors sufficient to improve brain function and / or regenerate neurons in the brain.

[0047] In one embodiment, the method comprises administering an effective dose or a therapeutically effective dose of the population and / or progeny and / or soluble factors.

[0048] In one embodiment, the method comprises administering 0.1×10 6 Up to 5×10 6 STRO-1 + Cells and / or their progeny. For example, the method comprises administering 0.3×10 6 Up to 2×10 6 STRO-1 + Cells and / or their progeny. For example, the method comprises administering 0.5×10 6 Up to 2×10 6 STRO-1 + Cells and / or their progeny. For example, the method comprises administering 0.5×10 6 to 1.5×10 6 STRO-1 + Cells and / or their progeny. For example, the method comprises administering about 5×10 5 STRO-1 + cells and / or their progeny or about 1.5×10 per kg 6 STRO-1 + Cells and / or progeny thereof. For example, the method comprises administering about 1.8×10 6 STRO-1 + cells and / or their progeny.

[0049] In one embodiment, the method described herein according to any embodiment comprises administering a low dose of STRO-1 + cells and / or their progeny. For example, low doses of STRO-1 + Cells and / or their progeny include 0.1×10 5 With 0.5×10 6 STRO-1 + cells and / or their progeny.

[0050] In one embodiment, the population and / or the progeny and / or the soluble factors are administered multiple times. For example, the population and / or the progeny and / or the soluble factors are administered once every four weeks or more.

[0051] In one embodiment, less than 25% of STRO-1 is expressed 28 days after administration. + The cells and / or their progeny remain in the brain (eg, cerebrum) of the subject. For example, less than 10% of STRO-1 cells are present 28 days after administration. + The cells and / or their progeny remain in the brain (eg, cerebrum) of the subject. For example, less than 5% or 3% of STRO-1 cells are present 28 days after administration. + The cell and / or its progeny remain in the brain (eg, cerebrum) of the subject.

[0052] In one embodiment, STRO-1 is enriched + The population of cells and / or progeny cells are autologous or allogeneic and / or soluble factors can be obtained from autologous or allogeneic cells. In one embodiment, the method described herein according to any embodiment includes isolating or enriching the population and / or progeny and / or isolating the soluble factors.

[0053] In embodiments of the methods described herein according to any embodiment, the STRO-1 is enriched + The group of cells and / or progeny cells have been cultured and expanded prior to administration and / or prior to obtaining the soluble factors. In one embodiment, the methods described herein additionally include culturing the group and / or progeny. In one embodiment, the methods additionally include storing the group and / or progeny.

[0054] In one embodiment, the population and / or its progeny cells and / or soluble factors obtained therefrom include STRO-1 + In one embodiment, the method described herein according to any embodiment additionally comprises formulating the population and / or progeny and / or soluble factors with a carrier and / or excipient.

[0055] In one embodiment, the method described herein according to any embodiment additionally includes testing the brain function of the experimenter after the treatment. For example, the method additionally includes testing the movement and / or strength and / or language and / or strength of the experimenter. In one embodiment, if the brain function of the experimenter is not significantly improved compared with before using the group and / or progeny and / or soluble factors, the method includes using the group and / or progeny and / or soluble factors of another dosage.

[0056] In one embodiment, the subject being treated is a mammal, such as a primate, eg, a human.

[0057] The present invention also provides a STRO-1-rich + A cell population of cells and / or their progeny and / or soluble factors obtained therefrom, which are used to improve brain function of a subject after stroke or treat movement disorders in a subject after stroke or regenerate brain neurons in a subject after stroke.

[0058] The present invention additionally provides STRO-1-enriched + Use of a cell population of cells and / or their progeny and / or soluble factors obtained therefrom for the manufacture of a medicament for improving brain function of a subject after stroke or treating movement disorders of a subject after stroke or regenerating brain neurons of a subject after stroke.

[0059] The present invention also provides a method comprising a STRO-1-rich + A kit of a cell population of cells and / or their progeny and / or soluble factors obtained therefrom, packaged together with instructions for use of the method described herein according to any embodiment.

[0060] For example, the present invention provides a kit comprising a composition comprising the population and / or progeny and / or soluble factors, packaged together with product information indicating the use of the composition in the method described herein according to any embodiment.

[0061] The present invention also provides a method for isolating or culturing STRO-1. + A kit of cells and / or their progeny packaged together with instructions for use of the methods described herein according to any embodiment.

[0062] The present invention also provides a method comprising providing a STRO-1-enriched protein using instructions for use of the method described herein according to any embodiment. + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0063] The present invention also provides a method comprising isolating or providing to isolate or store or providing to store a protein rich in STRO-1 + The cell population of cells and / or their progeny and / or soluble factors obtained therefrom are for use later in the methods described herein according to any embodiment.

[0064] The methods described herein should be applicable, mutatis mutandis, as a means of reducing the risk of further stroke.

[0065] As non-limiting examples, the present application provides the following embodiments:

[0066] 1. A method for improving brain function or preventing brain function loss in a subject suffering from stroke, the method comprising administering to the subject a protein rich in STRO-1 + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0067] 2. The method of embodiment 1, wherein improving the subject's brain function improves the subject's movement disorder.

[0068] 3. A method for treating or preventing movement disorders in a subject suffering from a stroke, the method comprising administering to the subject a drug rich in STRO-1 + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0069] 4. The method of embodiment 2 or 3, wherein the movement disorder is paralysis, partial paralysis, slurred speech, uncoordinated movements, muscle weakness, hypotonia, hypertonia, or involuntary abnormal movements.

[0070] 5. A method for regenerating brain neurons in a subject suffering from a stroke, the method comprising administering to the subject a protein rich in STRO-1 + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0071] 6. A method as described in embodiment 5, wherein the brain neurons are in the cerebral cortex.

[0072] 7. A method as described in embodiment 6, wherein the brain neurons are in the motor cortex and / or sensory cortex.

[0073] 8. The method of any one of embodiments 1 to 7, wherein the stroke is an ischemic stroke.

[0074] 9. The method of embodiment 8, wherein the ischemic stroke is caused by occlusion of the subject's middle cerebral artery.

[0075] 10. The method of any one of embodiments 1 to 9, comprising administering to the subject a STRO-1-enriched + A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0076] 11. The method of embodiment 10, comprising administering to the subject a STRO-1-rich - A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0077] 12. The method of any one of embodiments 1 to 11, comprising administering a STRO-1-enriched 亮 A cell population of a cell and / or its progeny and / or soluble factors obtained therefrom.

[0078] 13. The method of embodiment 12, wherein the cells additionally express tissue non-specific alkaline phosphatase (TNAP) and / or heat shock protein 90β (HSP90P) and / or CD146.

[0079] 14. The method of any one of embodiments 1 to 13, wherein the cell population is obtained from bone marrow or dental pulp.

[0080] 15. The method of any one of embodiments 1 to 8, wherein the STRO-1-enriched + The population of cells and / or their progeny and / or soluble factors derived therefrom are administered systemically.

[0081] 16. The method of any one of embodiments 1 to 14, wherein the STRO-1-enriched + The population of cells and / or progeny thereof and / or soluble factors derived therefrom are administered locally to the brain of the subject.

[0082] 17. The method according to embodiment 16, wherein the STRO-1-enriched + The population of cells and / or progeny thereof and / or soluble factors derived therefrom are administered locally to the brain of a subject.

[0083] 18. The method of any one of embodiments 1 to 17, comprising administering a dose of the population and / or the progeny and / or the soluble factors sufficient to improve brain function and / or regenerate brain neurons.

[0084] 19. The method of any one of embodiments 1 to 17, wherein the population and / or the progeny and / or the soluble factors are administered multiple times.

[0085] 20. The method of embodiment 19, wherein the population and / or the progeny and / or the soluble factors are administered once every four weeks or more.

[0086] 21. The method of any one of embodiments 1 to 20, comprising administering 0.1×10 6 Up to 5×10 6 STRO-1 + cells and / or their progeny.

[0087] 22. The method of any one of embodiments 1 to 20, comprising administering 0.3×10 6 Up to 2×10 6 STRO-1 + cells and / or their progeny.

[0088] 23. The method of any one of embodiments 1 to 20, comprising administering a low dose of STRO-1 + cells and / or their progeny.

[0089] 24. The method according to embodiment 23, wherein the low dose of STRO-1 + Cells and / or their progeny contain 0.1×10 5 to 0.5×10 6 STRO-1 + cells and / or their progeny.

[0090] 25. The method of any one of embodiments 1 to 24, wherein the STRO-1-enriched + The population of cells and / or progeny cells may be autologous or allogeneic and / or the soluble factors may be obtained from autologous or allogeneic cells.

[0091] 26. The method of any one of embodiments 1 to 25, wherein the STRO-1-enriched + The population of cells and / or progeny cells have been expanded in culture prior to administration and / or prior to obtaining the soluble factors.

[0092] 27. The method of any one of embodiments 1 to 26, wherein the STRO-1 + Cells and / or progeny cells thereof and / or soluble factors obtained therefrom to contain the STRO-1 + The cells and / or their progeny cells and / or soluble factors obtained therefrom are administered in the form of a composition with a carrier and / or an excipient.

[0093] 28. A protein rich in STRO-1 +A cell population of cells and / or their progeny and / or soluble factors obtained therefrom, which are used to improve brain function of a subject after stroke or treat movement disorders in a subject after stroke or regenerate brain neurons in a subject after stroke.

[0094] 23. Rich in STRO-1 + Use of a cell population of cells and / or their progeny and / or soluble factors obtained therefrom for the manufacture of a medicament for improving brain function of a subject after stroke or treating movement disorders of a subject after stroke or regenerating brain neurons of a subject after stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 .TNAP (STRO-3) and mesenchymal progenitor cell marker STRO-1 亮 Co-expression in adult bone marrow mononuclear cells (BMMNCs). Two-color immunofluorescence and flow cytometry were performed by incubating MACS-selected BMMNCs and indirectly labeled with STRO-1 (x-axis) with goat anti-mouse IgM antibody coupled to FITC and STRO-3 mAb (mouse IgG1) (y-axis) with goat anti-mouse IgG coupled to PE. The dot matrix histograms represent 5 × 10 4 events. The vertical and horizontal lines were set at reactivity levels less than 1.0% of the mean fluorescence obtained with isotype-matched control antibodies 1B5 (IgG) and 1A6.12 (IgM) treated under the same conditions. The results show that STRO-1 亮 A smaller population of cells co-expressed TNAP (upper right quadrant), while the rest expressed STRO-1 + The cells failed to react with STRO-3 mAb.

[0096] Figure 2 . Cultured and expanded STRO-1 亮 STRO-1 by MPC 亮 or STRO-1 暗 Gene expression profiles of progeny. Single cell suspensions of ex vivo expanded bone marrow MPCs were prepared by trypsin / EDTA treatment. Cells were stained with STRO-1 antibody, which was then visualized by incubation with goat anti-mouse IgM-fluorescein isothiocyanate. After fluorescence activated cell sorting (A), purified STRO-1 暗 or STRO-1 亮The group of expressing cells prepares total cell RNA.Use RNAzolB extraction method and standard procedure, separate total RNA from each subgroup and use as the template of cDNA synthesis.Use standard scheme as described previously (Gronthos etc. J Cell Sci.77 (5:1827-1835,2003) by PCR amplification to assess the expression of various transcripts.The primer set used in this research is shown in Table 2.After amplification, each reaction mixture is analyzed by 1.5% agarose gel electrophoresis, and is observed by ethidium bromide staining (B).Use ImageQant software to assess the relative gene expression (C) of each cell marker with reference to the expression of housekeeping gene GAPDH.

[0097] Figure 3 . Cultured and expanded STRO-1 + STRO-1 by MPC 亮 The offspring expressed high levels of SDF-1, STRO-1 暗 The offspring cannot. (A) FACS analysis of the STRO-1 region 亮 and STRO-1 暗 / 深 STRO-1 isolated by MACS + The BMMNC preparations were divided into different STRO-1 subsets. Total RNA was prepared from each STRO-1 subset and used to construct the STRO-1 亮 Subtractive hybridization library (BC). Replication has been performed using STRO-1 亮 Nitrocellulose filter spotted with representative PCR products amplified from cDNA-transformed bacterial clones. The filter was then stained with [ 32 P] deoxycytidine triphosphate (dCTP)-labeled STRO-1 亮 (B) or STRO-1 暗 / 深 (C) Subtractive cDNA probing. Arrows indicate differential expression of 1 clone containing a cDNA fragment corresponding to human SDF-1. (D) Reverse transcriptase (RT)-PCR analysis showing relative expression of SDF-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in total RNA prepared from a BMMNCSTRO-1 population freshly isolated by MACS / FACS before culture. bp indicates base pairs.

[0098] Figure 4Differentiation of human DPSCs into neuron-like cells. (A) Reproduction of a micrograph showing that human foreskin fibroblasts (HFFs) are unresponsive to neuronal induction medium in vitro. (B) Reproduction of a micrograph showing that hDPSCs differentiate into a neuronal phenotype in vitro in neuronal induction medium. (C) Graph showing Na recorded in differentiated DPSCs in response to a 30 ms duration, spanning a range of -44 mV to 36 mV, with a step size of 10 mV. + Representative examples of current traces. (D) Graphical representation showing Na obtained in differentiated (D) and undifferentiated (ND) DPSCs in response to a voltage step. + Average IV plot of the peak current.

[0099] Figure 5 .A series of representations depict the process for treating a stroke model. Figure A shows a representative rat brain coronal section stained with 2,3,5-triphenyltetrazolium chloride to visualize the infarct (white) tissue 1 day after middle cerebral artery occlusion (MCAo). Figure B shows a representative rat brain coronal section stained with anti-human mitochondrial antigen antibody and hematoxylin. The black dots in Figures A and B represent the first striatal injection site located at anterior-posterior-0.40mm, medial-lateral-4.00mm relative to bregma, and the first dorsal-ventral-5.50mm from the dura mater, and the second cortical injection site located at a distance from the dorsal-ventral-1.75mm of the dura mater. Figure C shows a graphical representation of the treatment regimen, in which animals were trained for 3 days before MCAo (denoted as Day 0), and the third segment was used as a preoperative baseline (Day -1). Animals were randomly assigned to receive human DPSCs or culture medium alone 24 hours after MCAo. Behavioral assessments were performed by an investigator unaware of the treatment group assignments on days 1, 7, 14, 21, and 28 after MCAo. All animals received daily subcutaneous injections of 10 mg / kg of cyclosporin A during the experimental period. Animals were sacrificed and their brains were processed for immunohistochemistry at the end of the neurobehavioral study. Scale bar = 2 mm (A, B). Abbreviations: Cx, cerebral cortex; DPSC, dental pulp stem cell; MCAo, middle cerebral artery occlusion; Str, striatum.

[0100] Figure 6A series of graphical representations show the effect of human DPSC treatment on neurobehavioral outcomes after MCAo. After MCAo, all animals showed functional deficits in all behavioral tests (day 1 or day 7 vs. day -1). Panel A shows that at 4 weeks after transplantation, DPSC-treated animals showed significant improvements in total neurological scores compared to vehicle-treated animals (p < 0.018 group × day interaction, repeated measures analysis of variance [ANOVA]; p < 0.05 and p < 0.01 between treatment groups at days 21 and 28, respectively; post hoc Fisher-protected minimally significant differences). As shown in Figure B, the DPSC-transplanted group also showed significantly improved forelimb sensorimotor ability (p = 0.045 total treatment effect), as demonstrated by a significantly greater number of steps performed with the contralateral forelimb in the stepping test. Panel C also shows that the DPSC-transplanted group also showed a significantly smaller proportion of time spent attempting to remove the label from the contralateral forepaw in the tape removal test compared to the vehicle-treated group (p = 0.049 total treatment effect). Differences in behavioral performance between treatment groups failed to reach statistical significance (p>0.05, repeated measures ANOVA) in the rotarod (Figure D) and cone / ledge balance beam walking (Figure E) tests. Stroke animals were unable to complete the balance beam walking task 1 day after MCAo due to circling behavior. The data have been plotted as box plots to visualize the distribution of values. The central asterisk in the box represents the median, and the box represents the middle 50% of the values ​​(i.e., it ranges from the 25th percentile to the 75th percentile). The whisker shows the range of the data, with extreme observations presented by the outer circles. Abbreviations: DPSC, dental pulp stem cells; MCAo, middle cerebral artery occlusion.

[0101] Figure 7 .Photographic representations of duplicates showing the distribution of surviving DPSCs in the brain after ischemia. Representative rat brain coronal sections show the survival and migration patterns of transplanted DPSCs (single cells indicated as black dots) at 4 weeks after transplantation. Sections were stained for human mitochondrial antigen to detect human cells from rat brain tissue. Note that the target migration of DPSCs is toward the stroke lesion and eventually accumulates around the occlusion. Scale bar = 2 mm. Abbreviations: B, bregma; CC, corpus callosum; Cx, cerebral cortex; DG, dorsal hippocampal dentate gyrus; LV, lateral ventricle; Str, striatum.

[0102] Figure 8.Photographic representations of copies showing the morphology of human DPSCs implanted 28 days after stroke. High magnification of human DPSCs in rodent brains 4 weeks after middle cerebral artery occlusion (human mitochondrial antigen-positive cells stained dark gray, right hand side of the figure) depicts accumulation in the immediate vicinity of the infarct core (Figure A) and marked migration along the CC (Figure B). Note the sparse distribution of DPSCs in the cerebral hemisphere contralateral to the injection and stroke sites. Dots indicate individual human DPSCs. Figure C shows that some cells obtained from DPSCs appear to be implanted in the walls of cerebral vessels, with a morphological appearance consistent with endothelial cells (arrows), pericytes, or smooth muscle cells (arrows). Figure D shows that the morphology of cells obtained from DPSCs suggests astrocytes (arrows) and neurons (arrows). Scale bars = 2 mm (Figures A, B) and 25 μm (Figures C, D and = 40 times magnification insets in Figures A, B). Abbreviations: B, bregma; CC, corpus callosum.

[0103] Fig. 9 .A series of representations shows the effect of DPSC treatment on contralateral CC atrophy. Figure A shows a representative rat brain slice at the level of the bregma, indicating two sites for measuring CC thickness at the midline of the brain (LI) and at the lateral edge of the lateral ventricle (L2) in both the ipsilateral and contralateral hemispheres. Figure B shows a trend of reduced corpus callosum atrophy in the contralateral hemisphere of DPSC-treated animals compared with vehicle-treated animals at 4 weeks after transplantation. Normalization of L2 (ipsilateral) to LI (midline) CC measurements failed to reach statistical significance (P> 0.05). There was no difference in contralateral CC thickness measurements between treatment groups. Data are shown as mean ± SEM. Scale bar = 100 μm. Abbreviations: CC, corpus callosum; Contra, contralateral; Cx, cerebral cortex; DPSC, dental pulp stem cell; Ipsi, ipsilateral; LV, lateral ventricle.

[0104] Fig.10 .Graphical representation shows representative flow cytometry histograms generated from single cell suspensions of culture-expanded bone marrow-derived cynomolgus macaque MPCs with positive cell surface expression of mesenchymal stem cell markers STRO-1, STRO-4, and CD146 (solid line) relative to isotype (IgM, IgG2a, and IgG1) negative controls (dashed lines) when detected using goat anti-mouse IgM or IgG conjugated FITC secondary antibodies. Representative histograms also show that cynomolgus macaque MPCs lack cell surface marker expression of monocytes / macrophages (CD14), hematopoietic stem / progenitor cells (CD34), and mature leukocytes (CD45). Fluorescence levels greater than 1% compared to isotype controls are indicated as positive.

[0105] Sequence Listing Annotations

[0106] SEQ ID NO: 1—Oligonucleotide used to amplify nucleic acid encoding GAPDH.

[0107] SEQ ID NO:2—Oligonucleotide for amplifying nucleic acid encoding GAPDH

[0108] SEQ ID NO:3 - Oligonucleotide for amplifying nucleic acid encoding SDF-1

[0109] SEQ ID NO:4 - Oligonucleotide for amplifying nucleic acid encoding SDF-1

[0110] SEQ ID NO:5—Oligonucleotide for amplifying nucleic acid encoding IL-1β

[0111] SEQ ID NO:6—Oligonucleotide for amplifying nucleic acid encoding IL-1β

[0112] SEQ ID NO:7 - Oligonucleotide for amplifying nucleic acid encoding FLT-1

[0113] SEQ ID NO:8—Oligonucleotide for amplifying nucleic acid encoding FLT-1

[0114] SEQ ID NO:9 - Oligonucleotide for amplifying nucleic acid encoding TNF-a

[0115] SEQ ID NO: 10 - Oligonucleotide for amplifying nucleic acid encoding TNF-a

[0116] SEQ ID NO: 11 - Oligonucleotide for amplifying nucleic acid encoding KDR

[0117] SEQ ID NO: 12 - Oligonucleotide for amplifying nucleic acid encoding KDR

[0118] SEQ ID NO: 13 - Oligonucleotide for amplifying nucleic acid encoding RANKL

[0119] SEQ ID NO: 14 - Oligonucleotide for amplifying nucleic acid encoding RANKL

[0120] SEQ ID NO: 15 - Oligonucleotide for amplifying nucleic acid encoding leptin

[0121] SEQ ID NO: 16 - Oligonucleotide for amplifying nucleic acid encoding leptin

[0122] SEQ ID NO: 17 - Oligonucleotide for amplifying nucleic acid encoding CBFA-1

[0123] SEQ ID NO: 18 - Oligonucleotide for amplifying nucleic acid encoding CBFA-1

[0124] SEQ ID NO: 19 - Oligonucleotide for amplifying nucleic acid encoding PPARγ2

[0125] SEQ ID NO:20 - Oligonucleotide for amplifying nucleic acid encoding PPARγ2

[0126] SEQ ID NO:21 - Oligonucleotide for amplifying nucleic acid encoding OCN

[0127] SEQ ID NO:22 - Oligonucleotide for amplifying nucleic acid encoding OCN

[0128] SEQ ID NO:23 - Oligonucleotide for amplifying nucleic acid encoding MyoD

[0129] SEQ ID NO:24 - Oligonucleotide for amplifying nucleic acid encoding MyoD

[0130] SEQ ID NO:25 - Oligonucleotide for amplifying nucleic acid encoding SMMHC

[0131] SEQ ID NO:26 - Oligonucleotide for amplifying nucleic acid encoding SMMHC

[0132] SEQ ID NO:27 - Oligonucleotide for amplifying nucleic acid encoding GFAP

[0133] SEQ ID NO:28 - Oligonucleotide for amplifying nucleic acid encoding GFAP

[0134] SEQ ID NO:29 - Oligonucleotide for amplifying nucleic acid encoding nestin

[0135] SEQ ID NO:30 - Oligonucleotide for amplifying nucleic acid encoding nestin

[0136] SEQ ID NO:31 - Oligonucleotide for amplifying nucleic acid encoding SOX9

[0137] SEQ ID NO:32 - Oligonucleotide for amplifying nucleic acid encoding SOX9

[0138] SEQ ID NO:33 - Oligonucleotide for amplifying nucleic acid encoding type X collagen

[0139] SEQ ID NO:34 - Oligonucleotide for amplifying nucleic acid encoding type X collagen

[0140] SEQ ID NO:35 - Oligonucleotide for amplifying nucleic acid encoding aggrecan

[0141] SEQ ID NO:36 - Oligonucleotide for amplifying nucleic acid encoding aggrecan Detailed Description

[0142] General techniques and selected definitions

[0143] In this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter are to be considered to include one or more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter, unless specifically stated otherwise or the context requires otherwise.

[0144] Unless otherwise stated, each embodiment of the present invention is applicable mutatis mutandis to each and every other embodiment.

[0145] Those skilled in the art will appreciate that the present invention allows for variations and modifications beyond those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions and compounds mentioned or indicated individually or collectively in this specification, as well as any and all combinations of the steps or features or any two or more thereof.

[0146] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0147] The present invention is performed using, without undue experimentation, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solution phase peptide synthesis, solid phase peptide synthesis, and immunology unless otherwise indicated. The procedures are described in, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 2nd edition (1989), Volumes I, II and III in their entirety; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, in their entirety; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, in their entirety, and especially Gait's paper therein, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Glover, ed., 1985), IRL Press, Oxford, in their entirety. Hames and SJ Higgins, 1985) IRL Press, Oxford, full text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, Academic Press, Inc.), full series; JF Ramalho Ortigao, "The Chemistry of Peptide Synthesis" see: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, RL (1976). Biochem. Biophys. Res. Commun.73 336-342; Merrifield, RB (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, RB (1979) In The Peptides (Gross, E. and Meienhofer, J. eds.), Vol. 2, pp. 1-284, Academic Press, New York 12. Wünsch, E. ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E. ed.), Vol. 15, 4th edition, Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. and Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Volumes I-IV (Edited by DM Weir and CC Blackwell, 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, 3rd Edition (Edited by John RW Masters, 2000), ISBN 0199637970, full text. .

[0148] In this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of stated steps or elements or integers or groups of steps or elements or integers, but not the exclusion of any other steps or elements or integers or groups of elements or integers.

[0149] As used herein, the term "derived from" shall be meant to indicate that the specified integer can be obtained from a particular source, even if not necessarily directly from that source. +In the context of soluble factors obtained from STRO-1 cells and / or their progeny cells, the term shall mean one or more factors, such as proteins, peptides, carbohydrates, etc., that are produced by in vitro culture of STRO-1 cells. + cells and / or their progeny cells during the course of the reaction.

[0150] As used herein, the term "stroke" should mean the loss of brain function, usually rapid progression, due to interference with blood flow to the brain or brainstem. This interference can be ischemia (lack of blood) caused by, for example, thrombosis or embolism, or can be due to hemorrhage. In one embodiment, loss of brain function is accompanied by neuronal cell death. In one embodiment, stroke is caused by interference or loss of blood from (to) the brain or one of its regions. In one embodiment, stroke is a neurological deficit (as defined by the World Health Organization) of cerebrovascular causes that lasts more than 24 hours or is interrupted due to death within 24 hours. Symptoms continue to distinguish stroke from transient ischemic attack (TIA) for more than 24 hours, and symptoms continue to be less than 24 hours in TIA. Symptoms of a stroke include hemiparesis (paralysis on one side of the body); hemiparesis (weakness on one side of the body); facial muscle weakness; numbness; decreased sensation; altered sense of smell, taste, hearing, or vision; loss of smell, taste, hearing, or vision; drooping eyelids (ptosis); detectable weakness of the eye muscles; decreased gag reflex; decreased ability to swallow; decreased pupil reactivity to light; decreased facial sensation; decreased balance; nystagmus; changes in breathing rate; changes in heart rate; sternocleidomastoid muscle weakness with decreased or inability to turn the head to one side; tongue weakness; aphasia (inability to speak or understand speech); apraxia (changes in voluntary movements); visual field loss; memory loss; hemineglect or hemispatial neglect (loss of attention to visual space on the side opposite the impairment); disorganized thinking; confusion; development of hypersexual postures; anosognosia (persistent denial of the existence of the impairment); difficulty walking; changes in motor coordination; vertigo; loss of balance; loss of consciousness; headache; and / or vomiting.

[0151] The skilled person will know that the "brain" includes the cerebral cortex (or cerebral hemispheric cortex), the basal ganglia (or basal ganglia), and the limbic system.

[0152] The term "brain function" includes:

[0153] Reasoning, planning, speech, movement, emotion, and problem-solving (related to the frontal lobe);

[0154] Movement, location, identification, and perception of stimuli (related to the parietal lobe);

[0155] Visual processing (associated with the occipital lobe); and

[0156] Perception and recognition of auditory stimuli, memory, and language (associated with the temporal lobe).

[0157] As used herein, the term "movement disorder" shall mean any change in a subject's ability to move compared to before the stroke. Exemplary movement disorders include paralysis, partial paralysis, slurred speech, uncoordinated movements, muscle weakness, hypotonia, hypertonia, or involuntary abnormal movements.

[0158] As used herein, "regenerating" neurons should mean that the subject's endogenous neurons are induced to grow (e.g., grow axons) and / or divide and / or endogenous neural stem cells are induced to grow and / or divide and / or differentiate to produce new neurons. This is different from the cells that differentiate into neurons that are administered. This term does not mean that the method of regenerating neurons cannot additionally include the differentiation of administered cells into neurons or other cell types.

[0159] The term "effective amount" as used herein shall mean an amount of STRO-1-rich protein sufficient to induce regeneration of neurons in the brain of a subject suffering from a stroke and / or differentiation into neurons in the subject. + The amount of a population of cells and / or progeny thereof and / or soluble factors obtained therefrom. An effective amount is not necessarily sufficient to provide a therapeutic benefit by itself, for example, multiple administrations of an effective amount of a population and / or cells and / or soluble factors may provide a therapeutic benefit.

[0160] As used herein, the term "therapeutically effective amount" shall mean an amount of STRO-1-rich protein sufficient to improve brain function and / or treat movement disorders and / or regenerate brain neurons in a subject suffering from a stroke. + A population of cells and / or progeny cells thereof and / or the amount of soluble factors obtained therefrom.

[0161] The term "low dose" as used herein should be understood to mean STRO-1 + The amount of cells and / or their progeny is less than 1×1O 6 , but still sufficient to be an "effective amount" as defined herein and / or a "therapeutically effective amount" as defined herein. For example, a low dose includes 0.5×10 6 or fewer cells, or 0.4 × 10 6 or fewer cells or 0.3×10 6 or fewer cells or 0.1×10 6 or fewer cells.

[0162] As used herein, the terms "treat" or "treatment" or "treating" are understood to mean administering an amount of soluble factors and / or cells and reducing the severity of a movement disorder.

[0163] The terms "prevent" or "preventing" or "prevention" as used herein are understood to mean administering an amount of soluble factors and / or cells and stopping or preventing or delaying the development or progression of movement disorders and / or loss of brain function following a stroke. Thus, the methods of the present invention shall mean, mutatis mutandis, methods suitable for preventing movement disorders or loss of brain function in a subject suffering from a stroke.

[0164] The term "soluble factor" as used herein is understood to refer to water-soluble factors derived from STRO-1. + Any molecule produced by a cell and / or its progeny, such as a protein, peptide, glycoprotein, glycopeptide, lipoprotein, lipopeptide, carbohydrate, etc. The soluble factor can be intracellular and / or secreted by the cell. The soluble factor can be a complex mixture (e.g., supernatant) and / or its components and / or can be a purified factor. In one embodiment of the invention, the soluble factor is a supernatant or is contained in a supernatant. Therefore, any embodiment herein involving the administration of one or more soluble factors should be understood to be applicable to the administration of a supernatant, mutatis mutandis.

[0165] The term "supernatant" as used herein refers to non-cellular material produced after in vitro culturing STRO-1+ cells and / or their progeny in a suitable medium (e.g., liquid medium). Typically, the supernatant is produced by culturing cells in a medium under suitable conditions and time, followed by removal of cellular material by processes such as centrifugation. Prior to administration, the supernatant may or may not be subjected to further purification steps. In one embodiment, the supernatant contains less than 10 5 more, e.g., less than 10 4 If less than 10 3 and, for example, no living cells.

[0166] As used herein, the term "normal or healthy individual" shall mean a subject that has not suffered from a stroke.

[0167] In this specification, the term "effects of stroke" will be understood to include and provide literal support for one or more of: improving brain function, treating or preventing movement disorders, and / or regenerating brain neurons.

[0168] STRO-r cells or progeny cells, and supernatants or one or more soluble factors obtained therefrom

[0169] STRO-1 +The cells are found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligament, tendon, skeletal muscle, dermis and periosteum; and are capable of differentiating into cells of the germ lineage such as mesoderm and / or endoderm and / or ectoderm. STRO-1 + An exemplary source of cells is from bone marrow and / or dental pulp.

[0170] In one embodiment, STRO-1 + The cells are multipotent cells that are able to differentiate into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage determination and differentiation path that these cells enter depends on a variety of influences from mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines) and / or local microenvironmental conditions established by the host tissue. STRO-1 + A multipotent cell is thus a non-hematopoietic progenitor cell that divides to produce daughter cells that are either stem cells or precursor cells that, in time, irreversibly differentiate to produce a phenotypic cell.

[0171] In one embodiment, STRO-1 + The cells are capable of differentiating into neurons or neuron-like cells.

[0172] In one embodiment, STRO-1 + The cells secrete one or more factors that induce or promote neuronal axon growth.

[0173] In one embodiment, STRO-1 + The cells are enriched from a sample obtained from a subject, such as a subject to be treated or a related subject or an unrelated subject (whether the same species or a different species). The term "enriched" or variations thereof are used herein to describe a cell population in which the proportion of a particular cell type or the proportion of multiple specific cell types is increased when compared to an untreated population of cells (e.g., cells in a natural environment). In one embodiment, the enriched STRO-1 + A population of cells comprises at least about 0.1% or 0.5% or 1% or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% STRO-1 + In this regard, the term “STRO-1-enriched + The term "cell population" will be understood as "comprising X% str + In certain embodiments, STRO-1 +The cells may form clonal colonies, for example, CFU-F (fibroblasts) or a subpopulation thereof (eg 50% or 60% or 70% or 70% or 90% or 95%) may have such activity.

[0174] In one embodiment, the cell population is a cell preparation enriched for STRO-r cells in a selectable form. In this regard, the term "selectable form" will be understood to mean that the cells express a protein that allows the selection of STRO-1. + A marker for a cell (e.g., a cell surface marker). The marker may be STRO-1, but need not be. For example, as described and / or exemplified herein, a cell (e.g., MPC) that expresses STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also expresses STRO-1 (and may be STRO-1 亮 ). Therefore, it is shown that the cells are STRO-1 + It is not intended that cells are selected based on their expression of STRO-1. In one embodiment, cells are selected based on their expression of at least STRO-3, e.g., they are STRO-3 + (TNAP + ).

[0175] Reference to selecting a cell or population thereof does not require selection from a particular tissue source. As described herein, STRO-1 + The cells can be selected or isolated or enriched from a wide variety of sources. That is, in certain embodiments, these terms are from any cell containing STRO-1 + Tissues containing cells (e.g., MPCs) or vascularized tissues or pericytes (e.g., STRO-1 + Support is provided by selecting from tissues containing pericytes or any one or more of the tissues described herein.

[0176] In one embodiment, the cells express one or more markers selected individually or collectively from the group consisting of: TNAP + 、VCAM-1 + 、THY-1 + 、STRO-2 + 、STRO-4 + (HSP-90β), CD45 + 、CD146 + 、3G5 + or any combination thereof.

[0177] In one embodiment, the cell expresses or the cell population is enriched for STRO-1 + (or STRO-1亮 ) and CD146 + of mesenchymal precursor cells.

[0178] "Individually" means that the present invention independently encompasses the marker or marker group, and although an individual marker or marker group may not be individually listed herein, the appended claims may still define the marker or marker group independently and separately from each other.

[0179] "Encyclopedia" means that the present invention covers any number of the aforementioned markers or peptide groups or combinations thereof, and although the aforementioned number of markers or marker groups or combinations thereof may not be specifically listed herein, the appended claims may still define the aforementioned combination or sub-combination independently and separately from any other combination of markers or marker groups.

[0180] In one embodiment, STRO-1 + STRO-1 亮 (with STRO-1 bri In one embodiment, relative to STRO-1 暗 or STRO-1 中间 Cell, Stro-1 bri Cells are preferentially enriched.

[0181] For example, STRO-1 亮 TNAP + 、VCAM-1 + 、THY-1 + 、 STRO-2 + 、STRO-4 + (HSP-90β) and / or CD146 + For example, cells are selected for and / or show expression of one or more of the aforementioned markers. In this regard, cells showing expression of a marker need not be specifically tested, but rather pre-enriched or isolated cells can be tested and subsequently used, and it can be reasonably assumed that the isolated or enriched cells also express the same marker.

[0182] In one embodiment, the mesenchymal precursor cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630. For example, the mesenchymal precursor cells express markers of perivascular cells, for example, the cells are STRO-1 + or STRO-1 亮 and / or 3G5 + In one embodiment, the cell is or was or is a progeny cell isolated from a vascularized tissue or organ or a portion thereof.

[0183] A cell that is referred to as "positive" for a given marker may express low (lo or dark) or high (bright, bright) levels of the marker, depending on the extent to which the marker is present on the cell surface, wherein the term relates to the intensity of the fluorescent or other marker used in the cell sorting process. The distinction between lo (or dark or deep) and bright should be understood in the context of the marker used on the particular cell population being sorted. A cell that is referred to as "negative" for a given marker is not necessarily completely absent from the cell. The term refers to a cell that expresses the marker at a relatively low level and produces a very low signal when detectably labeled or is undetectable above background levels, such as levels detected using an isotype control antibody.

[0184] As used herein, the term "bright" refers to a marker on the cell surface that produces a relatively high signal when detectably labeled. While not wishing to be bound by theory, it is proposed that "bright" cells express more target marker protein (e.g., an antigen recognized by STRO-1) than other cells in the sample. For example, when labeled with a FITC-conjugated STRO-1 antibody, STRO-1 as determined by fluorescence activated cell sorting (FACS) analysis may be detected. 亮 cells produced more than non-bright cells (STRO-1 深 / 暗 ) high fluorescence signal. For example, the "bright" cells constitute at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, the "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In one embodiment, STRO-1 亮 The surface expression of STRO-1 in cells is relative to the background (i.e., STRO-1 - In contrast, STRO-1 暗 and / or STRO-1 中间 The cells have less than 2 logs of surface expression of STRO-1 compared to "background", typically less than about 1 log compared to "background".

[0185] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses liver isoforms (LAP), bone isoforms (BAP), and kidney isoforms (KAP). In one embodiment, TNAP is BAP. In one embodiment, TNAP as used herein refers to a molecule that can bind to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC under the provisions of the Budapest Treaty on December 19, 2005 under the deposit number PTA-7282.

[0186] In addition, in one embodiment of the present invention, STRO-1 + The cells were able to give rise to clonal CFU-F.

[0187] In one embodiment, a large proportion of STRO-1 + Pluripotent cells are preferably capable of differentiating into at least two different germ lines. Non-limiting examples of the lineages that pluripotent cells can develop into include bone precursor cells; hepatocyte progenitor cells, which have the multipotency of biliary epithelial cells and hepatocytes; neural restricted cells, which can produce glial cell precursors that develop into oligodendrocytes and astrocytes; neuronal precursors, which develop neurons; myocardial and myocardial cells, precursors of glucose-responsive insulin-secreting pancreatic β cell lines. Other lineages include, but are not limited to, odontoblasts, odontogenic cells and chondrocytes, and the following precursor cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, ureteral epithelial cells, smooth muscle and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, myocardial, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelial, glial, neurons, astrocytes and oligodendrocytes.

[0188] In another embodiment, STRO-1 + The cells were unable to generate hematopoietic cells when grown in culture.

[0189] In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques and used to obtain supernatant or soluble factors or expanded cells for administration to the subject in the form of autologous or allogeneic compositions. In an alternative embodiment, cells of one or more established human cell lines are used. In another applicable embodiment, cells of non-human animals (or if the patient is not human, from another species) are used.

[0190] The present invention also encompasses the use of STRO-1 produced by in vitro culture. + The supernatant or soluble factors obtained or obtained from cells and / or their progeny cells (the latter are also referred to as expanded cells). The expanded cells of the present invention can have a variety of phenotypes depending on the culture conditions (including the amount and / or type of stimulating factors in the culture medium), the number of passages, etc. In certain embodiments, the progeny cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages from the parent population. However, the progeny cells can be obtained after any number of passages from the parent population.

[0191] Daughter cells can be obtained by culturing in any suitable culture medium. The term "culture medium" as used herein when referring to cell culture includes components of the cell surrounding environment. Culture medium can be a mixture of solid, liquid, gaseous or phase and material. Culture medium includes liquid growth medium and liquid culture medium that does not support cell growth. Culture medium also includes colloidal culture medium, such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous culture medium includes the gas phase exposed to cells grown on a petri dish (petri dish) or other solid or semi-solid supports. The term "culture medium" also refers to materials intended for cell culture, even if it has not yet contacted with cells. In other words, the nutrient-rich liquid prepared for bacterial culture is a culture medium. The powder mixture that becomes suitable for cell culture when mixed with water or other liquids can be called a "powdered culture medium".

[0192] In one embodiment, progeny cells suitable for the methods of the present invention are isolated or enriched for TNAP from bone marrow using magnetic beads labeled with STRO-3 antibodies. + STRO-1 + The isolated cells are isolated and then expanded in culture (for examples of suitable culture conditions, see Gronthos et al., Blood 85:929-940, 1995).

[0193] In one embodiment, the expanded cells (progeny) (eg, after at least 5 passages) may be TNAP - 、CC9 + ,I + HLA class II - HLA-like, CD14 - 、CD19 - 、CD3 - 、 CD11a - c - 、CD31 - 、CD86 - 、CD34 - and / or CD80 - However, under culture conditions different from those described herein, the expression of different markers may vary. Furthermore, while cells with these phenotypes may predominate in an expanded cell population, this does not mean that a smaller percentage of cells do not have this phenotype (e.g., a small percentage of expanded cells may be CC9 - In one embodiment, the expanded cells still have the ability to differentiate into different cell types.

[0194] In one embodiment, the expanded cell population used to obtain the supernatant or soluble factors or the cells themselves comprises cells in which at least 25% (such as at least 50% of the cells) are CC9 + .

[0195] In another embodiment, the expanded cell population used to obtain the supernatant or soluble factors or the cells themselves comprises at least 40% (such as at least 45%) of the cells are STRO-1 + .

[0196] In another embodiment, the expanded cells may express one or more markers selected from the group consisting of LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a / CD49b / CD29, CD49c / CD29, CD49d / CD29, CD 90, CD29, CD18, CD61, integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2 = leptin-R), RANKL, STRO-4 (HSP-90P), STRO-1 亮 and CD146 or any combination of these markers.

[0197] In one embodiment, the progeny cell is a pluripotent expanded STRO-1 as defined and / or described in WO 2006 / 032092. + Pluripotent cell progeny (MEMP). Preparation of STRO-1 cells that can be used to obtain progeny + Methods for enriching populations of pluripotent cells are described in WO 01 / 04268 and WO 2004 / 085630. In an in vitro setting, STRO-1 + Pluripotent cells rarely exist in the form of absolutely pure preparations, but generally exist together with other cells as tissue-specific committed cells (TSCCs). WO01 / 04268 proposes to collect such cells from the bone marrow at a purity level of about 0.1% to 90%. The population containing MPCs from which progeny can be obtained can be collected directly from a tissue source, or it can be a population that has been expanded ex vivo.

[0198] For example, progeny can be obtained from substantially purified STRO-1 +The collected and unexpanded population of pluripotent cells comprises at least about 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 95% of the total cells in the population in which they are present. This level can be determined, for example, by selecting for at least one gene individually or collectively selected from TNAP, STRO-4 (HSP-90P), STRO-1 亮 、3G5 + This was achieved by selecting cells that were positive for a panel of markers consisting of , VCAM-1, THY-1, CD146 and STRO-2.

[0199] MEMPS and freshly collected STRO-1 + Pluripotent cells can be distinguished by their expression of the marker STRO-1 亮 In contrast, freshly isolated STRO-1 + Pluripotent cells for STRO-1 亮 In one embodiment of the invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have the phenotype STRO-1. 亮 , ALP - In another embodiment, the MEMPS are positive for one or more of the markers Ki67, CD44 and / or CD49c / CD29, VLA-3, α3β1. In another embodiment, the MEMPs do not show TERT activity and / or are negative for the marker CD18.

[0200] STRO-1 + The starting population of cells may be derived from any one or more of the tissue types described in WO 01 / 04268 or WO 2004 / 085630, i.e. bone marrow, dental pulp cells, adipose tissue and skin, or perhaps more broadly from adipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles, intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletal muscle.

[0201] It should be understood that when performing the methods described herein, the separation of cells with any given cell surface marker can be achieved by a variety of different methods, however, exemplary methods rely on the binding of a binding agent (e.g., an antibody or an antigen binding fragment thereof) to the marker of interest, followed by separation of those cells that show binding (either high level binding or low level binding) or no binding. The most suitable binding agent is an antibody or an antibody-based molecule, such as a monoclonal antibody or a monoclonal antibody-based molecule (e.g., a protein comprising an antigen binding fragment thereof), because these latter binding agents have specificity. Antibodies can be used in both steps, however other agents can also be used, so ligands for these markers can also be used to enrich cells with the marker or lacking the marker.

[0202] The antibody or ligand can be connected to a solid support to allow for a rough separation. In one embodiment, the separation technique maintains the viability of the portion to be collected to the greatest extent. Various techniques with different efficacies can be used to obtain a relatively rough separation. The specific technique used will depend on the separation efficiency, the associated cytotoxicity, the ease and speed of execution, and the necessity of complex equipment and / or technical skills. Separation procedures may include, but are not limited to, magnetic separation using magnetic beads coated with antibodies, affinity chromatography, and "panning" using antibodies connected to a solid matrix. The technology providing accurate separation includes, but is not limited to, FACS. The method for performing FACS is apparent to those skilled in the art.

[0203] Antibodies to each of the markers described herein are commercially available (eg, monoclonal antibodies against STRO-1 are commercially available from R&D Systems, USA), obtained from the ATCC or other depository organizations, and / or can be generated using art-recognized techniques.

[0204] In one embodiment, STRO-1 is isolated + The method for cells, for example, comprises a first step, which is a solid phase sorting step using, for example, magnetic activated cell sorting (MACS) that recognizes high levels of expression of STRO-1. This can then be followed by a second sorting step, if necessary, to obtain higher levels of precursor cell expression, as described in patent specification WO 01 / 14268. This second sorting step may involve the use of two or more markers.

[0205] Obtaining STRO-1 + The method of cells may also include collecting the source cells before the first enrichment step using known techniques. Thus, the tissue will be removed surgically. The cells constituting the source tissue are then separated into a so-called single cell suspension. This separation can be achieved by physical and or enzymatic means.

[0206] Once a suitable STRO-1 is obtained+ The cell population can be cultured or expanded by any suitable means to obtain MEMP.

[0207] In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques and used to obtain supernatants or soluble factors or expand cells for administration to the subject in the form of autologous or allogeneic compositions. In an alternative embodiment, cells of one or more established human cell lines are used to obtain supernatants or soluble factors. In another applicable embodiment, cells of non-human animals (or if the patient is not human, from another species) are used to obtain supernatants or soluble factors.

[0208] The present invention can be implemented using cells from any non-human animal species, including but not limited to non-human primate cells, ungulates, canines, felines, lagomorphs, rodents, avian and fish cells. Primate cells that can be used to implement the present invention include but are not limited to cells of chimpanzees, baboons, cynomolgus macaques and any other New World or Old World monkeys. Ungulates cells that can be used to implement the methods of the present invention include but are not limited to cells of cattle, pigs, sheep, goats, horses, buffaloes and bison. Rodent cells that can be used to implement the methods of the present invention include but are not limited to cells of mice, rats, guinea pigs, hamsters and gerbils. Examples of lagomorph species that can be used to implement the methods of the present invention include rabbits, jackrabbits, hares, cottontail rabbits, snow hares and pikas. Chickens (Gallus gallus) are an example of avian species that can be used to implement the methods of the present invention.

[0209] In one embodiment, the cell is a human cell.

[0210] Cells suitable for the methods of the present invention may be stored prior to use or obtaining supernatants or soluble factors. Methods and protocols for preserving and storing eukaryotic cells and especially mammalian cells are known in the art (see, for example, Pollard, JW and Walker, JM (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, NJ; Freshney, RI (2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, NJ). Any method for maintaining the biological activity of isolated stem cells (such as mesenchymal stem cells / progenitor cells) or their progeny may be used in conjunction with the present invention. In one embodiment, cells are maintained and stored by using cryopreservation.

[0211] Genetically modified cells

[0212] In one embodiment, STRO-1 + Cells and / or their progeny cells are genetically modified, for example, to express and / or secrete a protein of interest. For example, cells are engineered to express a protein useful in treating movement disorders or other effects of stroke, such as a nerve growth factor or polypeptide described, for example, in Meade et al., J Exp Stroke TransI Med 2:22-40,2009.

[0213] The method for genetically modifying cells is obvious to those skilled in the art. For example, the nucleic acid intended to be expressed in the cell is operably connected to a promoter for inducing expression in the cell. For example, the nucleic acid is connected to a promoter operable in a variety of cells of the subject, such as a viral promoter, for example a CMV promoter (for example a CMV-IE promoter) or a SV-40 promoter. Other suitable promoters are known in the art and are understood to be applicable to embodiments of the present invention mutatis mutandis.

[0214] In one embodiment, nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid having the ability to confer expression to an operably connected nucleic acid (e.g., a reporter gene and / or a counter-selectable reporter gene) in a cell. In the context of the present invention, it will be appreciated that an expression construct may comprise or be a plasmid, a phage, a phagemid, a cosmid, a viral subgenome or a genomic fragment or other nucleic acids that can maintain and / or replicate heterologous DNA in an expressible format.

[0215] Methods for constructing expression constructs suitable for practicing the present invention will be apparent to those skilled in the art and are described in, for example, Ausubel et al. (see: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al. (see: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 3rd edition 2001). For example, the components of the expression construct are amplified from a suitable template nucleic acid using, for example, PCR and then cloned into a suitable expression construct (such as a plasmid or phagemid).

[0216] Suitable vectors for the expression construct are known in the art and / or described herein. For example, suitable expression vectors for use in the methods of the invention in mammalian cells are, for example, vectors of the pcDNA vector set supplied by Invitrogen, vectors of the pCI vector set (Promega), vectors of the pCMV vector set (Clontech), pM vectors (Clontech), pSI vectors (Promega), VP 16 vectors (Clontech) or vectors of the pcDNA vector set (Invitrogen).

[0217] Those skilled in the art will recognize other vectors and sources of such vectors, such as Life Technologies, Clontech, or Promega.

[0218] The mode for introducing the nucleic acid molecule of separation or the gene construct comprising the nucleic acid into the cell for expression is known to those skilled in the art. The technology for specifying the organism depends on known successful technology. The mode for introducing the recombinant DNA into the cell comprises microinjection, DEAE-dextran-mediated transfection, liposome (such as using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA)) mediated transfection, PEG-mediated DNA absorption, electroporation and microparticle bombardment (such as using tungsten particles or gold particles (Agracetus Inc., WI, USA) coated with DNA) and other modes.

[0219] Alternatively, the expression construct of the present invention is a viral vector. Suitable viruses are known in the art and are commercially available. Conventional viral-based systems for delivering nucleic acids and integrating the nucleic acids into the host cell genome include, for example, retroviral vectors, lentiviral vectors, or adeno-associated viral vectors. Alternatively, adenoviral vectors are suitable for introducing nucleic acids that remain in free form into host cells. Viral vectors are effective and versatile methods for gene transfer in target cells and tissues. In addition, high transduction efficiencies have been observed in a variety of different cell types and target tissues.

[0220] For example, retroviruses typically contain cis-acting long terminal repeats (LTRs) with the capacity to package up to 6-10 kb of foreign sequences. Minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the expression construct into target cells to provide long-term expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739 (1992); Johann et al., J. Virol. 65:1635-1640 (1992); Sommerfelt et al., Virol. 76:58-59 (1990); Wilson et al., J. Virol. 63:274-2318 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700; Miller and Rosman BioTechniques 7:980-990, 1989; Miller, AD Human Gene Therapy 7:5-14, 1990; Scarpa et al. Virology 75:849-852, 1991; Burns et al. Proc. Natl. Acad. Sci USA 90:8033-8037, 1993).

[0221] Various adeno-associated virus (AAV) vector systems have been developed for nucleic acid delivery. AAV vectors can be readily constructed using techniques known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 and WO 93 / 03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka. Current Topics in Microbiol, and Immunol. 755:97-129, 1992; Kotin, Human Gene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169, 1994; and Zhou et al. J. Exp. Med. 779:1867-1875,1994.

[0222] Other viral vectors suitable for delivery of the expression constructs of the invention include, for example, vectors derived from the poxvirus family, such as vaccinia virus and fowlpox virus or alphavirus or conjugate viral vectors (eg, those described in Fisher-Hoch et al., Proc. Natl Acad. Sci. USA 56:317-321, 1989).

[0223] Determination of therapeutic / preventive potential of cells and soluble factors

[0224] Methods for determining the ability of cells or soluble factors to improve brain function and / or treat movement disorders and / or regenerate neurons in the brain will be apparent to those skilled in the art.

[0225] For example, cells or soluble factors are cultured with neurons and the ability of the cells or soluble factors to induce axonal growth is assessed, e.g., microscopically. Similar assays can be performed in vivo by injecting cells and / or soluble factors into the brain of chicken embryos and assessing axonal growth, e.g., as exemplified herein.

[0226] In one embodiment, neurons are exposed to oxygen and / or glucose deprivation prior to or during incubation with cells and / or soluble factors. Oxygen and glucose deprivation is a model of ischemia.

[0227] In another embodiment, the cells are grown under conditions that induce neuronal differentiation, such as comprising butylated hydroxyanisole and valproic acid or 5-azacytidine or indomethacin and isobutylmethylxanthine and insulin. Cells that differentiate into neurons are considered suitable for therapy.

[0228] In yet another embodiment, cells or soluble factors (e.g., factors or mixtures of individual factors or factor fragments (e.g., obtained by affinity purification or chromatography)) are administered to a stroke model and the effects on brain function, brain neuron regeneration, and / or movement disorders are assessed.

[0229] There are various known techniques for inducing ischemic stroke in non-human animal subjects, such as aorta / vena cava occlusion, external neck tourniquet or cuff, hemorrhage or hypotension, intracranial hypertension or common carotid artery occlusion, two-vessel occlusion and hypotension, four-vessel occlusion, unilateral common carotid artery occlusion (only in certain species), endothelin-1-induced arterial and venous constriction, middle cerebral artery occlusion, spontaneous cerebral infarction (spontaneously hypertensive rats), macrosphere embolization, blood clot embolization or microsphere embolization. Hemorrhagic stroke can be modeled by infusing collagenase into the brain.

[0230] In one embodiment, the stroke model includes middle cerebral artery occlusion to produce ischemic stroke.

[0231] To test the ability of a population and / or progeny and / or secreted factors to treat the effects of stroke, the population and / or progeny and / or secreted factors are administered after induction of stroke, e.g., within 1 hour to 1 day of stroke. Brain function and / or movement disorders are assessed several times after administration.

[0232] Methods for assessing brain function and / or movement disorders will be apparent to those skilled in the art and include, for example, rotarod, elevated plus maze, open field test, Morris water maze, T-maze, eight-arm maze, motor assessment (e.g., area covered in a certain time), tail flick, or De Ryck's behavioral test (De Ryck et al., Stroke. 20:1383-1390, 1989). Additional tests will be apparent to those skilled in the art and / or are described herein.

[0233] In another embodiment, the effects of the population and / or progeny and / or secreted factors on the brain are assessed by imaging. For example, apoptosis, autophagy, and neurovascular units can be detected by in vivo optical imaging (Liu et al., Brain Res. 2011 Apr. 27), and PET, MRI, or CT can be used to assess various factors such as the size of the infarct, the size of the penumbra, and / or the extent of brain damage.

[0234] The effect of groups and / or progeny and / or secreted factors on brain neurons is also assessed. For example, a region of the brain or its brain is removed and the number of neurons or the subtype of neurons is assessed or estimated. Depending on the model used, this number can be compared with the number in control animals, or if only focal ischemia is induced, then compared with a control region of the brain of the same animal.

[0235] Cell composition

[0236] In one embodiment of the present invention, STRO-1 + The cells and / or their progeny cells are administered in the form of a composition. In one embodiment, the composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0237] The terms "carrier" and "excipient" refer to compositions of matter routinely used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mac Publishing Company (1980). The carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., unable to react with other components of the carrier. In one embodiment, the carrier does not produce significant local or systemic adverse effects in the recipient at the dosages and concentrations used for treatment.

[0238] Carriers suitable for use in the present invention include those conventionally used carriers, such as water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronic acid and glycols are exemplary liquid carriers, especially (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

[0239] In another embodiment, the carrier is a medium composition, such as a medium composition in which cells are grown or suspended. In one embodiment, the medium composition does not induce any adverse effects in the subject to which it is administered.

[0240] Exemplary vehicles and excipients do not adversely affect cell viability and / or the ability of the cells to reduce, prevent or delay the effects of stroke.

[0241] In one embodiment, the carrier or excipient provides buffering activity to maintain cells and / or soluble factors at a suitable pH, thereby exerting biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows rapid release of cells and factors, in which case the composition of the invention can be manufactured as a liquid for direct application, for example, by injection, to the bloodstream or tissue or to an area surrounding or adjacent to the tissue.

[0242] STRO-1 + Cells and / or their progeny cells can also be incorporated into or embedded in a framework that is compatible with the recipient and degrades into products that are harmless to the recipient. The framework provides support and protection for cells intended to be transplanted into the recipient's body. Natural and / or synthetic biodegradable frameworks are examples of the framework.

[0243] A variety of different skeletons can be successfully used to implement the present invention. Exemplary skeletons include, but are not limited to, biodegradable skeletons. Natural biodegradable skeletons include collagen, fibronectin, and laminin skeletons. Synthetic materials suitable for cell transplant skeletons should be able to support a wide range of cell growth and cell function. The skeletons can also be absorbed. Suitable skeletons include polyglycolic acid skeletons, such as Vacanti et al., J. Ped. Surg. 23: 3-9 1988; Cima et al., Biotechnol. Bioeng. 35: 145 1991; Vacanti et al., Plast. Reconstr. Surg. 88: 153-9 1991, or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

[0244] In another embodiment, cells may be administered in a gel matrix such as Gelfoam from Upjohn Corporation.

[0245] Cell compositions suitable for use in the methods described herein can be administered alone or as a mixture with other cells. Cells that can be administered in conjunction with the compositions of the present invention include, but are not limited to, other pluripotent or multipotent cells or stem cells, or bone marrow cells. Different types of cells can be mixed with the compositions of the present invention just before or shortly before administration, or they can be co-cultured together for a period of time before administration.

[0246] In one embodiment, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about 1×10 5 STRO-1 + Cells / kg is about 1×10 7 STRO-1 + cells / kg or about 1×1O6 STRO-1 + Cells / kg: about 5×10 6 STRO-1 + The exact amount of cells to be administered depends on a variety of factors, including the age, weight, and sex of the patient and the extent and severity of the stroke and / or the site of the stroke.

[0247] In one embodiment, a low dose of cells is administered to a subject. Exemplary doses include about 0.1×10 cells per kilogram. 4 and 0.5×10 6 cells, for example, about 0.1×10 per kilogram 5 and 0.5×10 6 cells, such as about 0.5×10 per kilogram 5 and 0.5×10 6 cells, for example, about 0.1×10 per kilogram 6 and 0.5×10 6 cells, for example, about 0.2×10 per kilogram 6 or 0.3×10 6 or 0.4×10 6 cells.

[0248] In some embodiments, the cells are contained in a chamber that does not allow the cells to leave and enter the subject's circulation, but allows factors secreted by the cells to enter the circulation. In this way, soluble factors can be administered to a subject by allowing the cells to secrete factors into the subject's circulation. The chamber can also be implanted in a certain part of the subject to increase the local level of soluble factors, such as in or near the pancreas.

[0249] In some embodiments of the present invention, it may not be necessary or desirable to immunosuppress the patient prior to initiating cell composition therapy. + The cells or their progeny may be tolerant in some circumstances.

[0250] However, in other cases, it may be necessary or appropriate to pharmacologically immunosuppress the patient and / or reduce the subject's immune response to the cell composition before starting cell therapy. This can be achieved by using systemic or local immunosuppressants, or this can be achieved by delivering cells in an encapsulation device. Cells can be encapsulated in a capsule that is permeable to nutrients and oxygen required by the cells and therapeutic factors, but impermeable to the cells to immunize humoral factors and cells. In one embodiment, the encapsulating agent has low allergenicity, is easily and stably located in the target tissue, and provides additional protection to the implanted structure. These and other ways to reduce or eliminate the immune response to transplanted cells are known in the art. As an alternative, cells can be genetically modified to reduce their immunogenicity.

[0251] Composition of soluble factors

[0252] In one embodiment of the present invention, STRO-1 + The supernatant or soluble factors obtained from the cells and / or from the progeny cells are administered in the form of a composition, for example, comprising a suitable carrier and / or excipient. In one embodiment, the carrier or excipient does not adversely affect the biological effects of the soluble factors or supernatant.

[0253] In one embodiment, the composition comprises a composition of matter that stabilizes soluble factors or supernatant components (eg, protease inhibitors). In one embodiment, the amount of the protease inhibitor included is insufficient to adversely affect the subject.

[0254] Contains STRO-1 + Compositions of cell-derived and / or progeny cell-derived supernatants or soluble factors can be prepared as a suitable liquid suspension, such as a suspension in a culture medium or in a stable carrier or buffer solution (e.g., phosphate buffered saline). Suitable carriers are described above. In another embodiment, a composition comprising STRO-1 + The suspension of the supernatant or soluble factors obtained from the cells and / or from the daughter cells is an oily suspension for injection. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. The suspension to be used for injection may also contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or polydextrose. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution.

[0255] Sterile injectable solutions can be prepared by incorporating the required amount of supernatant or soluble factors in an appropriate solvent with one or a combination of the above ingredients, followed by filtered sterilization as required.

[0256] In general, dispersions are prepared by incorporating the supernatant or soluble factors into a sterile vehicle containing a basic dispersion medium or other desired ingredients from the above listed ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution thereof. According to alternative aspects of the invention, the supernatant or soluble factors may be formulated with one or more other compounds that enhance its solubility.

[0257] Other exemplary carriers or excipients are described in, e.g., Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0258] The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, it is desirable to include an isotonic agent, such as sugar, polyols (such as mannitol, sorbitol) or sodium chloride in the composition. Extended absorption of injectable compositions can be achieved by including an agent (e.g., monostearate and gelatin) that delays absorption in the composition. In addition, soluble factors can be administered in a time-release formulation, for example, in a composition comprising a slow-release polymer. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid (PLG) can be used. Various methods for preparing such formulations are patented or generally known to those skilled in the art.

[0259] The supernatant or soluble factors may be administered in combination with, for example, an appropriate matrix that provides slow release of the soluble factors.

[0260] Additional components of the composition

[0261] STRO-1 + Cell-derived supernatant or soluble cytokines, STRO-1 +The cells or their progeny can be administered together with other beneficial drugs or biomolecules (growth factors, nutritional factors). When administered together with other agents, they can be administered together in a single pharmaceutical composition or in separate pharmaceutical compositions, simultaneously with the other agents or sequentially (before or after the administration of the other agents). Bioactive factors that can be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetics, TPO, IGF-I and IGF-II, HGF, cysteine ​​protease inhibitors); anti-inflammatory drugs (e.g., p38 MAPK inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANLAST, REMICADE, SIROLIMUS, and NSAIDs (nonsteroidal anti-inflammatory drugs, such as TEPOXALIN, TOLMETIN, SUPROFEN); immunosuppressive / immunomodulatory agents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); antiproliferative agents (e.g., azathioprine oxaliplatin, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); antibodies such as monoclonal anti-IL-2α receptor antibodies (e.g., basiliximab, daclizumab), polyclonal anti-T cell antibodies (e.g., anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3)); antithrombotic agents (e.g., heparin, heparin derivatives, urokinase, PPack (dextrorotatory phenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors and platelet inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), and local anesthetics.

[0262] In one embodiment, a composition as described herein according to any embodiment comprises an additional factor for improving brain function and / or regenerating brain neurons and / or treating movement disorders, for example, a nerve growth factor.

[0263] Alternatively or additionally, the cells, secreted factors and / or compositions as described herein according to any embodiment are combined with known treatments for the effects of stroke, for example, physical therapy and / or speech therapy.

[0264] In one embodiment, a pharmaceutical composition as described herein according to any embodiment comprises a compound for treating the effects of stroke. Alternatively, a method of treatment / prevention as described herein according to any embodiment of the invention further comprises administering a compound for treating the effects of stroke. Exemplary compounds are described herein and are understood to apply mutatis mutandis to these embodiments of the invention.

[0265] In another embodiment, the composition as described herein according to any embodiment further comprises a factor that induces or enhances the differentiation of progenitor cells into vascular endothelial cells. Exemplary factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF, such as PDGF-BB) and FGF.

[0266] In another embodiment, the composition as described herein according to any embodiment further comprises tissue specific committed cells (TSCC). In this regard, International Patent Application No. PCT / AU2005 / 001445 demonstrates the administration of TSCC and STRO-1 + The cells may result in increased proliferation of TSCC. In one embodiment, the TSCC is a vascular cell. Administration of the composition to a subject may result in increased generation of blood vessels, for example, resulting in increased delivery of nutrients to the affected tissue.

[0267] Medical Devices

[0268] The present invention also provides a medical device for use or when used in a method as described herein according to any embodiment. For example, the present invention provides a medical device comprising STRO-1 as described herein according to any embodiment. + A syringe or catheter or other suitable delivery device of cells and / or their progeny and / or their soluble factors and / or compositions. Optionally, the syringe or catheter is packaged with instructions for the method as described herein according to any embodiment.

[0269] In another embodiment, the invention provides a STRO-1 comprising a STRO-1 as described herein according to any embodiment. + The implant comprises cells and / or their progeny and / or their soluble factors and / or compositions. Optionally, the implant is packaged with instructions for use according to any embodiment of the method as described herein. Suitable implants can be formed with a scaffold, for example, as described above, and STRO-1 + Cells and / or their progeny cells and / or their soluble factors.

[0270] Mode of administration

[0271] By STRO-1 +Cell-derived supernatant or soluble factors, STRO-1 + The cells or their progeny can be surgically implanted, injected, delivered (eg, by means of a catheter or syringe), or otherwise administered directly or indirectly to a site in need of repair or augmentation (eg, an organ) or into a fat pad.

[0272] In one embodiment, STRO-1 + The supernatant or soluble factors obtained from cells, STRO-1 + The cells or their progeny are delivered into the bloodstream of a subject. For example, + Cell-derived supernatant or soluble factors, STRO-1 + The cells or their progeny are delivered parenterally. Exemplary routes of parenteral administration include, but are not limited to, peritoneal, intraventricular, intraventricular, intrathecal or intravenous. In one embodiment, STRO-1 + Cell-derived supernatant or soluble factors, STRO-1 + The cells or their progeny are delivered intra-arterially into the aorta, into an atrium or ventricle of the heart, or into a blood vessel, such as into a vein.

[0273] In the case of cell delivery to the atria or ventricles of the heart, the cells may be administered to the left atrium or ventricle to avoid complications that may arise from rapid delivery of cells to the lungs.

[0274] In one embodiment, the population and / or progeny and / or soluble factors are administered into the carotid artery.

[0275] In one embodiment, the population and / or progeny and / or soluble factors are administered to the brain of a subject, e.g., intracranially. For example, the population and / or cells and / or soluble factors are administered to the brain and / or optionally to the striatum.

[0276] In one embodiment, the population and / or cells and / or soluble factors are administered to the ischemic site and / or the periphery of the ischemic site. The site can be determined using methods known in the art, such as magnetic resonance imaging and / or computed tomography and / or ultrasound.

[0277] The choice of administration regimen for a therapeutic formulation depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, and the immunogenicity of the entity.

[0278] In one embodiment, STRO-1 + The supernatant or soluble factors obtained from cells, STRO-1 +The cells or their progeny are delivered as a single bolus dose. Alternatively, the STRO-1 cells may be administered by continuous infusion, or by doses spaced, for example, once a day, once a week, or 1-7 times per week. + Cell-derived supernatant or soluble factors, STRO-1 + Cells or their progeny. An exemplary dosage regimen is one that involves a maximum dose or a dosage frequency that avoids significant undue side effects. The total weekly dose depends on the type and activity of the factor / cell used. The appropriate dose is determined by a clinician, for example, using parameters or factors known or suspected to affect treatment or predicted to affect treatment in the art. In general, the dose begins with an amount slightly less than the optimal dose and is increased in smaller increments thereafter until the desired or optimal effect is achieved relative to any negative side effects.

[0279] The present invention includes the following non-limiting examples. Example

[0280] Example 1: By selecting STRO-3 + Immunoselection of MPCs

[0281] Bone marrow (BM) was collected from healthy normal adult volunteers (20-35 years old). Briefly, 40 ml of BM was aspirated from the posterior iliac crest into a tube containing lithium heparin anticoagulant.

[0282] Lymphoprep was used as previously described (Zannettino, AC et al. (1998) Blood 92:2613-2628). TM BMMNCs were prepared by density gradient separation (Nycomed Pharma, Oslo, Norway). After centrifugation at 400×g for 30 min at 4° C., the buffy coat was removed with a transfer pipette and washed three times in “HHF” consisting of Hank’s balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS; CSL Limited, Victoria, Australia).

[0283] STRO-3 was subsequently isolated by magnetic activated cell sorting as previously described (Gronthos et al. (2003) Journal of Cell Science 116: 1827-1835; Gronthos, S. and Simmons, PJ (1995) Blood 85: 929-940). + (or TNAP + ) cells. Briefly, place approximately 1-3 × 10 8BMMNCs were incubated in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF for 20 min. The cells were incubated with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer for 1 h on ice. The cells were then washed twice in HHF by centrifugation at 400×g. A 1 / 50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer was added and the cells were incubated on ice for 1 h. The cells were incubated in MACS buffer (Ca-free supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as above. 2+ And no Mn 2+ The cells were washed twice in PBS (0.5% PBS) and resuspended in a final volume of 0.9 ml MACS buffer.

[0284] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice and resuspended in 0.5 ml of MACS buffer and then loaded onto a micro MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) on December 19, 2005, with the deposit number PTA-7282 - see International Publication No. WO 2006 / 108229). After adding 1 ml of MACS buffer, the column was removed by a magnet and TNAP was separated by positive pressure. + Cells. Cell aliquots from each fraction were stained with streptavidin-FITC and assessed for purity by flow cytometry.

[0285] Example 2: Cells selected by STRO-3 mAb are STRO-1 亮 cell

[0286] Experiments were designed to demonstrate the use of STRO-3 mAb as a tool for isolating cellular STRO-1 亮 Possibility of single reagent for cells.

[0287] Since the isotype of STRO-3 (IgG1) is different from that of STRO-1 (IgM), the STRO-3 and STRO-1 proteins separated using the MACS procedure were analyzed by two-color FACS. - The ability of STRO-3 to identify cloned CFU-F was evaluated by co-expression of cells ( Figure 1 ). The dot matrix histogram represents 5 × 10 4 The vertical and horizontal lines were set at reactivity levels less than 1.0% of the mean fluorescence obtained with isotype-matched control antibodies 1B5 (IgG) and 1A6.12 (IgM) treated under the same conditions. 亮 A smaller population of cells co-expressed TNAP (upper right quadrant), while the rest expressed STRO-1 + The cells failed to react with STRO-3 mAb. The CFU-F incidence of cells isolated by FACS was then determined for all four quadrants (Table 1).

[0288] Table 1: Enrichment of human bone marrow cells based on co-expression of cell surface markers STRO-1 and TNAP by two-color FACS analysis (ref. Figure 1 ). FACS sorted cells were cultured under standard cloning conditions in αMEM supplemented with 20% FCS. Data represent 10 5 The mean number of colony forming cells (CFU-F) on day 14 was ± SE (n = 3 different bone marrow aspirates). These data indicate that human MPCs are restricted to the TNAP-positive portion of the BM, which clearly co-expresses the STRO-1 antigen.

[0289]

[0290] Example 3: STRO-1 深 and Stro-1 亮 Relative gene and surface protein expression of cells

[0291] In the first series of experiments, semiquantitative RT-PCR analysis was used to examine the expression of STRO-1 isolated by fluorescence activated cell sorting. 深 or STRO-1 亮 Gene expression profiles of various lineage-associated genes expressed by the population ( Figure 2 A) In a second series of experiments, flow cytometry and mean channel fluorescence analysis were used to examine STRO-1 isolated by fluorescence activated cell sorting. 深 or STRO-1 亮 Surface protein expression profiles of various lineage-associated proteins expressed by the population.

[0292] Total cellular RNA was collected from 2×10 6 STRO-1 亮 or STRO-1 深Sorted primary cells, chondrocyte pellets and other induced cultures were prepared and lysed using the RNAzolB extraction method (Biotecx Lab. Inc., Houston, TX) according to the manufacturer's recommendations. RNA isolated from each subpopulation was then used as a template for cDNA synthesis using a first-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden). The expression of various transcripts was assessed by PCR amplification using the standard protocol described above (Gronthos et al., J. Bone and Min. Res. 74: 48-57, 1999). The primer sets used in this study are shown in Table 2. After amplification, each reaction mixture was analyzed by 1.5% agarose gel electrophoresis and observed by ethidium bromide staining. RNA integrity was assessed by the expression of GAPDH.

[0293] ImageQant software was used to assess the relative gene expression of each cell marker with reference to the expression of the housekeeping gene GAPDH ( Figure 2 B. Figure 2 C) In addition, the protein expression profile of ex vivo expanded MPCs was examined using two-color flow cytometry analysis based on the expression of various cell lineage-related markers in combination with STRO-1 antibody. 深 and STRO-1 亮 An overview of the general phenotype of gene and protein expression in cultured cells is presented in Table 3. The data show that STRO-1 亮 MPCs showed differentially higher expression of markers associated with perivascular cells, including angiopoietin 1, VCAM-1, SDF-1, IL-1p, TNFα, and RANKL. 深 and STRO-1 亮 Comparison between the protein and gene expression profiles of the cultured cells is summarized in Tables 3 and 4 .

[0294] Subtractive hybridization studies were also performed to identify 亮 Briefly, STRO-1 was isolated as described above. 深 and STRO-1 亮 (See Figure 3 A) STRO-1 RNA was pooled from 5 different bone marrow samples using the RNA STAT-60 system (TEL-TEST). 深 and STRO-1 亮Total RNA was prepared from cells. First strand synthesis was performed using the SMART cDNA Synthesis Kit (Clontech Laboratories). The resulting mRNA / single-stranded cDNA hybrid was amplified by long-range PCR (Advantage 2 PCR Kit; Clontech) using specific primer sites on the 3' and 5' initial ends formed during the initial RT according to the manufacturer's instructions. STRO-1 was digested with RsaI. 亮 After cDNA was obtained, two aliquots were used to ligate different specific adapter oligonucleotides using the Clontech PCR-Select cDNA Subtraction Kit. STRO-1 was used according to the manufacturer's protocol. 亮 (Subject) and STRO-1 深 Two rounds of subtractive hybridization were performed using cDNAs of STRO-1 (driver) and vice versa. 亮 STRO-1 drives cDNA hybridization 深 The test cDNA was subjected to this procedure in reverse.

[0295] To identify STRO-1 亮 Genes uniquely expressed by a population, using STRO-1 亮 cDNA was subtracted to construct a construct containing 200 randomly selected STRO-1 clones ligated into a T / A cloning vector. 亮 Duplicate low-density microarray filters of cDNA-transformed bacterial clones were subtracted. 32 P]dCTP-labeled STRO-1 亮 or STRO-1 深 Subtractive cDNA Probing Microarray ( Figure 3 B-3C). A total of 44 clones were identified in different screenings, which were 深 With STRO-1 亮 The expression of 104 genes was highly differential between subpopulations. DNA sequencing of all differentially expressed clones revealed that only 1 clone represented a known stromal cell mitogen; namely, platelet-derived growth factor (PDGF) (Gronthos and Simmons, Blood. 85: 929-940, 1995). Interestingly, 6 of the 44 clones were found to contain a DNA insertion corresponding to the chemokine stromal-derived factor 1 (SDF-1). Human STRO-1 亮 The high abundance of SDF-1 transcripts in cells was confirmed by the isolation of freshly sorted STRO-1 亮 、STRO-1 深 and STRO-1 阴性 Semiquantitative RT-PCR of total RNA prepared from bone marrow subpopulations was confirmed ( Figure 3 D and Table 3).

[0296] Table 2. RT-PCR primers and conditions for specific amplification of human mRNA

[0297]

[0298]

[0299] Table 3. STRO-1 亮 and STRO-1 深 Summary of relative gene expression in the population. Presented are the relative expression of STRO-1 when measured by reverse transcription-PCR. 亮 With STRO-1 深 List of genes showing measurable and differential expression between populations. Values ​​represent relative gene expression to the reference housekeeping gene GAPDH.

[0300]

[0301]

[0302] To correlate protein surface expression with the density of STRO-1 expression, single cell suspensions of cells obtained from bone marrow MPCs expanded in vitro were prepared by trypsin / EDTA separation and subsequently incubated with STRO-1 antibodies in combination with antibodies that identify a variety of cell lineage-associated markers. STRO-1 was identified using goat anti-mouse IgM-fluorescein isothiocyanate, while all other markers were identified using goat anti-mouse or anti-rabbit IgG-phycoerythrin. For those antibodies that identify intracellular antigens, cell preparations were first labeled with STRO-1 antibodies, fixed with 70% cold ethanol to permeabilize the cell membrane and then incubated with intracellular antigen-specific antibodies. Isotype-matched control antibodies were used under the same conditions. Dual-color flow cytometric analysis was performed using a COULTER EPICS flow cytometer and list mode data were collected. The dot plots represent 5,000 list mode events indicating the fluorescence intensity levels of each lineage cell marker (y-axis) and STRO-1 (x-axis). Vertical and horizontal quadrants were established with reference to isotype-matched negative control antibodies.

[0303] Table 4. STRO-1 亮 and STRO-1 深 Summary of relative protein expression in the population. Presented are the relative protein expression in STRO-1 as measured by flow cytometry. 亮 With STRO-1 深 List of proteins showing differential expression between groups. Values ​​represent the relative mean fluorescence intensity of staining.

[0304]

[0305]

[0306] Example 4: Differentiation of human dental pulp stem cells (hDPSC) into neurons

[0307] Isolation of DPSCs and human foreskin fibroblasts

[0308] DPSC and human exfoliated deciduous teeth (SHED) were separated as described by Gronthos et al., Proc Natl Acad Sci USA 97: 13625-13630, 2000. Briefly, the third impacted molars or exfoliated teeth (7-8 years old) discarded from adults (19-35 years old) were collected and routinely extracted from patients with informed consent at the Dental Clinic of the University of Adelaide under the approved guidelines formulated by the University of Adelaide and the Institute of Medical and Veterinary Science Human Subjects Research Committees (H-73-2003). The tooth surface was cleaned and cracked using a vice to expose the pulp cavity. The pulp tissue was gently separated from the crown and root, and then digested in a solution of 3 mg / ml type I collagenase and 4 mg / ml dispase at 37°C for 1 hour. Single cell suspensions were obtained by passing the cells through a 70-μM filter. Cultures were established by the following procedures: Single cell suspensions (1-2×10 5 ) were inoculated into growth medium (a-modified Eagle's medium supplemented with 20% fetal bovine serum, 100 μM 1-ascorbic acid 2-phosphate, 2 mM 1-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin) in a T-25 flask and then incubated at 37°C, 5% CO2.

[0309] Human foreskin fibroblasts (HFFs) were isolated by collagenase / dispase digestion of foreskin biopsy explants from male neonatal donors with informed consent from the parents.

[0310] Neural induction assay

[0311] Coat tissue culture flasks or wells with polyornithine (final concentration 10 μg / ml) overnight at room temperature. Wash flasks and wells twice with water and then coat with laminin (final concentration 5 μg / ml) overnight in a humid incubator at 37°C. Wash flasks and wells with phosphate buffered saline (PBS) and then with growth medium before adding cells. Thaw cells and grow in T25-coated flasks until 80% confluent. Re-seed cells at a density of 1.5×105 cells / well in 2 ml / well growth medium in 6-well coated plates for 3 days, or at 2×103 cells on 8-well coated chamber culture slides. Cells are then cultured in medium A or medium B for 3 weeks. Medium A consisted of Neurobasal A medium (10888-022; Invitrogen, Carlsbad, CA, USA), 100 U / ml penicillin, 1× B27 supplement, 100 μg / ml streptomycin, 20 ng / ml epidermal growth factor (EGF, 100-15; PeproTech, Rocky Hill, NJ, USA), and 40 ng / ml basic fibroblast growth factor (FGF, No. 104FGFB01; Prospec TanyTechnoGene, Rehovot, Israel). Medium B contained three separate medium conditions over a 3-week period: the first incubation was medium A for 7 days, followed by a change to medium consisting of a 50:50 ratio of Dulbecco's modified Eagle's medium (DMEM, 12100-046; Invitrogen) to F12 medium (21700-075; Invitrogen), with insulin-transferrin-sodium selenite supplement (ITTS, 11074547001; Roche Diagnostics, Basel, Switzerland), 100 U / ml penicillin, 100 μg / ml streptomycin, and 40 ng / ml FGF for 7 days. The final 7-day incubation consisted of a medium containing a 50:50 ratio of DMEM to F12 medium, ITTS, 100 U / ml penicillin and 100 μg / ml streptomycin, 40 ng / ml of FGF, and 0.5 μM retinoic acid (RA, R2625; Sigma-Aldrich, St. Louis, USA). Control samples were maintained in growth medium for the duration of the neuronal induction assay. The medium for all conditions was changed twice a week.

[0312] Electrophysiology

[0313] At the end of the 3-week neuronal differentiation assay, cells were released with trypsin and cultured at 2 × 104 Cells were plated at a concentration of 10 cells / 500 μL on glass coverslips treated with hydrochloric acid and incubated for 24 h. Whole-cell patch clamping was performed at room temperature using a computer-based patch clamp amplifier (EPC-9; HEKA Electronics, Lambrecht / Pfalz, Germany) and PULSE software (HEKA Electronics). The bath solution contained (in mM): NaCl, 140; KCl, 4; CaCl2, 2; MgCl2, 2; Glucose, 10; and HEPES, 10; the pH was adjusted to 7.4 with NaOH. To block K + Channels and separation of Na + Current, potassium glutamate and KCl in the internal solution were replaced with equimolar amounts of Cs-glutamate and CsCl. The membrane transfer pipette was pulled from borosilicate glass and fire-polished; the transfer pipette resistance ranged between 2 MΩ and 4 MΩ. All voltages shown were corrected for the liquid junction potential (for K based + and Cs + The internal solution was -14 mV and -18 mV, respectively), estimated by JP Calc (provided by Dr. PH Barry, University of New South Wales, Sydney, Australia, 1994). The holding potential was -90 mV throughout.

[0314] Avian embryo injection

[0315] Chicken eggs (white Leghorn chickens; HICHICK Breeding Company, Bethal, Australia) were placed lengthwise in egg boxes and incubated at 37°C in a humidified incubator for approximately 40 hours to reach the 10-12 stage before injection. Green fluorescent protein (GFP)-transduced adult DPSCs or HFFs (5×10 3 2 μL) was injected into the developing avian embryo. Fast green dye (2 μL) was added to the cells to visualize it during the injection procedure. Embryos were prepared by wiping eggs with 70% ethanol, extracting albumin from the eggs using a needle, cutting a window at the top of the egg, and injecting India ink (Winsor and Newton, Middlesex, England) below the embryo (1:10 in Ringer's solution [7.2 g NaCl in 1 L distilled water) to obtain the embryo. 2 , 0.17 g CaCl 2 , 0.37g KCl, 0.115g Na 2 HP0 4, pH adjusted to 7.4, filter sterilized]) to visualize the embryo. Ringer's solution was placed on top of the embryo to keep it moist. The vitelline membrane was removed from around the embryo's head with a tungsten wire. The cells were placed in a glass capillary needle (GC100TF-10; SDR Clinical Technology, Sydney, Australia), which was attached to a micromanipulator and a high-pressure syringe (Narishage, Tokyo), set at 25 volts. The micromanipulator was used to guide the needle into the area directly adjacent to rhombomere 2 in the developing hindbrain. The cells were injected into the embryo using a foot pump attached to the high-pressure syringe. The glass needle was removed and a few drops of Ringer's solution were placed on top of the embryo. The eggs were sealed with tape and returned to a humid incubator. After the incubation period, the embryos were cut out of the eggs and placed in cold PBS; GFP-injected cells were visualized using a fluorescent dissecting microscope. The embryos were then dissected; the head was cut open like an open book, i.e., cut down the length of the head from the nose to the hindbrain along the flank. Dissected embryos were fixed in 4% PFA for 2 h at room temperature or overnight at 4 °C and then washed twice with PBS before immunofluorescence staining.

[0316] Immunocytochemistry of cultured cell preparations

[0317] The chamber culture slides were fixed with 4% PFA for 30 minutes at room temperature and then washed with PBS and 0.1% Tween 20 (Sigma-Aldrich) (PBS-T). The cultures were blocked (10% horse serum in PBS-T) for 30 minutes at room temperature and then incubated with primary antibodies (nestin (611658; BD Biosciences, San Diego, USA) at 1:100; polysialic acid-neural cell adhesion molecule (PSA-NCAM) at 1:500 (kindly provided by Dr. T. Seki), β-III tubulin clone, TUJ1 (MMS-435P, 1 mg / ml; Covance, Princeton, NJ, USA) at 1:200; NF-M (13-0700, 0.5 mg / ml; Zymed, South San Diego, CA) at 1:500. Francisco, CA); 1:500 NF-H (AB1991; Chemicon, Temecula, CA, USA) were incubated in blocking solution at 4 ° C overnight. Mouse, goat and rabbit (Ig) controls were treated under the same conditions. After washing, secondary antibodies (1:200 goat anti-mouse Alexa 488 [A11029, 1.5 mg / ml; Jackson ImmunoResearch Laboratories, West Grove, PA, USA]; 1:200 goat anti-rabbit Alexa 488 [A11034, 2 mg / ml [Molecular Probes Inc., Eugene, OR, USA]) were added to the blocking solution and continued for 2 hours at room temperature in the dark. Finally, the slides were washed and the coverslips were covered with Prolong gold antifade with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, P36931; Invitrogen).

[0318] result

[0319] like Figure 4 As shown in , when hDPSCs are grown in neuronal induction medium, they differentiate into multi-process neuron-like cells with the biophysical properties of conducting sodium currents. In addition, when hDPSCs are transplanted into the brain of chicken embryos, they differentiate into neuron-like cells expressing neurofilaments.

[0320] Example 4: hDPCS treatment improves forelimb motor function after stroke in a rodent model

[0321] The therapeutic efficacy of hDPSCs was assessed in the middle cerebral artery occlusion (MCAO) rat model causing ischemic stroke. MCAO is the most common manifestation of human ischemic stroke, and this preclinical rodent model of MCAO reproduces the human condition. In this model, MCAO is manufactured by the following operation: inserting a nylon thread into the internal carotid artery, the thread is advanced forward to occlude the middle cerebral artery at the distal end of the internal carotid artery.

[0322] method

[0323] Human DPSC culture and preparation

[0324] Human DPSCs were isolated from adult impacted (failed to fully erupt to its expected position) third molars by digestion of dental pulp tissue and reverse transcription transduction with the fluorescent marker green fluorescent protein (GFP). DPSCs at passages 3-5 were cryopreserved in fetal bovine serum containing 10% dimethyl sulfoxide. The cells were thawed and passaged at least once before use for intracerebral transplantation. DPSCs were cultured at 37°C in 5% CO2 in modified Eagle's medium (Sigma-Aldrich, St. Louis, http: / / www.sigmaaldrich,com) supplemented with 20% fetal bovine serum, 100 DM of L-ascorbic acid 2-phosphate (Wako, Richmond, VA), 2 mM of L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin. The cells were dissociated with trypsin and cultured at 1.5×10 5 Resuspend the cells at a final concentration of 10 cells / μl. Cell viability was determined by trypan blue dye exclusion.

[0325] Middle cerebral artery occlusion and intracerebral transplantation in rodents

[0326] Fifty-four adult male Sprague-Dawley rats weighing 300 ± 20 g were maintained on a 12-h light / dark cycle with ad libitum access to food and water.

[0327] Rats were anesthetized with isoflurane and subjected to transient focal cerebral ischemia by intraluminal proximal middle cerebral artery occlusion (MCAo) [essentially as described in Aspey et al., Neuropathol Appl Neurobiol., 26:232-242, 2000 and Spratt et al., J Neurosci Methods, 155:285-290, 2006]. Rectal temperature was maintained at 37±1°C by a heating pad, and oxygen saturation and pulse rate were monitored and maintained within physiological parameters using a pulse oximeter (model 8500AV; Nonin Medical, Plymouth, MN). Briefly, the right common carotid artery, external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery were exposed by blunt dissection, followed by cauterization and ligation of the ECA. A 4-0 monofilament nylon thread (Dynek, Hendon, SA, Australia) and a silicone-coated tip (0.35 mm diameter, 5 mm length) were advanced from the ECA stump, through the ICA, and up to the right middle cerebral artery (MCA). The thread was secured in place and the incision was closed. After 2 hours of occlusion, the rats were re-anesthetized and the thread was removed to allow reperfusion. The rats were allowed to recover from surgery, and at 2 hours, contralateral forelimb flexion was assessed by briefly suspending the animals by the tail and circling behavior on the floor to verify MCAo. 24 hours after MCAo, animals were randomly selected to receive human DPSC (n=31) or vehicle (n=23) treatment (see description of treatment regimens). Figure 5 The total volume of the cell suspension (6×10 5 Cells) were injected (1 μL / min) into the ipsilateral parenchyma at two stereotaxic coordinates (anterior-posterior, medial-lateral relative to bregma; dorsal-ventral from dura mater). A stereotaxic instrument (Kopf Instruments, Tujunga, CA) was used to inject 2 μl into the striatum (-0.40, +4.00; -5.50 mm) and another 2 μl into the cortex (-0.40, +4.00; -1.75 mm). Vehicle animals were treated similarly, using α-modified Eagle's medium as a vehicle. All animals received daily subcutaneous injections of 10 mg / kg of cyclosporin A (Novartis, Basel, Switzerland).

[0328] 2,3,5-Triphenyltetrazolium chloride staining

[0329] 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to confirm ischemia. Fresh brains were cut into 2-mm slices and incubated in 2% TTC (Sigma-Aldrich) at 37° C. in the dark for 10 min. Brain slices were imaged in color using a LiDE210 flatbed scanner (Canon, Tokyo).

[0330] Functional assessment

[0331] Animals were trained for 3 consecutive days before MCAo, and the third segment (-1 day) was used as a preoperative baseline. Blind evaluation of behavior was performed during the light cycle of the 7th, 14th, 21st, and 28th days (three trials for each test) after the 1st day (before DPSC transplantation), MCAo (Fig. 5C). Animals that failed neurobehavioral training before MCAo (being that animals did not reach baseline scores in each test) and animals that showed almost no to no or very serious functional deficits at least 1 week after MCAo were excluded. The experiment was carried out under randomized treatment allocation and blind outcome evaluation, which complies with the guidelines of the Stroke Treatment Academic Industry Roundtable (STAIR) and similar guidelines.

[0332] Neurological score

[0333] The overall neurological deficit in each animal after MCAo was assessed using a scoring system modified from previous studies [Barry et al., Brain Res, 1389: 143-151, 2011; Bederson Stroke, 17: 472-476, 1986; and Thai et al., J. Neurol. Sci, 286: 150-159, 2008]. Higher scores indicated more severe neurological deficits.

[0334] Tape Removal Test

[0335] Adhesive labels (15 mm2) were affixed to the medial side of the rat's forepaws [Bouet et al., Nat Protoc 4: 1560-1564, 2009]. The order in which the adhesive labels were placed on the contralateral or ipsilateral forepaws was randomized. Upon return to the test cage, the time spent attempting to remove the adhesive label and the total time spent removing the label (maximum value allocated was 2 minutes) were recorded for each paw. The duration spent removing (or attempting to remove) the tape was expressed as a percentage of the total time spent.

[0336] Step test

[0337] Each rat was firmly grasped using one hand to gently lift the rat's trunk and hind limbs and the other hand to control one forepaw so that the body weight was loaded on the other forepaw that was not restrained [Schallert et al., Exp Neurol, 64:33-43, 1979]. The rat was then moved sideways (30 cm) so that the weight-bearing forepaw stepped in the backhand direction to adjust the body movement. The order of forepaw testing was randomized. The number of steps performed by each forelimb was recorded.

[0338] Rotarod

[0339] Rats were placed on a rotarod apparatus consisting of a motorized rotating assembly of 18 × 1-mm rods

[28] . The animals were required to walk on the apparatus as the rotation speed increased from 0 to 24 rpm over 2 min. The time it took for the animal to complete the trial, fall off the apparatus, or grip a rung and rotate two consecutive rotations without attempting to walk on the rung was recorded.

[0340] Cone / Ledge Balance Beam Walking

[0341] Animals were required to cross a high balance beam that tapered along its length and ended up in a dark box [Schallert T, Woodlee MT. Orienting and placing. In: Whishaw IQ, Kolb B ed. The Behavior of the Laboratory Rat: A Handbook with Tests. Oxford, UK: Oxford University Press 2005; 129–140], and their performance was videotaped. The number of foot slips produced by each hind limb on the lower hanging ledge (2 cm below the upper balance beam surface) was calculated as a percentage of the total number of steps spent crossing the balance beam. Due to the circling behavior of stroke animals, balance beam walking could not be completed 1 day after MCAo.

[0342] Immunohistochemistry and imaging

[0343] At the end of the neurobehavioral assessment, rats were deeply anesthetized, 1,000 U of heparin was injected directly into the heart, and perfused transcardially with ice-cold saline followed by phosphate-buffered saline (PBS) containing 4% (wt / vol) paraformaldehyde. The brain was removed and postfixed overnight at 4°C in the same fixative. The brain was embedded in paraffin and cut into serial 5-μm coronal sections for colorimetric detection of viable human DPSCs. Sections were blocked with PBS containing 3% normal horse serum and washed twice in PBS before incubation with mouse anti-human mitochondria (1:1,500; Millipore, Billerica), followed by biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA) and streptavidin horseradish peroxidase-conjugated antibodies (1:1,000; Thermo Scientific, Rockford, IL). Antibodies were diluted in blocking solution. Antibody staining was visualized with 3,3-diaminobenzidine (Sigma-Aldrich). Bright field images were acquired and analyzed using a NanoZoomer digital pathology system (Hamamatsu Photonics, Shizuoka, Japan).The number of transplanted cells remaining in rodent brains (n=3) 4 weeks after transplantation was counted in every second 5 μm coronal section, and the distribution of transplanted cells was analyzed.

[0344] For double immunofluorescence studies, 100 μm free-floating sections were blocked with PBS containing 10% normal horse serum with 0.3% Triton X-100 before identification of human DPSCs using goat anti-GFP (1:250; Rockland, Gilbertsville, PA), mature neurons using mouse anti-neuronal nucleus (NeuN; 1:500; Millipore), or astrocytes using rabbit anti-glial fibrillary acidic protein (GFAP; 1:250; DAKO, Glostrup, Denmark). Other primary antibodies used were mouse anti-β-III tubulin (1:500; Millipore), mouse anti-nestin (1:100; Abeam, Cambridge, UK), mouse anti-polysialic acid-neural cell adhesion molecule (PSA-NCAM; 1:200; Millipore), and mouse anti-04 (1:500; Sigma-Aldrich). Secondary antibodies were Cy2-conjugated anti-goat IgG, Cy3-conjugated anti-rabbit IgG, or Cy3-conjugated anti-mouse IgG (1:300; Jackson Immunoresearch Laboratories, West Grove, PA). Antibodies were diluted in blocking solution. All sections were mounted with ProLong Gold antifade (Invitrogen, Carlsbad, CA). To estimate the percentage of GFP-positive cells co-expressing NeuN or GFAP, z-stack images (n=3 animals) were taken at a magnification of D40 in 5 fields (consisting of 2 cortical and 3 striatal areas in the ipsilateral hemisphere at the level of DPSC transplantation). Confocal images were sequentially acquired using a TCS SP5 confocal microscope and processed using its LAS AF software (Leica Microsystems, Wetzlar). Orthogonal projections were performed using ImageJ (NIH).

[0345] Corpus callosum thickness measurement

[0346] Paraffin sections at the level of bregma and bregma ± 0.1 mm were stained with 0.1% cresyl violet (n = 6 DPSC-treated, n = 2 vehicle-treated). Corpus callosum thickness measurements were made at the midline (midpoint between the two hemispheres) and lateral edges of the ventricles of both hemispheres of the brain. Measurements were expressed as a percentage of the corresponding midline measurements and were performed using Axio Vision imaging software (Carl Zeiss, Oberkochen, Germany).

[0347] Statistical analysis

[0348] GenStat (version 13; VSN International, Hemel Hempstead, UK) was used for analysis. The effects of treatment on behavioral test scores were analyzed using repeated measures analysis of variance (ANOVA), using the Greenhouse-Geisser correction to account for associations between serial measurements of individual rats, and adjusting pre-stroke data as a covariate to allow for possible differences in the initial abilities of the rats. If the initial ANOVA was significant, a post hoc Fisher-protected least significant difference (LSD) test was performed. Differences were considered statistically significant at p < 0.05. Data were presented as box plots using R (version 2.10.1; R Development Core Team, Vienna, Austria) or as bar graphs using Prism (version 5.02, GraphPad Software, Inc., San Diego). Reported values ​​are mean ± SEM.

[0349] result

[0350] Transplanted human DPSCs enhance forelimb sensorimotor recovery after stroke

[0351] The ability of human DPSC treatment to improve neurobehavioral function over time after focal cerebral ischemia was explored. With regard to morbidity or mortality, no deleterious effects were observed following DPSC treatment compared to controls. The mortality rate obtained following intracerebral transplantation was comparable in both treatment groups (approximately 9%).

[0352] The clinically relevant rodent model of unilateral MCAo induces substantial ischemic damage to the dorsolateral striatum and overlying frontal / parietal cortex of the ipsilateral hemisphere of the rodent brain. As a result, animals developed neurobehavioral deficits as evidenced by an increase in neurological scores of 11.7 ± 0.4 points on day 1 post-stroke ( Figure 6 A). In addition, marked forelimb somatosensory and motor asymmetries were observed in the stepping test ( Figure 6 B, The reduction in the number of steps collected by the contralateral forelimb on day 1 after MCAo was 82.2 ± 6.6%), and the tape removal test ( Figure 6 C; The time spent attempting to remove external stimuli from the contralateral forepaw was reduced by 93.7 ± 2.8% on day 1 after MCAo.

[0353] In experimental stroke, the rotarod test ( Figure 6 Loss of motor balance and coordination was also observed in the rats (D; time spent on the rotarod decreased by 53.0 ± 2.0% on day 1 after MCAo).

[0354] Stroke-affected animals showed hindlimb asymmetry in foot slipping of the contralateral hindlimb during beam walking at 1 week after MCAo ( Figure 6 E; increased to 21.7±6.2%). The ipsilateral limbs had normal post-stroke neurobehavioral function.

[0355] All animals showed progressive recovery of neurobehavioral function within 4 weeks after stroke ( Figure 6 DPSC-treated animals showed significantly improved overall neurobehavioral function compared to vehicle-treated animals ( Figure 6 A; p<0.018 group × day interaction, repeated measures ANOVA) and significantly decreased neurological scores, especially on days 21 (p<0.05, post hoc Fisher-protected LSD) and 28 (pO.01) after stroke. In limb-specific neurobehavioral studies, in the stepping test ( Figure 6 B; p<0.045 overall group effect) and tape removal test ( Figure 2 C; p<0.049 overall group effect), DPSC-treated animals exhibited significantly increased sensorimotor recovery of the ipsilateral (stroke-affected) forelimb compared to vehicle controls. Overall, DPSC-treated animals displayed reduced post-stroke deficits in the use of the contralateral forelimb on all measured days.

[0356] There were no significant differences in ipsilateral limb function between treatment groups. Figure 6 D) and cone / ledge balance beam walking ( Figure 6 E) There were no statistically significant differences in performance in the tests (p < 0.05). One possible explanation for the differences in increased functional improvement between limbs is that this rodent MCAo model produces greater damage to the sensorimotor cortex in the rodent brain that represents forelimb function due to the preferential vascular supply to this area [Salo et al., Soc Neurosc Abstr 13: 1268, 1987]. In support of this, there was a severe loss of forelimb function in the animals of this study as measured by the stepping and tape removal tests, but the impairment of hindlimb function as assessed by the cone / ledge balance beam walking test immediately after MCAo was less obvious. Rotarod running and balance beam walking assess motor coordination and hindlimb function, respectively [Hicks et al., Cell stem Cell, 5: 139-140, 2009], and these tests may not be sensitive enough to detect post-stroke deficits. Compared with vehicle-treated animals, the increased functional improvement after DPSC treatment became apparent in the forelimb analysis at 4 weeks, but not in hindlimb function.

[0357] Another explanation may involve the animals' learned compensatory motor behaviors. In the rotating rod running and cone / ledge balance beam walking tests, through repeated testing, rodents may have learned the following novel ways: the limbs or tail deviations that are not affected by the stroke can be used to compensate for the loss of function of the limbs affected by the stroke [Schaar et al., Exp TranslStroke Med, 2:13, 2010]. As measured by the stepping and tape removal tests, the forelimb-specific tasks allow the least use of compensatory behaviors to overcome the loss of sensorimotor function after stroke.

[0358] The foregoing data indicate that hDPSC transplantation affects the normal function of the host nervous system after stroke and produces improved neurological function compared to control animals. Without being bound by theory or mode of action, this improvement may occur through changes in neuroplasticity in the brain after stroke. For example, as shown above, STRO-1 + The cells expressed SDF-1 (CXCL-12). hDPSCs were also shown to express this chemokine. Furthermore, hDPSCs chemotactic axons from the host trigeminal ganglia after transplantation into the head of chick embryos caused these animals to show significant functional improvements at later time points.

[0359] Survival of DPSCs in ischemic brain at 4 weeks after transplantation

[0360] Findings in neurobehavioral studies following DPSC transplantation indicated peak benefits at later time points in neurological scores and forelimb sensorimotor function. Subsequent studies explored whether the underlying cellular mechanisms explaining these findings were due to neural cell replacement following transplantation of DPSCs within the stroke brain. For example, the number of donor cells surviving at the study endpoint when peak benefits were observed was determined. Specific antibodies against human mitochondrial antigens that do not cross-react with rat tissue were used to visualize individual DPSCs within the rodent brain. In each second brain coronal section, i.e., at 10-μm intervals, the number of labeled cells was manually counted. The total number of human mitochondrial-positive cells detected in the brain of each animal at 4 weeks after transplantation ranged from 9,458 to 22,394 cells (Table 5). This gives an average number of 13,807-4,293 remaining human DPSCs and an initial transplantation of 6×10 5 Our data demonstrated that cell transplantation was successful and resulted in long-term viability of DPSC-derived cells in the postischemic brain, albeit in small numbers.

[0361] Without being bound by theory or mode of action, since only an average of 2.3% of the initially transplanted hDPSCs survived to 28 days after stroke, neuronal replacement is unlikely to be the primary mechanism of action for hDPSCs to improve neurological function. However, the neuronal replacement may provide some therapeutic benefit. Alternative or additional mechanisms that may contribute to this improvement include neuroprotection, angiogenesis, immunomodulation, and neuroplasticity.

[0362] Table 5: Survival of transplanted human DPSCs at 4 weeks

[0363]

[0364] DPSC migration to targets of stroke damage

[0365] The spatial distribution of human DPSCs after transplantation into ischemic rat brains was also analyzed. 24 hours after transplantation, DPSCs were found to aggregate around the injection site. After 4 weeks, human mitochondrial labeled cells were found to be primarily in the region surrounding the infarct core ( Figure 7 , Figure 8 A, Figure 8B), indicating that transplanted cells preferentially migrated from the transplantation site to the ischemic infarct and localized in the ischemic border zone (IBZ). Figure 7 , Figure 8 A. Figure 8 B) and in the corpus callosum ( Figure 8 B) A small number of DPSCs were observed. During the 4-week period, cells had migrated to at least 4 mm in the contralateral hemisphere. Compared with the healthy hemisphere, cells derived from DPSCs were widely distributed in the contralateral hemisphere in low numbers, and DPSCs did not significantly migrate to specific target areas in the contralateral hemisphere. In rare cases, cells derived from DPSCs merged into and surrounded endothelial cells ( Figure 4 C, arrows), pericytes, or smooth muscle cells ( Figure 4 C, arrows) Consistent morphological appearance of brain microvascular walls.

[0366] Transplanted human DPSCs differentiate into neurons and astrocytes in vivo

[0367] The morphology of human mitochondrial antigen-positive cells in the brain 4 weeks after ischemia was then examined. Cells that exhibited morphology consistent with astrocytes showed a star-like appearance ( Figure 8 D, arrows), while putative neurons exhibit long processes ( Figure 8D, arrow). To verify these phenotypes, double immunofluorescence analysis and confocal imaging were used. Transplanted human DPSCs had previously been reverse-transcribed and transduced to express GFP, which allowed co-labeling with NeuN or GFAP to identify mature neurons or astrocytes, respectively. The co-localization of GFAP and GFP expression in cells within the glial scar confirmed that the cells derived from DPSCs had differentiated into astrocytes. In the ipsilateral peri-infarct cortical region, NeuN and GFP double-positive cells indicated that the transplanted DPSCs differentiated into neurons. Astrocytes and neurons are unevenly distributed in the ipsilateral cerebral hemisphere after ischemia. At the IBZ in the striatal region, astrocytes derived from DPSCs accounted for the majority (51.0±8.6% GFAP / GFPD double-positive cells, expressed as the percentage of GFP single-labeled cells), while neurons derived from DPSCs accounted for a smaller proportion (8.7±6.1% NeuN / GFPD cells) in comparison. In the cortical area around the infarct, the proportions of astrocytes and neurons appear to be similar (38 ± 0.6% GFAP / GFP cells and 32.1 ± 1.8% NeuN / GFPD cells, respectively). It was found that transplanted DPSCs differentiated mainly into astrocytes along the IBZ in the striatum, which seemed to be incorporated into the glial scar. Compared with the striatum, there are more neurons derived from DPSC in the cortical peri-infarct area. Interestingly, about 1 / 3 of the GFP cells in the peri-infarct cortical area are also labeled with the early / intermediate neuron marker β-III tubulin. However, in the glial scar along the IBZ, many GFP cells also co-express the neural progenitor marker nestin. There is almost no evidence of nestin / GFPD cells in the cortex. In the peri-infarct cortex, some GFP cells are also positive for the migrating neuroblast marker PSA-NCAM. In rare cases, oligodendrocytes (04 / GFPD cells) derived from DPSC were observed in the rodent brain at 4 weeks after transplantation.

[0368] DPSC treatment reduces corpus callosum atrophy after stroke

[0369] The effect of DPSC treatment on the loss of interhemispheric connectivity across the corpus callosum following stroke lesions was also explored. At 4 weeks post-stroke, there was a non-significant reduction in ipsilateral corpus callosal atrophy in DPSC-treated animals compared with vehicle-treated animals (DPSC-treated, 67.6 ± 12.5%, L2 measurements expressed as a percentage of the corresponding midline L1 measurements; vehicle-treated, 56.6 ± 3.0%; p > .05) ( Fig. 9). In DPSC-treated brains, the thickness of the corpus callosum in the ipsilateral hemisphere showed a trend toward better protection and achieved similar corpus callosum thickness in the contralateral (unaffected by stroke) hemisphere. There was no difference in corpus callosum thickness in the contralateral hemisphere between treatment groups (DPSC-treated, 66.1±14.0%; vehicle-treated, 66.5±1.6%).

[0370] Example 5: Cynomolgus monkey STRO-3 + Characterization of MPCs

[0371] The simian bone marrow progenitor cells (from cynomolgus macaques; cyno-MPC) are separated from about 15 ml of bone marrow aspirates collected from female cynomolgus macaques (macacafascicularis). The bone marrow aspirate suspension is separated and washed to remove anucleated cells (erythrocytes) using a Ficoll gradient. Nucleated cells are counted and then separated by connecting CA12 antibodies (anti-STRO-3) and Dynalbeads. Cells connected with antibodies and beads are positively selected by the magnetic field of the MPC-1 magnet. Positively selected cells are counted and inoculated into the growth medium in a T-flask as passage (p.) 0. Preselected, positive and negative cells are used in colony formation assays (CFU-F).

[0372] Feed cyno-MPC cells with growth medium. Feed all cultures (pO–p.5) every 2 to 4 days until they reach the desired confluence. Then wash with HBSS and then passage or harvest the cells using collagenase and trypsin / Versene in sequence. Count p.1 cells and seed into T-flasks. When p.1 cyno-MPC cells reach the desired confluence, harvest the cells and cryopreserve using a controlled rate freezer.

[0373] Passage 1 cryopreserved cyno-MPCs were thawed and inoculated into T-flasks (p.2). p.2 cells were passaged to p.3 in a cell factory. p.3 cells were harvested and passaged to p.4 in a cell factory. Additional p.3 cells were cryopreserved. p.4 cells were passaged to p.5 in a 6× cell factory. When p.5 cyno-MPC cells reached the desired confluence, cells were harvested and cryopreserved using a controlled rate freezer. Cells were cryopreserved in 50% αMEM, 42.5% Profreeze, and 7.5% DMSO. Samples were tested for CFU-F assay, FACS, sterility, mycoplasma, and endotoxin.

[0374] Representative flow cytometric analysis of the immunophenotype of cultured cyno-MPCs is shown in Figure 10. As shown, these cells are STRO-1 + 、STRO-4 + and CD146 + .

[0375] Example 6 Prospective Isolation of Non-Human Primate (nhp) DPSCs:

[0376] The use of magnetic bead sorting allows the purification of scarce DPSCs from the fibrous pulp tissue. After pulp tissue harvesting and enzymatic digestion, approximately 1-2 × 10 DPSCs can be recovered from an adult third molar. 5 Cells were collected from multiple teeth from the same animal. Single cell suspensions of nonhuman primate dental pulp were incubated with antibodies to human MPC-associated antigens that we have previously shown to react with nonhuman primate bone marrow cells ( Fig.10 ), including STRO-1 (mouse IgM anti-human MPC; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); CD146 (mouse IgG2a anti-human CD146 / MUC-18; 9); or STRO-4 (mouse IgG1 anti-human heat shock protein-β). Labeled dental pulp cells were incubated with sheep anti-mouse IgG-conjugated or rat anti-mouse IgM-conjugated magnetic beads Dynabead (4 beads per cell: Dynal, Oslo, Norway). Magnetic bead-positive cells were collected using an MPC-1 magnetic particle concentrator (Dynal) according to the manufacturer's recommended protocol. STRO-1, STRO-4, or CD146 magnetic bead-positive cells were concentrated at approximately 3×10 4 cells / cm 2 Primary cultures were generated in standard growth medium (α-MEM supplemented with 20% (v / v) FBS, 2 mM L-glutamine, 100 mM L-ascorbic acid-2-phosphate, 50 U / ml penicillin, 50 mg / ml streptomycin). After reaching approximately 90% confluence, primary DPSCs were plated at approximately 0.5-1×10 4 cells / cm 2 The inoculation density was used for subculture.

[0377] Example 7 Assessment of nhpDPSC Colony Frequency: Using Colony-Forming Fibroblast Assay

[0378] The efficiency of STRO-1, STRO-4, or CD146 antibodies as single agents to purify nhpDPSCs was assessed. Unfractionated and STRO-1, STRO-4, or CD146 positive and negative selected cell fractions were in triplicate at 0.3, 1.0, and 3.0 × 10 4 Cells / well were seeded into standard growth medium in 6-well culture plates and incubated at 37°C, 5% CO2 and >90% humidity for 12 days. Aggregates of >50 cells were scored as fibroblastic colony-forming units (CFU-F). GLP-grade nhpDPSCs were generated using an antibody selection protocol that resulted in the highest incidence of CFU-F colony formation for functional characterization studies.

[0379] Example 8 Characterization of nhpDPSCs

[0380] Flow cytometry is used to characterize the immunophenotype of DPSCs expanded in vitro. The expression of mesenchymal and non-mesenchymal stem cell-related surface markers at different cell generations is evaluated as described above. When allogeneic nhpDPSCs are used to treat ischemic stroke, the immunomodulatory ability of these cells to inhibit activated immune cells in mixed lymphocyte reactions or in vitro co-culture with mitogen-stimulated lymphocytes is evaluated (e.g., as described in Wada et al., J Cell Physiol, 219: 667-676, 2009).

[0381] The ability of DPSC to generate mesodermal and ectodermal tissues (including those similar to those obtained therefrom) is a hallmark feature of these cells. The ability of DPSC to differentiate into different cell lineages in vitro was explored by culturing under inductive conditions. The ability of multi-lineage differentiation into osteoblasts (alizarin red staining of mineral deposits), adipocytes (oil red O staining of lipid droplets) and chondrocytes (toluidine blue staining of sulfated proteoglycans and type II collagen expression) was assessed using nhpDPSC expanded in vitro, for example, as described in Grontos et al., J Cell Scl., 116: 1827-2835, 2003.

[0382] Ex vivo expanded nhpDPSC single cell suspensions pre-labeled with GFP were additionally transplanted subcutaneously into the dorsal flank of severely compromised immunodeficient (NOD / SCID) mice (n=3) in combination with ceramic hydroxyapatite / tricalcium phosphate (HA / TCP) particles as a carrier vehicle for a period of approximately 8 weeks. After 8 weeks post-transplantation, recipient animals were sacrificed to retrieve the grafts for histological analysis.

[0383] In vitro differentiation of nhpDPSC into neural cells was assessed on poly-ornithine-coated chamber culture slides under neural induction conditions as described previously.

[0384] Real-time PCR and immunohistochemistry were used to assess early (NeuroD, nestin, NeuN, b-III tubulin) and late (GFAP, neurofilament medium and heavy chains) gene and protein expression.

[0385] Patch clamp techniques were performed to determine the ability of differentiated nhpDPSCs to initiate action potentials in vitro.

[0386] In addition, the neurogenic and neuroplastic potential of GFP-labeled nhpDPSCs was determined by in ovo injection of these cells into chick embryos (eg, as described previously).

[0387] The electrophysiological properties of implanted GFP-labeled DPSCs were assessed by measuring the presence of voltage-dependent sodium and potassium channels on 300 mm brain tissue sections at days 7 and 14 after implantation.

[0388] Together, these studies define an allogeneic nhpDPSC population for treating stroke in preclinical studies in nonhuman primates.

[0389] Example 9: Use of nhpDPSCs to improve motor function in a non-human primate ischemic stroke model

[0390] The similarities between humans and nonhuman primates in sophisticated cognitive abilities, remarkable social complexity, and the fine complexity of brain organization make them excellent models for studying neurological diseases and disorders.

[0391] The non-human primate ischemic stroke model was generated by MCAO method using nylon suture occlusion via the internal carotid artery (e.g., as described in Freret et al., J Cereb Blood Flow Metab., 28:786-796, 2008). The non-human primate model was generated in cynomolgus monkeys (an old world monkey (Macaca fascicularis)). The following is the experimental design procedure for ischemic stroke studies:

[0392] (i) Baseline neurobehavioral testing was performed on all animals before stroke.

[0393] (ii) Induced MCAO ischemic stroke model.

[0394] Briefly, monkeys were anesthetized, intubated, and mechanically ventilated while intensively monitoring blood oxygen levels, core body temperature, blood pressure, ECG, and laser Doppler flowmetry of intracranial blood flow. The detailed surgery was performed by an experienced neuroscientist exposing the right common carotid artery at the bifurcation of the external and internal carotid arteries. The external carotid artery and other branch arteries were cauterized to ensure smooth passage of the silk thread along the internal carotid artery to the origin of the middle cerebral artery. A 0.54 mm diameter × 3 mm length silicone rubber-coated nylon silk thread was inserted into the internal carotid artery and advanced until resistance was felt, indicating that the silk thread had occluded the middle cerebral artery. The silk thread was fixed in place by a ligature. Three hours after MCAO, the silicone rubber-coated nylon silk thread was withdrawn to induce reperfusion, a process similar to human middle cerebral artery ischemic stroke.

[0395] (iii) MRI was performed on day 7 after MCAO to determine whether an ischemic stroke of appropriate size and location was induced.

[0396] (iv) The cell therapy design consisted of administering 5×10 5 or 1.5 × 10 6 Intrastriatal injection of nhpDPSC or culture medium alone (without cells). As in rodents, due to increased intracranial pressure leading to morbidity, the volume of fluid that can be safely injected into the brain after stroke is limited. This is estimated to be 12 μL in macaques. Therefore, all intrastriatal injections will be 12 μL, with different cell concentrations. As described above, the monkeys are anesthetized and a burr hole is made in the skull for stereotactic intrastriatal injection.

[0397] (iv) Neurobehavioral testing was performed weekly during the 6-week study period. To fully explore the extent of sensorimotor deficits following MCA ischemic stroke, the following tests were used: (Cambridge Neurophysiological Test Automated Battery), neurological score, Hill-and-Valley Staircase, and Tactile forelimb stimulation tests (e.g., as described in Bihel et al., J Cereb Blood Flow Metab., 30: 273-285, 2010).

[0398] (vi) The study was completed at 6 weeks after stroke.

[0399] Example 10: Interaction between implanted nhpDPSCs and NHP brain tissue

[0400] 10.1 Treatment of ischemic penumbra:

[0401] Surrounding ischemic stroke is a region of ischemic penumbra tissue that is at risk of death but can be treated. - Cell therapy may interact with this ischemic penumbra and thereby limit the size of the stroke.

[0402] The monkeys were anesthetized and positioned prone on a 0.0T Siemens Trio scanner, and T2-W turbo spin echo images (T2WI, TR / TE=4800 / 86ms) were collected with a certain field of view (180×180mm) and an image acquisition matrix of 0.5×0.5 in plane resolution. Twelve T2WI coronal images with a slice thickness of 2mm at an interval of 0.2mm were collected. The infarct volume of each monkey on T2WI was depicted using Siemens MRI software.

[0403] 10.2 Neuronal Replacement and Connectivity in NHP Stroke Brains

[0404] Infarction results in the loss of approximately 2 / 3 of neurons and glia in one cerebral hemisphere. The goal of this series of studies is to determine: (1) the number of surviving nhpDPSCs, (2) the number of nhpDPSCs that differentiate into neurons or glia, and (3) whether neurons derived from nhpDPSCs produce functional connections. To do this, nhpDPSCs were transduced with retrovirus to express GFP and cultured in BrdU (approximately 20 μM for 24 hours) before transplantation for nuclear labeling. These two labeling methods provide formal identification of nhpDPSCs implanted in the host brain.

[0405] The number of nhpDPSCs that differentiated into a neural phenotype was estimated stereologically by determining the density of nhpDPSCs in a defined volume. From rodent studies, we found that at 4 weeks, hDPSCs migrated into the stroke site approximately 1 mm from the injection site in the anterior-posterior direction (this was evident from the scars on the brain surface). Therefore, a monkey brain with a thickness of 8 mm from the anterior-posterior axis to the injection site included most of the transplanted human cells by 6 weeks after stroke. Serial coronal sections of 100 mm were taken along this axis (approximately 4×103 mm tissue width at 100 mm = 40 coronal sections per treated animal). A random sampling scheme was constructed (using Image J) to count the number of BrdU-positive cells in standardized tissue volumes. Direct 3-dimensional counting of positively stained human cells was performed using a Bio-Rad MRC-1000UV confocal microscope (Adelaide Microscopy). Confocal microscopy identified GFP-positive or BrdU-positive nhpDPSCs that co-localized with cytoplasmic antigens expressed by differentiated cells such as neurons using antibodies against neurofilament medium chain (NF-M) or β-III tubulin, astrocytes expressing glial fibrillary acid protein GFAP or tyrosine hydroxylase.

[0406] The electrophysiological properties of the neurons obtained from nhpDPSC implanted in monkey stroke were also evaluated. First, the biophysical properties of the neurons obtained from nhpDPSC isolated from rodent stroke brain were evaluated. Briefly, the animals were decapitated and the brain was taken out and placed in ice-cold oxygenated saline. The brain was cut into slices of 300-500 mm thickness using a vibrating tissue slicer, washed and then digested by a mixture of trypsin and collagenase. Single neurons were obtained by carefully aspirating tissue, washed and inoculated on the glass coverslips treated with collagen. After 24 hours of recovery, cells were used for whole-cell patch clamp techniques. The neurons obtained from nhpDPSC were identified as GFP positive cells. The types of voltage-gated channels expressed by these cells were identified using appropriate voltage schemes and channel inhibitors.

[0407] The ability of the neurons obtained from nhpDPSC to generate action potentials (AP) and to form connections with other neurons was explored by the patch clamp technique and imaging of cells in brain slices. The brain near the injection site was cut into slices of 250-400 mm thickness on a vibrating microtome. After recovering for 1 hour at room temperature and constant oxygenation, the single slice was transferred to the recording chamber installed on an upright BX51WI Olympus microscope with fluorescent attachment IR and DIC optics. First, nhpDPSC was identified using GFP fluorescence, and then IR filters (720-850nm) and Rolera-XR IR-sensitive cameras were used to observe. The whole-cell patch clamp technique of neurons in brain slices was carried out using an EPC-10 computer-controlled amplifier and related instruments. Ap was caused by injection of depolarizing current and recorded in current clamp mode.

[0408] To investigate whether APs generated by nhpDPSCs can propagate to surrounding neurons through tight junctions or synaptic connections, Fura-2 fluorescence combined with patch clamp technology was used to observe intracellular Ca 2+ The concentration of Fura-2 was changed. Briefly, the brain slices were loaded with membrane-permeable Fura-2AM, washed and placed in a recording chamber. Implanted nhpDPSC neurons were identified by GFP fluorescence and patch clamped using IR. The fluorescence settings were then changed to those suitable for Fura-2 measurement. Ap in nhpDPSC was induced by passing a depolarizing current and fluorescence was measured in surrounding rodent host neurons.

[0409] 10.3 Functional MRI (fMRI) and Diffusion Tensor Imaging (DTI) of Stroke Brains in Nonhuman Primates to Explore Neuroplasticity:

[0410] These techniques allow for non-invasive exploration of neuroplasticity following nhpDPSC treatment as compared to control monkeys. In an ischemic stroke model, fJVIRI was used to assess forelimb motor control and determine if there was greater activation of the motor cortex following nhpDPSC treatment. fJVIRI studies were performed 3 and 6 weeks after treatment. 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Claims

1. Use of a composition comprising a population of human dental pulp stem cells (hDPSCs) in the manufacture of a medicament for regenerating brain neurons in a human subject who has suffered an ischemic stroke for 24 hours to 1 week, thereby reducing motor function loss.

2. The use as claimed in claim 1, wherein the regenerated brain neurons are in the cerebral cortex.

3. The use as claimed in claim 2, wherein the regenerated brain neurons are in the motor cortex and / or sensory cortex.

4. The use according to any one of claims 1 to 3, wherein the cell population is a culture-expanded cell population.

5. The use according to any one of claims 1 to 3, wherein the cell population is an allogeneic cell population.

6. The use according to any one of claims 1 to 3, wherein the cell population is an autologous cell population.

7. The use of any one of claims 1-3, wherein the human subject has movement disorders due to stroke or is at risk of developing movement disorders due to stroke.

Images