WO2007142651A1

WO2007142651A1 - Methods and compositions for the treatment of neuropathy

Info

Publication number: WO2007142651A1
Application number: PCT/US2006/022624
Authority: WO
Inventors: Young-Sup Yoon
Original assignee: Caritas St. Elizabeth Medical Center Of Boston, Inc.
Priority date: 2006-06-09
Filing date: 2006-06-09
Publication date: 2007-12-13

Abstract

The invention features compositions and methods that are useful for the prevention or treatment of neuropathy or for enhancing angiogenesis in a neural tissue.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF

NEUROPATHY

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by the following grants from the National Institutes of Health, Grant No. IROl HL 079137-01. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

In the United States alone, more than 18 million people suffer from diabetes. Neuropathy is the most common complication of diabetes, affecting 1 to 7 million people including 7% within 1 year of diagnosis and up to 60% of long-standing diabetic patients. Even with intensive insulin therapy, the incidence of new clinically detected neuropathy per patient-year was as high as 7.0%; with conventional therapy, the incidence of neuropathy increased to as much as 16.1% of the 18 million diabetics in the United States. When loss of sensation is compounded by loss blood flow due to combined peripheral artery obstructive diseases (PAOD), length of hospitalization, surgical complications, the rate of limb loss and hospital mortality is significantly increased. Thus, peripheral neuropathy presents a serious toll on patient well-being and health care costs. It has also been reported that up to 90% of patients have subclinical levels of neuropathy. Symptoms are typically dominated by sensory defects. Loss of sensation in the feet is the most frequent manifestation of diabetic neuropathy and promotes significant risk to the limb. Due to decreased tactile and pain perception, injuries to the feet are often undetected by the patient and thus may go unchecked and untreated. Successive injuries manifest as non-healing foot ulcerations, and progressive damage to the feet can ultimately result in amputation. Indeed, some reports indicate that 20% of all hospital admissions among diabetic patients in the US are for foot problems. This dire sequence of events accounts for 60% of all lower extremity amputations in the general population. Despite the devastating effects and widespread occurrence of diabetic neuropathy, virtually no effective therapies exist to treat or prevent diabetic neuropathy.

SUMMARY OF THE INVENTION

The invention features compositions and methods that are useful for the prevention or treatment of neuropathy or for enhancing angiogenesis in a neural tissue. In one aspect, the invention generally provides methods for increasing angiogenesis in a neural tissue of a subject in need thereof. The method involves administering a cell (e.g., a multipotent stem cell, a hematopoietic stem cell, an endothelial progenitor cell, mononuclear cell, mesenchymal stem cell, or a progenitor or progeny cell thereof) having the potential to differentiate into an endothelial cell to a subject, thereby increasing angiogenesis in a neural tissue of the subject.

In another aspect, the invention provides a method for preventing or ameliorating a neuropathy in a subject in need thereof. The method involves administering to the subject a cell having the potential to differentiate into an endothelial cell; and increasing angiogenesis in a neural tissue of the subject, thereby ameliorating a neuropathy in the subject.

In yet another aspect, the invention provides a method for increasing the level of a therapeutic polypeptide in a neural tissue of a subject in need thereof. The method involves locally administering a cell having the potential to differentiate into an endothelial cell to a subject, thereby increasing the level of a therapeutic polypeptide in a neural tissue of the subject.

In yet another aspect, the invention provides method for preventing or ameliorating diabetic neuropathy in a subject in need thereof. The method involves locally administering to a neural tissue of the subject a cell having the potential to differentiate into an endothelial cell, thereby ameliorating diabetic peripheral neuropathy in the subject. In yet another aspect, the invention features a packaged pharmaceutical comprising a therapeutically effective amount of a cell having the potential to differentiate into an endothelial cell, and instructions for use in treating a subject having a neuropathy.

In yet another aspect, the invention provides packaged pharmaceutical comprising a therapeutically effective amount of a cell having the potential to differentiate into an endothelial cell, and instructions for use in treating or preventing a diabetic neuropathy in a subject.

In various embodiments of the previous aspects, the cell is genetically modified. In other embodiments of the previous aspects, the pharmaceutical further contains a therapeutic polypeptide. In one embodiment, the invention features a method for identifying an agent useful for the treatment of neuropathy. The method involves contacting a multipotent stem cell with the agent; providing the cell to a host; and measuring an increase in angiogenesis in a neural tissue of the host, wherein an increase in angiogenesis relative to a reference identifies the agent as useful for the treatment of neuropathy. In another embodiment, the method further involves measuring Schwann cell proliferation, endothelial cell proliferation, neural conductance, or pain responsiveness in the host, wherein measuring an increase relative to a reference identifies the agent as useful for the treatment of neuropathy. In another embodiment, the method further involves measuring apoptosis, wherein measuring a decreasi in apoptosis identifies the agent as useful for the treatment of neuropathy.

In another aspect, the invention provides a method for identifying an agent useful for the treatment of neuropathy. The method involves contacting a cell with at least one inhibitory nucleic acid molecule; providing the cell to a host; and identifying a decrease in angiogenesis in a host tissue relative to the tissue of a corresponding control host, thereby identifying the target of the inhibitory nucleic acid molecule as an agent useful for the treatment of neuropathy. In one embodiment, the inhibitory nucleic acid is identified using proteomics, bioinformatics, or genomics. In another embodiment, the method further involves measuring Schwann cell proliferation, endothelial cell proliferation, neural conductance, or pain responsiveness in the host, wherein measuring an increase relative to a reference identifies the agent as useful for the treatment of neuropathy. In another embodiment, the method further involves measuring apoptosis, wherein measuring a decreasi in apoptosis identifies the agent as useful for the treatment of neuropathy.

In yet another aspect, the invention provides method for preventing or ameliorating neuropathy in a subject in need thereof. The method involves administering to the subject a genetically modified cell having the potential to differentiate into an endothelial cell, thereby preventing or ameliorating diabetic peripheral neuropathy in the subject. In one embodiment the genetically modified cell contains an expression vector containing a nucleic acid sequenα encoding a therapeutic polypeptide (e.g., acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF-2, VEGFl 65, epidermal growth factor, transforming growti factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I) Hypoxia inducible factor (HIF-I) and nitric oxide synthase) that supports endothelia or neuronal cell survival, proliferation, or function.

In various embodiments of any of the previous aspects, the subject (e.g., a human patient) has or has a propensity to develop diabetes. In other embodiments, the subject has oi has a propensity to develop a neuropathy (e.g., peripheral neuropathy, toxic neuropathy, diabetic dementia, or autonomic neuropathy). In yet other embodiments, the cell is derived from bone marrow, peripheral blood, or umbilical cord blood, e.g., is an endothelial progenitor cell, a mesenchymal stem cell, or a mononuclear cell. In still other embodiments of the previous aspects, the administration increases neural tissue expression of a therapeutic polypeptide (e.g., neurotrophic factor, angiogenic factor, or cytokine). In still other embodiments, the therapeutic polypeptide is any one or more of the following: acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF -2, VEGF 165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I), Hypoxja inducible factor (HIF-I) and nitric oxide synthase. In yet other embodiments, the method reduces apoptosis in the neural tissue or increases proliferation in the neural tissue (e.g., Schwann cell or endothelial cell proliferation. In yet other embodiments, the cell is integrated into the microvasculature of the neural tissue. In yet other embodiments, the subject has or has a propensity to develop a diabetic neuropathy.

In various embodiments of any of the previous aspects, the cell is locally administered (e.g., is directly or indirectly administered, for example, by injection into a muscle cell, into the neural tissue, or into the circulation supplying the neural tissue). In other embodiments of the above aspects, the cell is integrated into the neural tissue. In still other embodiments, the cell is isolated and expanded in vitro to obtain a cell population enriched in bone marrow- derived stem or progenitor cells prior to being administered to the host subject. In still other embodiments, the cell is genetically modified. In still other embodiments, the cell is a stem cell isolated from bone marrow of a donor subject or isolated from peripheral blood of a donor subject, where the donor and the subject receiving the cell are the same or different individuals.

In various embodiments of any of the previous aspects, the cell is a human multipotent stem cell having reduced levels of a marker selected from the group consisting of: CD90, CDl 17, CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD 133, MHC class I receptor and MHC class II receptor. In other embodiments of any of the previous aspects, the cell expresses reduced (e.g., undetectable in a standard cell marker detection assay) levels of at least two, three, four, five, seven, eight, nine, ten, eleven, twelve, thirteen, or more markers (e.g., all markers).

In various embodiments of any of the previous aspects, the method further involves administering to the host subject a therapeutic polypeptide or a nucleic acid encoding a therapeutic polypeptide (e.g., a polypeptide supports endothelial or neuronal survival, function, or proliferation) that increases angiogenesis or vascularity in a neural tissue. In various embodiments of any of the above aspects the neural tissue includes a support cell (e.g., a cell that supports the neural tissue, such as a glial cell, a Schwann cell, or a cell of the vasculature supply the tissue). Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "a cell having the potential to differentiate into an endothelial cell" is meant any cell that can when cultured or implanted under suitable conditions give rise to cells having an endothelial cell phenotype, expressing one or more endothelial cell markers, or having an endothelial cell function.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By_ "angiogenesis" is meant.the growth of new blood vessels.- Such growth may originate from an existing blood vessel or by the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells. These stem cells can be recruited from bone marrow endogenously or implanted therapeutically. Methods for measuring angiogenesis are standard, and are described, for example, in Jain et al. (Nat. Rev. Cancer 2: 266-276, 2002). Angiogenesis can be assayed by measuring the number of non- branching blood vessel segments (number of segments per unit area), the functional vascular density (total length of perfused blood vessel per unit area), the vessel diameter, or the vessel volume density (total of calculated blood vessel volume based on length and diameter of each segment per unit area). Methods for measuring angiogenesis are standard in the art and are described, for example, in Jain et al., (Nat. Rev. Cancer 2: 266-276,2002). By "derived from" is meant the process of obtaining a progeny cell.

By "engraft" is meant the process of cellular contact and incorporation into an existing tissue of interest (e.g., a blood vessel or microvasculature) in vivo.

By "genetically modified" is meant comprising a heterologous polynucleotide, such as an expression vector. By "increase in angiogenesis" is meant a positive change in blood vessel formation as measured by standard assays such as those described herein. Desirably, an agent that modulates blood vessel formation will increase blood vessel formation (e. g., angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network) in a neural tissue or organ or microvascular scaffold.

By "increase" is meant any positive change. Exemplary increases include 5%, 10%, 20%, 25%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or even by as much as 100%, 150%, or 200% compared to a control.

By "integrated" is meant incorporated into a tissue (e.g., a neural tissue).

By "locally administered" is meant provided to a cell, extracellular space, tissue, organ, or circulatory vessel supplying such a cell, tissue, or organ, under conditions suitable to achieve a therapeutic effect. Typically, a cell of the invention that is "locally administered" is injected into a muscle tissue comprising a neuron under conditions that provide for an increase in angiogenesis or vascularity in the neuron.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "multipotent stem cell" is meant a cell having the potential to differentiate into one or more cell types (e.g., endothelial cells, smooth muscle cells, muscle cells).

By "neural tissue" is meant a cellular, extracellular, or molecular component of the nervous system including a support cell (e.g., any cell that supports the growth, proliferation, or survival of the tissue). The nervous system includes the central, peripheral, and autonomic nervous system. Exemplary support cells include cells of the vasculature or microvasculature, glial cells and Schwann cells.

By "neuropathy" is meant any pathology that disrupts neural function.

By "neuronal function" is meant any functions of the nervous system, e.g. neural signaling, neural conductance, sensorimotor function or cognitive function.

By "reference" is meant a standard or control condition. By "peripheral blood derived stem cell" is meant a multipotent cell obtained from peripheral blood.

By "positioned for expression" is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence which directs transcription and, for proteins, translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule).

By "potential to differentiate into an endothelial cell" is meant a cell having the ability to produce one or more endothelial cells under the appropriate in vitro or in vivo conditions. By "propensity" is meant at risk for developing pathology. Such risk can be genetic, environmental, or behavioral.

By "expansion" is meant the propagation of a cell or cells prior to or following terminal differentiation.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene or polypeptide.

By "isolation phenotype" is meant the structural and/or functional characteristics of a stem cell upon isolation. . . By "expansion phenotype" is meant the structural and/or functional characteristics of a stem cell during or following expansion. The expansion phenotype can be identical to the isolation phenotype, or alternatively, the expansion phenotype can be more differentiated than the isolation phenotype. In one embodiment, the expansion or isolation phenotype is characterized by an alteration in the expression of a marker. By "differentiation" is meant the developmental process of commitment to a particular cell fate. Differentiation to a particular cell fate typically includes the acquisition of characteristic markers, phenotypes, or functions (e.g., endothelial cell markers or functions).

By "isolated" is meant separated from the molecular and/or cellular components that naturally accompany the cell, polypeptide, or polynucleotide.

By "mesenchymal stem cell" is meant a cell derived from the mesodermal layer that is pluripotent and can develop into a connective or supporting tissue, smooth muscle, vascular endothelium, or blood cells. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. By "progenitor cell" is meant a multipotent stem cell that is capable of generating

(e.g., by differentiation or division) a differentiated cell. An endothelial progenitor cell that is capable of generating an endothelial cell may express this capability when grown under appropriate in vitro or in vivo conditions, such as those described herein.

By "progeny" is meant a cell derived from a multipotent stem cell of the invention. Progeny include without limitation progenitor cells, differentiated cells, and terminally differentiated cells.

By "tissue" is meant a collection of cells having a similar morphology and function. As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

By "therapeutic polypeptide" means a protein or analog thereof that has the potential of positively affecting the function of an organism. A therapeutic polypeptide may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease, disorder, or in an organism. In various embodiments, therapeutic polypeptides (e.g., angiogenic factors, neurotrophic factors, pleiotrophic factors) support neuronal or endothelial cell survival, growth, or proliferation.

By "therapeutically effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a neuropathy varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

By "agent" is meant a polypeptide, polynucleotide or small compound. Polypeptide agents include growth factors, cytokines, hormones or small molecules, or to genetically- encoded products that modulate cell function (e.g., induce cell fate, increase expansion, inhibit or promote cell growth and survival). For example, "expansion agents" are agents that increase proliferation and/or survival of stem cells. "Differentiation agents" are agents that induce differentiation into committed cell lineages. By "subject" is meant any warm-blooded animal including but not limited to a human, cow, horse, pig, sheep, goat, bird, mouse, rat, dog, cat, monkey, baboon, or the like.

By "proteomics, bioinformatics, or genomics" is meant a method that involves the use of a database comprising polypeptide, biological, or genetic information. Typically such methods involve the use of algorithms to identify polypeptides, polynucleotides or fragments thereof as of interest.

The term "obtaining" as in "obtaining the agent" is intended to include purchasing, synthesizing or otherwise acquiring the agent (or indicated substance or material). The terms "comprises", "comprising", and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean "includes", "including" and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA and IB show that diabetic neuropathy is characterized by decreased vascularity and can be reversed by VEGF. Figures 1 A-a to 1 A-f show representative fluorescence photomicrographs of longitudinal views of whole-mounted rat sciatic nerves (Figures lA-a-lA-c) and their respective cross sections (Figures lA-d- Figures lA-f) four weeks after treatment. Before sacrifice, in vivo perfusion with FITC-conjugated BS-I lectin, was performed. Figures lA-a and d show samples taken from a nondiabetic saline-injected control rat having a normal pattern of vascularity. Figures 1 A-b and 1 A-e show samples taken from a diabetic rat, 4 weeks after saline injection and having reduced vascularity. The total network of vasa nervorum is markedly reduced, resulting in an irregular distribution pattern and areas of nonvascularized nerve tissue. Figures lA-d, lA-e, and 1 A-f show stained endoneurial vessels in the cross-sectional image. Figures lA-c and 1 A-f show samples from a diabetic rat 4 weeks after VEGF-I gene transfer. Vascularity appears well preserved, and the number of visible vessels appears similar to that of a normal sciatic nerve. Figure IB shows in vivo LDPI of blood flow in rat sciatic nerve 4 weeks after gene transfer. Nerves were surgically exposed from the sciatic notch to the knee level before three repeated LDPI measurements were obtained from the region of interest. Figure lB-a is a bar graph that summarizes results of LDPI measurements taken from both sides of five rats per study group. *P < 0.01 versus nondiabetic saline-injected. #P < 0.01 vs diabetic saline-injected. Figures 1 A-b shows a representative color-coded LDPI. Lowest blood flow is indicated in at the left, maximum blood flow in at the right. Diabetic rats receiving VEGF gene transfer show substantial restoration of perfusion. Figures 2A and 2B show that Sonic hedgehog induces functional recovery in diabetic peripheral neuropathy (DPN). Figure 2 A shows representative fluorescent photomicrographs of BS-I lectin-perfused rat sciatic nerves (cross-section). SHh-treated rats showed increased vascularity. Figure 2B is a graph showing that the total number of epineurial/perineurial vessels was decreased in saline-treated diabetic rats; however, in SHh-treated diabetic rats, the number of vessels was similar to nondiabetic controls. *P<0.01 vs nondiabetic plus saline, #P<0.05 vs diabetic plus saline.

Figures3A and 3B are graphs showing that motor and sensory nerve conduction (MCV and SCV, respectively) is restored by SHh treatment. Before treatment (week 0), both MCV and SCV in diabetic rats (n=l 1) were significantly decreased compared with age- matched nondiabetic rats (n=22). Four weeks after treatment with SHh protein (n=13), both MCV and SCV were improved significantly.

Figure 4 is a series of six graphs showing that SHh induces in vivo expression of multiple angiogenic and neurotrophic cytokines: RT-PCR. Tissue samples were harvested 1 week after treatment and mRNA expression was examined in dorsal root ganglia [GIi-I (SHh receptor protein), BDNF, IGF-I: upper] and sciatic nerves (VEGF-I, Ang-1 and 2: lower). Expression of all factors was significantly reduced in the saline-treated diabetic rats. SHh induced expression of multiple angiogenic cytokines and neurotrophic factors, whereas VEGF-2 gene therapy had more limited effects. All experiments were repeated at least five times and results of 3 representative experiments are shown.

Figures 5A and 5B are graphs showing that mRNA expression of angiogenic and neurotrophic factors is reduced in sciatic nerves of diabetic rats examined by real-time RT- PCR. n = 5, each group.

Figures 6A-6C shows the quantification of endothelial progenitor cells (EPC). Figures 6A-a and 6A-b are photomicrographs showing the number of circulating EPCs, identified as double positive (light gray) by BS-I lectin (medium gray) and Dil-acLDL uptake (dark gray, right), was decreased at 12 months after DM compared to baseline (0 mo). Figure 6C is a graph showing that EPCs started to decrease between 2 and 4 months after the onset of diabetes mellitus (DM) and progressively decreased over 12 months, n = 6, each group. Each of the terms used in the figures has the following meaning: *P < 0.05; P < 0.001; DM, diabetes Figure 7 is a series of 11 graphs showing that the mRNA expression of paracrine factors is significantly increased in acutely infarcted myocardium in EPC transplanted rats compared to PBS or endothelial cell (Endo) injected rats.

Figure 8 is a schematic diagram showing the experimental design. Figures 9A-9C show that intramuscular transplantation of mononuclear cells (MNCs) improves histopathologic and functional abnormalities of DPN. Figure 9A shows representative fluorescence images of whole-mounted rat sciatic nerves following in vivo perfusion with FITC-conjugated BS-I lectin. At 4 weeks after MNC transplantation, these sections revealed notably increased vasa nervorum networks (left panel) and robust engraftment of Dil-labeled MNCs (middle and right panels). A nerve sample from a saline injected diabetic rat shows markedly decreased vascular networks. Bar, 500 μm. Figure 9B shows cross-sectional images of the above MNC transplanted nerve demonstrate that a portion of transplanted MNCs (red fluorescence) was colocalized with BS-I lectin positive cells (green fluorescence) indicative of MNC differentiation into endothelial cells. Figure 9C is a pair of graphs showing that before treatment (week 0), both MCV and SCV in diabetic (DM) rats were significantly decreased compared to those in nondiabetic rats. Four weeks later, MNC transplanted rats demonstrated significantly increased MCV and SCV compared with those injected with saline(n=9,eachgroup).

Figures 1OA and 1OB show the effect of endothelial progenitor cell transplantation on neural conduction velocity in mice with streptozotocin-induced diabetes. Figure 1OA is a time course showing motor conduction velocity in control and endothelial progenitor cell transplanted animals. Figure 1OB is a time course showing motor and sensory conduction velocity. The abbreviations used in the figures have the following meanings: NonDM, nondiabetes mellitus; EPC, endothelial progenitor cells; MSC mesenchymal stem cell. Figure 11 is a graph showing the results of a tail flick test in mice with streptozotocin induced diabetes mellitus or control mice.

Figures 12A and 12B show sciatic nerve blood flow in control and endothelial progenitor or mesenchymal stem cell transplanted mice assayed by laser Doppler perfusion imaging (LDPI). The panel on the left in each figure shows a representative LDPI image and the graph on the right shows a quantitation of the imaging results.

Figure 13 is a set of three fluorescent micrographs showing a comparison of sciatic nerve vascularity in control or experimental mice eight weeks after transplantation of endothelial progenitor cells. Figure 14 shows a comparison of sciatic nerve vascularity in control and experimental animals eight weeks after endothelial progenitor cell transplantation. The top panel provides a set of three photomicrographs showing endothelial vessels in cross-section The bottom panel is a graph quantitating vascularity. Figure 15 is a set of six photomicrographs showing engraftment of endothelial progenitor cells in sciatic nerves. Di-I labeled endothelial progenitor cells co-localized with lectin.

Figure 16 is a series of fluorescent micrographs showing the differentiation of endothelial progenitor cells into endothelial cells. Figure 17 shows that Schwann cell proliferation increased at four weeks following transplantation. The upper panel on the left shows Schwann cells in sciatic nerves from control mice and endothelial progenitor transplanted mice with streptozotocin-induced diabetes mellitus. The lower panel on the left shows BrdU positive Schwann cells double- stained with the nuclear marker DAPI. The graph on the right shows a quantitation of these assays.

Figure 18 shows that endothelial progenitor cell transplantation increased endothelial cell proliferation in sciatic nerve 4 weeks after transplantation. Cells were stained with BrdU, an antibody against ILB4, and Dapi. A merged image is shown on the far right.

Figure 19 shows that apoptosis decreased in the sciatic nerve 1 week after mice with streptozotocin induced diabetes mellitus were transplanted with endothelial progenitor cells. The abbreviations used in the figures have the following meanings: DM-S, saline-injected mice with diabetes mellitus; DM-EPC, endothelial progenitor cell transplants.

Figure 20 is a graph showing that cytokine levels are increased four weeks after EPC transplantation.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for the prevention or treatment of neuropathy. In one embodiment, the invention is particularly useful for the treatment of diabetic peripheral neuropathy (DPN).

Neuropathy

Neuropathies are pathologies that disrupt neural function. Exemplary neuropathies include but are not limited to diabetic neuropathy, ischemic neuropathy, toxic neuropathy, diabetic dementia. Symptoms of neuropathy vary depending on whether the affected nerves are sensory, motor, or autonomic. Neuropathy can affect any one or a combination of all three types of nerves. Typically, symptoms of neuropathy include pain, loss of sensation, or inability to control muscles. Methods of assaying neuropathy include electromyography, nerve conduction velocity tests, nerve biopsy, and other standard clinical assays for neurological function. Peripheral neuropathy is characterized by an abnormal neurological exam, subjective symptoms, abnormal biothesiometry, or abnormal nerve conduction study. Autonomic neuropathy is characterized by an abnormal R-R interval, orthostatic hypotension, or resting tachycardia.

Neuropathy may be caused by a hereditary disorder (e.g., Charcot-Marie-Tooth disease, Friedreich's ataxia), an infectious or inflammatory conditions (e.g., rheumatoid arthritis, lyme disease, AIDs), exposure to agents that are toxic to neurons (e.g., heavy metals, such as lead), or systemic or metabolic disorders, such as diabetes.

Diabetic peripheral neuropathy Diabetic peripheral neuropathy is pathogenetically associated with a marked reduction of the microvasculature (vasa nervorum). Impaired angiogenesis, in particular, attenuation of the vasa nervorum, has been noted in models of diabetes and is associated with diabetic neuropathy. Microvascular insufficiency and neurotrophic factor deficiency plays a role in the development and progression of diabetic peripheral neuropathy. As does ischemia -in diabetic nerves (Dyck PJ₅ 1989, Neurology; Steven EJ, 1994, Diabetologia), inactivation of proteins critical to neural function (Cullum NA, 1991, Diabetologia) and altered neural polyol metabolism (Greene DA, 1987, NEJM; Cameron NE, 1994, Diabetes Metab. Rev). Diabetic neuropathy can be reversed by agents promoting angiogenesis such as VEGF-I and -2 (Schratzberger et al., J Clin Invest. May 2001;107(9):1083-1092), sonic hedgehog (SHh) (Kusano et al., Arterioscler Thromb Vase Biol. Nov 2004;24(l l):2102-2107), and a statin (Ii et al., Circulation. July 5, 2005 2005;112(l):93-102). Furthermore, promising results from a pilot clinical trial (Simovic et al., Arch Neurol. May 2001;58(5):761-768; Isner et al., Hum Gene Ther. Aug 102001; 12(12): 1593-1594) using a plasmid encoding human VEGF (phVEGF) via gene therapy approach for patients with DPN also support the importance of vascular supply in the pathogenesis of DPN.

As reported in more detail below, the present invention provides compositions and methods for treating or preventing neuropathies, including diabetic neuropathy, by increasing angiogenesis in the microvasculature of nerves. The invention is based, in part, on the discovery that the local implantation of multipotent stem cells, such as endothelial progenitor cells, mesenchymal stem cells, and peripheral blood mononuclear cells, increases vascularity, conduction velocity, cytokine or therapeutic polypeptide expression, Schwann and endothelial cell proliferation, and decreases apoptosis in nerves affected by neuropathy.

Multipotent Stem Cells

Multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and their progenitor cells or progeny cells are isolated by standard means known in the art for the separation of stem cells from the peripheral blood or from the bone marrow.

In one embodiment, stem cells useful in the methods of the invention are obtained from the bone marrow of a human patient. Typically, the method includes at least one or more of the following steps: a) collecting bone marrow cells from a mammal (e.g., a young adult), where the cells have a size of less than about 100 microns, less than about 50 microns, or about 40 microns or less, b) culturing (expanding) the collected cells in medium under conditions that select for adherent cells, c) selecting the adherent cells and expanding those cells in medium to semi- confluency, d) serially diluting the cultured cells into chambers with conditioned medium, the dilution being sufficient to produce a density of less than about 1 cell per chamber to make clonal isolates of the expanded cells; and e) culturing (expanding) each of the clonal isolates and selecting chambers having expanded cells to make the population of isolated bone marrow cells. In another embodiment, cells having the potential to differentiate into endothelial cells (e.g., multipotent stem cells, endothelial progenitor cells, mononuclear cells, mesenchymal stem cells, and their progenitor or progeny cells) are obtained by extracting fresh unprocessed bone marrow cells from young donors. The cells are typically separated from blood cells by centrifugation, hemolysis and related standard procedures described herein. The bone marrow cells are washed in an acceptable buffer such as DPBS and filtered to collect cells having a size less that about 100 microns, less than about 50 microns, or about 40 microns. Methods for size selection are known in the art. In one embodiment, a standard nylon filter is used. Once isolated, cells of the selected size are grown on a complete culture medium with low glucose (e.g., DMEM) that contains a rich source of growth factors and cytokines. Fetal bovine serum (FBS) is typically used in the culture medium. Cells are cultured (i.e. expanded) for less than about two weeks, preferably about a week or less such as four to six days. The conditioned medium is then replaced with fresh medium; adherent cells are removed from the culture dishes and resuspended in fresh medium to select cells for expansion. The selected cells are grown to semiconfluency (between 50% to 90% confluent) and again, adherent cells are selected. Such cells are then reseeded in complete medium in a tissue culture flask at a density of about 10⁴ cells per centimeter. After the cells reach semiconfluency, they are reseeded (serially) into the flasks at the same or similar density. The cultures are preferably passaged more than one time, typically less than five times and preferably about two times to continue selection for expanding cells. Selected cells are then serially diluted into single well chambers (e.g., standard 96 well plate) at a density of less than about 1 cell per chamber, preferably ¹A a cell per chamber. Preferably, the cells are cultured with conditioned media to promote growth to sub confluence (i.e. less then 50% confluent). Wells with expanded cell clones are expanded and replated as needed.

If desired, cell clones are selected that fail to express detectable levels of at least one of the following markers: CD90, CDl 17, CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD133, MHC class I receptor, MHC class II receptor or other cell marker as described herein. Methods for performing the selection include any of the suitable assays disclosed herein. In embodiments in which larger amounts of cells are needed a more automated or semi-automated method will often be preferred such as fluorescence activated cell sorting (FACS). Selected cells desirably are able to be propagated in culture for long periods of time without becoming polyploidy or losing mulipotency.

Peripheral blood derived cells of the invention, their progenitors or their progeny, are obtained by methods known in the art, including methods for harvesting umbilical cord blood. In general, peripheral blood mononuclear cells (PBMCs) are taken from a patient using standard techniques. By "peripheral blood mononuclear cells" or "PBMCs" herein is meant lymphocytes (including T-cells, B-cells, NK cells), monocytes and stem cells. Prior to harvest, patients may be treated with agents known in the art to increase mobilization of stem cells from the bone marrow into the peripheral blood. Mobilizing agents include but are not limited to GCSF or GMCSF. In some embodiments of the invention, only PBMCs are taken, either leaving or returning red blood cells and polymorphonuclear leukocytes to the patient. This is done as is known in the art, for example using leukophoresis techniques. In general, a 5 to 7 liter leukophoresis step is done, which essentially removes PBMCs from a patient, returning the remaining blood components. Collection of the cell sample is preferably done in the presence of an anticoagulant such as heparin, as is known in the art.

Peripheral blood derived stem cells of the invention can, if needed, be purified from peripheral blood, including umbilical cord blood. Human umbilical cord blood ("cord blood") is a rich source of mesenchymal stem cells (MSCs). Methods of isolating such cells are known in the art. Briefly, a 1 ml portion of umbilical cord is placed in a well containing RPMI and 20% FBS. The matrix cells migrate out from the cord and adhere to the plastic well. Such cells have a fibroblast morphology. The supernatant and tissue are discarded after several days in culture. The cells remaining in the well are trypsinized and transferred to a secondary culture for expansion. See, for example, Connealey et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 9836-9841, September 1997; and Meagher and Klingemann et al., J Hematother Stem Cell Res. 2002 Jun;l l(3):445-8. J Hematother Stem Cell Res. 2002 Jun;l l(3):445-8. While particular examples are directed to bone marrow-derived cells, one skilled in the art appreciates that any hematopoietic stem cell may be used in the methods of the invention.

Therefore, cells that can be used in the methods of the invention can comprise a purified sub-population of cells including, but not limited to stem cells, or any cell having the ability to give rise to endothelial cells under suitable conditions in vitro or in vivo. "Suitable conditions" are empirically determined by culturing or implanting a cell of the invention then subsequently identifying endothelial cells in the culture or implant (e.g., cells having endothelial morphology, function, or expressing one or more endothelial cell markers). Purified cells can be collected and separated, for example, by flow cytometry. Peripheral blood derived cells of the invention can be autologous (obtained from the subject) or heterologous (e.g., obtained from a donor). Heterologous cells can be provided together with immunosuppressive therapies known in the art to prevent immune rejection of the cells.

Purified peripheral blood derived cells or their progenitors can be obtained by standard methods known in the art, including cell sorting by FACs. Isolated peripheral blood can be sorted using flow cytometers known in the art (e.g., a BD Biosciences FACScalibur cytometer) based on cell surface expression of Sca-1 (van de Rijn et al., (1989) Proc. Natl. Acad. Sci. USA 86, 4634-4638) and/or c-Kit (Okada et al., (1991) Blood 78, 1706-1712); (Okada et al., (1992) Blood 80, 3044-3050) following an initial immunomagnetic bead column-based fractionation step to obtain lineage-depleted (Kn^") cells (Spangrude et al., (1988) Science 241, 58-62); (Spangrude and Scollay, (1990) Exp. Hematol. 18, 920-926), as described (Shen et al., (2001) J. Immunol. 166, 5027-5033); (Calvi et al., (2003) Nature 425, 841-S46).

For serial passage-based enrichment of peripheral blood stem cells or their progenitors in-vitro (Meirelles and Nardi, (2003) Br. J. Haematol. 123, 702-711); (Tropel et al., (2004) Exp. Cell Res. 295, 395-406), isolated peripheral blood can be plated on plastic in Dulbecco's modified Eagle's medium (Fisher Scientific, Pittsburgh, PA) with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin, streptomycin, L-glutamine and amphotericin- B. About forty-eight hours after the initial plating, the supernatants containing non-adherent cells can be removed and replaced with fresh culture medium after gentle washing. The cultures can then be maintained and passed once confluence is reached (e.g., for a total of about three times over the span of about 6 weeks) at which time the cultures can be terminated to collect adherent cells for analysis.

In one embodiment, a method for isolating stem cells of the invention (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) includes generation of a fraction that comprises cells expressing reduced levels of any one or more of the following markers CD90, CDl 17, CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD133, MHC class I receptor and MHC class II receptor as determined by standard cell marker detection assay. Additional selection means based on the unique profile of gene expression can be employed to further purify populations of cells capable of generating an endothelial cell. Compositions comprising an endothelial progenitor cell can be isolated and subsequently purified to an extent where they become substantially free of the biological sample from which they were obtained.

Multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells and their progenitor cells or progeny can be obtained from bone marrow or peripheral blood and then expanded in culture. Thus, the progenitor cells can be cells having an "expansion phenotype" characterized by the reduced expression of any one or more of the following markers CD90, CDl 17, CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD133, MHC class I receptor and MHC class II receptor. Alternatively, a differentiated endothelial cell expresses one or more characteristic endothelial cell markers that provide for its identification, such markers include, but are not limited to, VE-Cadherin, CD34, FIk-I, Tie2 and CD31, VonWillebrand Factor or factor 8. Endothelial Progenitor Cells

Endothelial progenitor cells exist in peripheral blood and bone marrow, and contribute to postnatal vasculogenesis, i.e., the de novo development of vessels from stem or progenitor cells. Recently, the therapeutic potential of bone marrow derived stem or progenitor cells has been widely explored in various cardiovascular diseases. Collectively, studies have demonstrated that in both animal models and early cohorts of patients, stem/progenitor cell therapy is safe and feasible, and the clinical outcomes are promising. Mechanistically, in these animal models, differentiation of endothelial progenitor cells into vasculature (vasculogenesis) has been considered as the major therapeutic mechanism. Additionally, studies have demonstrated that paracrine effects of endothelial progenitor cells can play a crucial role for mediating therapeutic effects. Endothelial progenitor cells contain abundant and multiple cytokines, such as VEGF, IGF-I and bFGF that can function as angiogenic and neurotrophic factors. Also, endothelial progenitor cells are involved in disease pathogenesis. Decreased availability and impaired function of endothelial progenitor cells in diabetes may contribute to the development of diabetic complications including cardiomyopathy and peripheral vascular diseases, which are characterized by defective neovascularization.

Angiogenesis and vasculogenesis are responsible for the development of the vascular system in embryos. Vasculogenesis refers to the de novo development of blood vessels from endothelial progenitor cells (EPCs) or angioblasts that differentiate into endothelial cells (ECs). In contrast, angiogenesis refers to the formation of new vasculature from preexisting blood vessels through proliferation, migration, and remodeling of fully differentiated ECs. The long held belief that vasculogenesis occurs exclusively during development and that in the adult, new vessels are formed solely by angiogenesis, was dismantled by the finding that circulating EPCs, isolated from adult species, could differentiate along an EC lineage in vitro, providing evidence for the existence of postnatal vasculogenesis. Initially, FIk-I and CD34, shared by angioblasts and hematopoietic cells were used to isolate putative angioblasts from the mononuclear cell fraction of the peripheral blood (Asahara et al., Science. 1991 ;215:964-

967). Meanwhile, EPCs subsequently were isolated from umbilical cord blood, bone marrow

+ +

(BM), and CD34 or CDl 33 hematopoietic stem cells (Asahara et al., drc Res. Aug 6 1999;85(3):221-228 ; Murohara et al., Journal of Clinical Investigation. 2000; 105: 1527- 1536; Shi et al., Blood. 1998;92:362-367; Rafii et al., Journal of Clinical Investigation. 2000;105:17-19). These cells differentiate into endothelial cells, as shown by expression of various endothelial proteins (KDR, von Willebrand factor, endothelial nitric oxidase synthase (eNOS), VE-cadherin, CD 146), uptake of Dil-acetylated low-density lipoprotein (DiI- acLDL) and binding of lectin. In animal models of ischemia, heterologous, homologous, and autologous EPCs have been shown to incorporate into sites of active neovascularization in ischemic tissue (Shi et al., Blood. 1998;92:362-367Asahara et al., EMBOJ. 1999;18:3964- 3972Hatzopoulos et al., Development. 1998;125(8): 1457-1468; Niklason et al., Science. 1999;286:1493-1494; Rekhter et al., Circulation Research. 1998;83:705-713; Gerber et al., Development. 1999;126:1149-1159; Gunsilius et al., Lancet. 2000;355:1688-1691)

As reported in more detail below, therapeutic intervention by transplantation of a stem cell of the invention (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or their progenitors or progeny) reversed or attenuated diabetic neuropathy by inducing vasculogenesis and supplying angiogenic and neurotrophic cytokines.

Endothelial cell promoting conditions Once isolated, stem cell useful in the methods of the invention may be maintained indefinitely in culture. In one approach, isolated stem cells are expanded in vitro to increase the number of cells suitable for therapeutic administration (e.g., cells having the potential to differentiate into an endothelial cell. Alternatively or subsequently, a cell of the invention (e.g., multipotent stem cells, endothelial progenitor cells, mononuclear cells, mesenchymal stem cells, or their progenitor or progeny cells) is incubated under conditions that promote endothelial cell differentiation. Examples of endothelial cell promoting conditions are known in the art. See, for example, US Patent No. 5,980,887; PCT/US99/05130 (WO 99/45775) and references cited therein. In one embodiment, the stem cells of the invention are contacted with any one or more of the following factors that promote or support neural or endothelial growth, proliferation, or cell differentiation: acidic and basic fibroblast growth factors (aFGF (GenBank Accession No. NP J 49127) and bFGF (GenBank Accession No. AAA52448)), vascular endothelial growth factor (VEGF-I, (GenBank Accession No. AAA35789 or NPJ)01020539)), VEGF-2, VEGF 165, epidermal growth factor (EGF)(GenBank Accession No. NP_001954)), transforming growth factor α and β (TGF-α (GenBank Accession No. NP J303227) and TFG-β (GenBank Accession No. 1109243 A)), platelet-derived endothelial cell growth factor (PD-ECGF)(GenBank Accession No. NPJ)01944)), platelet-derived growth factor (PDGF) (GenBank Accession No. 1109245 A), tumor necrosis factor α (TNF- α) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF) (GenBank Accession No. BAA 14348), insulin like growth factor (IGF) (GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF) (GenBank Accession No. AAB59527),, Sonic hedgehog (SHh, GenBank Accession No. NPJ)OO 184), granulocyte/macrophage CSF (GM-CSF (GenBank Accession No. NP_000749)), angiopoetin-1 (Angl (GenBank Accession No. NPJ)Ol 137)), angiopoietin-2 (Ang-2, GenBank Accession No. NPJ)Ol 138), stromal cell derived factor (GenBank Accession No. NP_008854), hypoxia inducible factor (HIF-I (GenBank Accession No. NPJ)01521), and nitric oxide synthase (NOS) (GenBank Accession No. AAA36365); and functional fragments thereof. See, also Klagsbrun, et al., Annu. Rev. Physiol., 53:217- 239 (1991); Folkman, et si., J. Biol Chem., 267:10931-10934 (1992) and Symes, et al., Current Opinion in Lipidology, 5:305-312 (1994). Muteins or fragments of a such factors may be used as long as they induce or promote formation of endothelial cells. In one embodiment, an endothelial cell promoting condition includes contact with VEGF, particularly VEGF-I, VEGF-2, and or VEGFl 65. Additionally preferred endothelial cell promoting conditions include contact with certain cell matrix proteins, such as fibronectin. Preferred angiogenic factors and mitogens (and methods of use) are disclosed herein as well as US Pat No. 5,980,887 and WO 99/45775.

Endothelial cells can be contacted with such angiogenic factors or mitogens prior to, during or following transplantation. Methods for making and using EPCs have been disclosed. See U.S. Pat. No. 5,980,887, for example. Typical methods can include isolating the EPCs from the mammal and contacting the EPCs with at least one angiogenic factor and/or mitogen ex vivo.

Administration Compositions comprising a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) can be provided locally to a neural tissue of interest (e.g., a sensory or motor neuron). Alternatively, compositions comprising a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) can be provided indirectly to the neural tissue of interest, for example, by local administration into a muscle comprising the neuron or into the circulatory system supplying the neuron. Following transplantation or implantation, the cells may engraft and differentiate into endothelial cells. "Engraft" refers to the process of cellular contact and incorporation into an existing tissue of interest in vivo. Expansion and differentiation agents can be provided prior to, during or after administration to increase production of endothelial cells in vivo.

Compositions of the invention include pharmaceutical compositions comprising a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, a cell having the potential to differentiate into an endothelial cell can be obtained from one subject, and administered to the same subject or a different, compatible subject.

A cell having the potential to differentiate into an endothelial cell (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, intramuscular injection, intraneural injection or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Compositions of the invention can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof.

The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

A method to potentially increase cell survival when introducing the cells into a subject in need thereof is to incorporate multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and their progenitor cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) of interest into a biopolymer or synthetic polymer. Depending on the subject's condition, the site of injection might prove inhospitable for cell seeding and growth because of scarring or other impediments. Examples of biopolymer include, but are not limited to, cells mixed with fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This could be constructed with or without included expansion factors, differentiation factors, endothelial cell promoting factors, neurotrophic factors, or angiogenic factors. Additionally, these could be in suspension, but residence time at sites subjected to flow would be nominal. Another alternative is a three-dimensional gel with cells entrapped within the interstices of the cell biopolymer admixture. Again, expansion or differentiation factors could be included with the cells. These could be deployed by injection via various routes described herein.

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the cells having the potential to differentiate into an endothelial cell. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein. Additionally, these could be in suspension, but residence time at sites subjected to flow would be nominal. Another alternative is a three-dimensional gel with cells entrapped within the interstices of the cell biopolymer admixture. Again, expansion or differentiation factors could be included with the cells. These could be deployed by injection via various routes described herein.

Cells having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) can be cultured, treated with agents and/or administered in the presence of polymer scaffolds. Polymer scaffolds are designed to optimize gas, nutrient, and waste exchange by diffusion. Polymer scaffolds can comprise, for example, a porous, non-woven array of fibers. The polymer scaffold can be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to the cells. Taking these parameters into consideration, one of skill in the art could configure a polymer scaffold having sufficient surface area for the cells to be nourished by diffusion until new blood vessels interdigitate the implanted engineered-tissue using methods known in the art. Polymer scaffolds can comprise a fibrillar structure. The fibers can be round, scalloped, flattened, star-shaped, solitary or entwined with other fibers. Branching fibers can be used, increasing surface area proportionately to volume.

Unless otherwise specified, the term "polymer" includes polymers and monomers that can be polymerized or adhered to form an integral unit. The polymer can be nonbiodegradable or biodegradable, typically via hydrolysis or enzymatic cleavage. The term "biodegradable" refers to materials that are bioresorbable and/or degrade and/or break down by mechanical degradation upon interaction with a physiological environment into components that are metabolizable or excretable, over a period of time from minutes to three years, preferably less than one year, while maintaining the requisite structural integrity. As used in reference to polymers, the term "degrade" refers to cleavage of the polymer chain, such that the molecular weight stays approximately constant at the oligomer level and particles of polymer remain following degradation.

Materials suitable for polymer scaffold fabrication include polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid- polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, and shape memory materials, such as poly(styrene-Wocλ:-butadiene), polynorbornene, hydrogels, metallic alloys, and oligo(ε-caprolactone)diol as switching segment/oligo(p-dioxyanone)diol as physical crosslink. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N. Y., 1989).

Factors, including but not limited to nutrients, growth factors, inducers of differentiation or de-differentiation, products of secretion, immunomodulators, cytokines, neurotrophic factors, angiogenic factors, inhibitors of inflammation, regression factors, hormones, or other biologically active compounds can be incorporated into or can be provided in conjunction with the polymer scaffold.

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and their progenitor cells as described in the present invention. One consideration concerning the therapeutic use of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and their progenitor cells of the invention is the quantity of cells necessary to achieve an optimal effect. In current human studies of autologous mononuclear peripheral blood cells, empirical doses ranging from 1 to 4 x 10⁷ cells have been used with encouraging results. The methods of the invention may require optimization of the amount of cells injected into a tissue of interest. Thus, the quantity of cells to be administered will vary depending on the neural tissue or the subject being treated. In one embodiment, between 10⁴ to 10⁸, 10⁶ to 10⁸, or 10⁵ to 10⁹ cells are implanted. In other embodiments, 10⁵ to 10⁷ cells are implanted. In still other embodiments, 3 x 10⁷ stem cells of the invention can be administered to a human subject. The precise determination of an effective dose may be based on factors individual to each patient, including their size, age, sex, weight, and condition. Therefore, dosages are determined empirically using no more than routine by those skilled in the art from this disclosure and the knowledge in the art. Multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells of the invention can comprise a purified population of stem cells having the potential to differentiate into an endothelial cell. Those skilled in the art can readily determine the percentage of such cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Desirable ranges of purity in mixed populations comprising multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, or progenitor cells of the invention cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More desirably, the purity is about 70 to about 75%, about 75 to about 80%, about 80 to about 85%; and still more desirably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Purity of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells of the invention can be determined according to the marker profile within a population. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).

The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions and to be administered in methods of the invention. Typically, any additives (in addition to the active stem cell(s) and/or agent(s)) are present in an amount of 0.001 to 50 % (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and still more preferably about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD₅₀ in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.

The number of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells of the invention can be increased by increasing the survival or proliferation of existing stem cells, or their progenitor cells.

Agents (e.g., expansion agents) which increase proliferation or survival of a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) include, but are not limited to, a hormone, growth factor, an Akt polypeptide, IGF-I, or telomerase.

Agents comprising growth factors are known in the art to increase proliferation or survival of stem cells. For example, U.S. Patent Nos. 5,750,376 and 5,851,832 describe methods for the in vitro culture and proliferation of stem cells using TGF. An active role in the expansion and proliferation of stem cells has also been described for BMPs (Zhu, G. et al, (1999) Dev. Biol. 215: 118-29 and Kawase, E. et al, (2001) Development 131: 1365) and Wnt proteins (Pazianos, G. et al, (2003) Biotechniques 35: 1240 and Constantinescu, S. (2003) J. Cell MoI. Med. 7: 103). U.S. Patent Nos. 5,453,357 and 5,851,832 describe proliferative stem cell culture systems that utilize FGFs. The contents of each of these references are specifically incorporated herein by reference for their description of expansion agents known in the art.

Agents comprising growth factors are also known in the art to increase mobilization of stem cells from the bone marrow into the peripheral blood. Mobilizing agents include but are not limited to GCSF or GMCSF. An agent that increases mobilization of stem cells into the blood can be provided before peripheral blood harvest or alternatively, to augment or supplement other methods of the invention where it would be desirable to increase circulating levels of stem cells (e.g., to increase targeting of the cells to the neural tissue). Agents comprising cell-signaling molecules are also known in the art to increase proliferation or survival of stem cells. For example, U.S. Patent Application No. 20030113913 describes the use of retinoic acid in stem cell self renewal in culture. The contents of each of these references are specifically incorporated herein by reference for their description of expansion agents known in the art. Agents comprising pharmacological or pharmaceutical compounds are also known in the art to increase production or survival of stem cells.

Agents comprising signaling molecules are also known to induce differentiation of endothelial cells. The contents of each of these references are specifically incorporated herein by reference for their description of differentiation agents known in the art.

Agents comprising pharmacological or pharmaceutical compounds are also known in the art to induce differentiation of stem cells.

Agents can be provided directly to a neural tissue of interest (e.g., motor neuron, sensory neuron). Alternatively, agents can be provided indirectly to the neural tissue of interest, for example, by local administration into the circulatory system or into the muscle that comprises the neural tissue.

Agents can be administered to subjects in need thereof by a variety of administration routes. Methods of administration, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, intraocular, buccal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, e.g., fibers such as collagen, osmotic pumps, or grafts comprising appropriately transformed cells, etc., or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic proteins. Other useful approaches are described in Otto, D. et al., J. Neurosci. Res. 22: 83 and in Otto, D. and Unsicker, K. J. Neurosci. 10: 1912. In vitro and ex vivo applications can involve culture of the multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells with the selected agent to achieve the desired result. For example, cultures of cells (from the same individual and from different individuals) can be treated with expansion agents to increase the number of cells of interest. Alternatively, the cultures are treated with differentiation agents of interest to stimulate the production of cells having the desired characteristics. Cells produced by these methods can then be used for a variety of therapeutic applications (e.g., localized implantation).

Multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells derived from cultures of the invention can be implanted into a host. The transplantation can be autologous, such that the donor of the stem cells is also the recipient of the stem cells. The transplantation can be heterologous, such that the donor of the stem cells is not the recipient of the stem cells. In one embodiment, once transferred into a host, the cells engraft in the micro vasculature of the host neural tissue. Agents of the invention may be supplied along with additional reagents in a kit. The kits can include instructions for the treatment regime or assay, reagents, equipment (test tubes, reaction vessels, needles, syringes, etc.) and standards for calibrating or conducting the treatment or assay. The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert. Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if whether a consistent result is achieved.

Methods of Treatment and Prophylaxis Cells having the potential to differentiate into endothelial cells (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) can be used in a variety of therapeutic or prophylactic applications. Accordingly, methods of the invention relate to, among other things, the use of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells,, mononuclear cells, or progenitors or progeny thereof for the treatment or prevention of neuropathy, particularly diabetic peripheral neuropathy. The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a neuropathy disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the neuropathy or symptom thereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, en2yme or protein marker, assay of neurological function, family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which neurological function or a deficit in neuronal angiogenesis or vascularity may be implicated.

In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., neurological screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a neuropathy, or a deficit in neural angiogenesis, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

In one embodiment, the present invention provides methods for treating neuropathy comprising providing a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) to a subject in need thereof, wherein the cell engrafts into a tissue (e.g., a muscle tissue, neural tissue) and releases cytokines that enhance angiogenesis in a neural tissue of interest. In one embodiment, the present invention provides methods for treating neuropathy comprising providing a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) to a subject in need thereof, wherein the cell engrafts into the microvasculature of a neural tissue of interest and increases angiogenesis, vascularity, or the biological function of the neural tissue. In yet another embodiment, the method provides a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) to a subject in need thereof, wherein the cell augments an inflammatory response in the neural tissue sufficient to exert a therapeutic effect (e.g., an increase in paracrine factors, neurotrophic factors, angiogenic factors, or an increase in angiogenesis).

The present invention also provides methods for restoring neural function in a diabetic subject having a loss of neural function (e.g., motor or sensory deficit), comprising providing a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) to the subject to enhance neural function. Peripheral blood derived stem cells

Peripheral blood derived stem cells of the invention, their progenitors or their in vitro- derived progeny, can be administered as previously described, and obtained by all methods known in the art. In general, the sample comprising the PBMCs can be pretreated in a wide variety of ways. Generally, once collected, the cells can be additionally concentrated, if this was not done simultaneously with collection or to further purify and/or concentrate the cells. The cells may be washed, counted, and resuspended in buffer transferred to a sterile, closed system for further purification and activation. The PBMCs are generally concentrated for treatment, using standard techniques in the art in a preferred embodiment, the leukophoresis collection step results in a concentrated sample of PBMCs, in a sterile leukopak, that may contain reagents or doses of a suppressive composition,. Generally, an additional concentration/purification step is done, such as Ficoll- Hypaque density gradient centrifugation as is known in the art. Separation or concentration procedures include but are not limited to magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents, either joined to a monoclonal antibody or used with complement, "panning", which uses a monoclonal antibody a to a solid matrix. Antibodies attached to solid matrices, such as magnetic beads, agarose beads, polystyrene beads, follow fiber membranes and plastic surfaces, allow for direct separation. Cells bound by, antibody can be removed or concentration by physically separating the solid support from the cell suspension. The exact conditions a and procedure depend on factors specific to the system employed. The selection of appropriate conditions is well within the skill in the art. Antibodies may be conjugated to biotin, which then can be removed with avidin or streptavidin bound to a support, or fluorochromes, which can be used with a fluorescence activated cell sorter (FACS), to enable cell separation. Any technique may be employed as long as it is not detrimental to the viability of the desired cells.

In a preferred embodiment, the PBMCs are separated in a automated, closed system such as the Nexell Isolex 30Oi Magnetic Cell Selection System. Generally, this is done to maintain sterility and to insure standardization of the methodology used for cell separation, activation and development of suppressor cell function.

Once purified or concentrated the cells may be aliquoted and frozen, preferably, in liquid nitrogen or used immediately as described below. Frozen cells may be thawed and used as needed. Cryoprotective agents, which can be used, include but are not limited to dimethyl sulfoxide (DMSO) (Lovelock, J. E. and Bishop, M. W. H., 1959, Nature 183:1394- 1395; Ashwood-Smith, M. J., 1961, Nature 190:1204-1205), hetastarch, glycerol, polyvinylpyrrolidine (Rinfret, A. P., 1960, Ann. N. Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter, H. A. and Ravdin, R. G., 1962, Nature 196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe, A. W., et al, 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender, M. A., et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and Bender, M. A, 1960, Exp. Cell Res. 20:851), methanol, acetamide, glycerol monoacetate (Lovelock. J. E., 1954, Biochem. J. 56:265), and inorganic salts (Phan The Tran and Bender, M. A., 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, M. A., 1961, in Radiobiology Proceedings of the Third Australian Conference on Radiobiology, Ilbery, P. L. T., ed., Butterworth, London, p. 59). Typically, the cells may be stored in 10% DMSO, 50% serum, and 40% RPMI 1640 medium. Methods of cell separation and purification are found in U.S. Pat. No. 5,888,499, which is expressly incorporated by reference. In a preferred embodiment, the PBMCs are then washed to remove serum proteins and soluble blood components, such as autoantibodies, inhibitors, etc., using techniques well known in the art Generally, this involves addition of physiological media or buffer, followed by centrifugation. This may be repeated as necessary. They can be resuspended in physiological media, preferably AIM-V serum free medium (Life Technologies) (since serum contains significant amounts of inhibitors of TGF-β) although buffers such as Hanks balancec salt solution (HBBS) or physiological buffered saline (PBS) can also be used.

Generally, the cells are then counted; in general from 1 X 10⁹ to 2 XlO⁹ white blood cells are collected from a 5-7 liter leukophoresis step. These cells are brought up roughly 200 mis of buffer or media.

Compositions comprising peripheral blood derived stem cells or their progenitors can be provided directly to a neural tissue of interest. Alternatively, compositions comprising stem cells or their progenitors can be provided indirectly to the neural tissue of interest, for example, by administration into the circulatory system or injection into a muscle comprising the neural tissue.

Genetically modified stem cells

Prior to administration, multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, their progenitors or their progeny, described herein can optionally be genetically modified, in vitro, in vivo or ex vivo, by introducing heterologous DNA or RNA or protein into the cell by a variety of recombinant methods known to those of skill in the art. These methods are generally grouped into four major categories: (1) viral transfer, including the use of DNA or RNA viral vectors, such as retroviruses (including lentiviruses), Simian virus 40 (SV40), adeno-associated virus, adenovirus, Sindbis virus_vand bovine papillomavirus, for example; (2) chemical transfer, including calcium phosphate transfection and DEAE dextran transfection methods; (3) membrane fusion transfer, using DNA-loaded membranous vesicles such as liposomes, red blood cell ghosts, and protoplasts, for example; and (4) physical transfer techniques, such as microinjection, electroporation, or direct "naked" DNA transfer. The stem cells of the invention (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells), their progenitors or their in progeny, can be genetically altered by insertion of pre-selected isolated DNA, by substitution of a segment of the cellular genome with pre-selected isolated DNA, or by deletion of or inactivation of at least a portion of the cellular genome of the cell. Deletion or inactivation of at least a portion of the cellular genome can be accomplished by a variety of means, including but not limited to genetic recombination, by antisense technology (which can include the use of peptide nucleic acids, or PNAs), or by ribozyme technology, for example. The altered genome may contain the genetic sequence of a selectable or screenable marker gene that is expressed so that the cell with altered genome, or its progeny, can be differentiated from cells having an unaltered genome. For example, the marker may be a green, red, yellow fluorescent protein, β- galactosidase, the neomycin resistance gene, A genetically altered stem cell, or its progeny, may contain DNA encoding a therapeutic protein (e.g., a protein that increases angiogenesis, increases endothelial cell or Schwann cell proliferation, or decreases apoptosis) under the control of a promoter that directs strong expression of the recombinant protein.

Alternatively, the cell may express a gene that can be regulated by an inducible promoter or other control mechanism where conditions necessitate highly controlled regulation or timing of the expression of a protein, en2yme, or other cell product. Such stem cells, when transplanted into a subject suffering from neuropathy characterized by a decrease in neural vascularity, produce high levels of the protein to confer a therapeutic benefit.

Proteins expressed in genetically modified cells include any protein capable of supporting or enhancing neural function or angiogenesis. Such proteins include angiogenic cytokines and neurotrophic factors. In one embodiment, the stem cell of the invention, its progenitor or its progeny, express heterologous DNA encoding an polypeptide or fragment thereof that encodes a therapeutic polypeptide (e.g., acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF-2, VEGF 165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet- derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I) Hypoxia inducible factor (HIF-I) and nitric oxide synthase). Insertion of one or more pre-selected DNA sequences can be accomplished by homologous recombination or by viral integration into the host cell genome. The desired gene sequence can also be incorporated into the cell, particularly into its nucleus, using a plasmid expression vector and a nuclear localization sequence. Methods for directing polynucleotides to the nucleus have been described in the art. Calcium phosphate transfection can be used to introduce plasmid DNA containing a target gene or polynucleotide into isolated or cultured stem cells or their progenitors and is a standard method of DNA transfer to those of skill in the art. DEAE- dextran transfection, which is also known to those of skill in the art, may be preferred over calcium phosphate transfection where transient transfection is desired, as it is often more efficient. Since the cells of the present invention are isolated cells, microinjection can be particularly effective for transferring genetic material into the cells. This method is advantageous because it provides delivery of the desired genetic material directly to the nucleus, avoiding both cytoplasmic and lysosomal degradation of the injected polynucleotide This technique has been used effectively to accomplish peripheral blood derived modificatioi in transgenic animals. Cells of the present invention can also be genetically modified using electroporation. Liposomal delivery of DNA or KNA to genetically modify the cells can be performed using cationic liposomes, which form a stable complex with the polynucleotide. For stabilization of the liposome complex, dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPQ) can be added. Commercially available reagents for liposomal transfer include Lipofectin (Life Technologies). Lipofectin, for example, is a mixture of the cationic lipid N-[l-(2, 3-dioleyloxy)propyl]-N-N-N- trimethyl ammonia chloride and DOPE. Liposomes can carry larger pieces of DNA, can generally protect the polynucleotide from degradation, and can be targeted to specific cells or tissues. Cationic lipid- mediated gene transfer efficiency can be enhanced by incorporating purified viral or cellular envelope components, such as the purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G). Gene transfer techniques which have been shown effective for delivery of DNA into primary and established mammalian cell lines using lipopolyamine- coated DNA can be used to introduce target DNA into the stem cells described herein.

Naked plasmid DNA can be injected directly into a tissue mass foπned of cells from the isolated peripheral blood or their progenitors. This technique has been shown to be effective in transferring plasmid DNA to skeletal muscle tissue, where expression in mouse skeletal muscle has been observed for more than 19 months following a single intramuscular injection. More rapidly dividing cells take up naked plasmid DNA more efficiently. Therefore, it is advantageous to stimulate cell division prior to treatment with plasmid DNA. Microprojectile gene transfer can also be used to transfer genes into stem cells either in vitro or in vivo. The basic procedure for microprojectile gene transfer was described by J. Wolff in Gene Therapeutics (1994), page 195. Similarly, microparticle injection techniques have been described previously, and methods are known to those of skill in the art. Signal peptides can be also attached to plasmid DNA to direct the DNA to the nucleus for more efficient expression. Viral vectors are used to genetically alter stem cells of the present invention and their progeny. Viral vectors are used, as are the physical methods previously described, to deliver one or more target genes, polynucleotides, antisense molecules, or ribozyme sequences, for example, into the cells. Viral vectors and methods for using them to deliver DNA to cells are well known to those of skill in the art. Examples of viral vectors that can be used to genetically alter the cells of the present invention include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), alphaviral vectors (e. g., Sindbis vectors), and herpes virus vectors.

Endothelial cell engraftment

As described in more detail below, the present invention provides methods for preventing or treating neuropathy by locally administering a cell having the potential to differentiate into an endothelial cell (e.g., multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof) directly or indirectly to a neural tissue. In one embodiment, this administration results in the incorporation of the cell into a neural tissue (e.g., a tissue comprising neural cells, extracellular matrix, and supporting cells, such as glial cells that ensheath the neuron, and cells of the vasculature that supply the neural tissue). Without wishing to be bound by theory, it is likely that cells of the invention home to neural tissue that lacks sufficient vascularity and are incorporated into that tissue. In one embodiment, the cells differentiate into mature endothelial cells and contribute to the microvasculature of the neural tissue. Methods for detecting differentiated endothelial cells are known in the art, see, for example, US Pat. No. 5,980,887 and WO 99/45775, which describe methods for detecting and monitoring endothelial cell function. A preferred assay involves detection of EC specific markers (e.g., VE-Cadherin, CD34, FIk-I, Tie2 and CD31, VonWillebrand Factor or factor 8).

Screening Assays

The invention provides methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, polynucleotides, small molecules or other drugs) that are useful for the treatment of neuropathy. Agents thus identified can be used to increase, for example, proliferation, survival, engraftment, or differentiation of a stem cell or its progenitor e.g., in a therapeutic protocol. In one embodiment, the agent modulates a cell of the invention thereby enhancing angiogenesis in a neural tissue of interest.

The test agents of the present invention can be obtained singly or using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. (1994) et al., J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sd. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engϊ. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992), Biotechniques I3_:412-421), or on beads (Lam (1991), Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865- 1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci 87:6378-6382; Felici (1991) J. MoI. Biol. 222:301-310; Ladner supra.).

Chemical compounds to be used as test agents (i.e., potential inhibitor, antagonist, agonist) can be obtained from commercial sources or can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock (1989) Comprehensive Organic Transformations, VCH Publishers; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Combinations of substituents and variables in compounds envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., transport, storage, assaying, therapeutic administration to a subject).

The compounds described herein can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. AU such isomeric forms of these compounds are expressly included in the present invention. The compounds described herein can also be represented in multiple tautomeric forms, all of which are included herein. The compounds can also occur in cis-or trans-or E-or Z-double bond isomeric forms. AU such isomeric forms of such compounds are expressly included in the present invention.

Test agents of the invention can also be peptides (e.g., growth factors, cytokines, receptor ligants) or polynucleotides encoding such peptides.

Screening methods of the invention identify agents that enhance or inhibit a biological activity of a cell of the invention. In one embodiment, a cell of the invention (e.g., multipotent stem cell, endothelial progenitor cell, mesenchymal stem cell, or other progenitor cell) is contacted with the agent prior to implantation in a host. In another embodiment, an agent is administered in combination with a cell of the invention. Desirably, the agent increases angiogenesis in a neural tissue of interest, increases Schwann cell proliferation, increases endothelial cell proliferation, increases neural conductance, increases pain- responsiveness, decreases apoptosis, or is otherwise useful for the treatment of a diabetic neuropathy.

In one embodiment, the cell is contacted with an agent that is an inhibitory nucleic acid molecule that decreases the expression of a target gene or polypeptide. The cell is subsequently implanted in a host. The level of angiogenesis, therapeutic polypeptide expression, or other clinical indicator of neuropathy is then measured. Inhibitory nucleic acid molecule that produce a decrease in the level of angiogenesis, therapeutic polypeptide expression or other indicator of neuropathy are selected. The gene or polypeptide targets of these inhibitory nucleic acid molecules are identified as useful in the methods of the invention. Overexpression of such genes or polypeptides is useful for the treatment of neuropathy.

In one embodiment, a cell of the invention is contacted with the agent in vitro prior to implantation in a host. The treated cell is then locally delivered to a neural tissue of interest. The biological function or vascularity of the neural tissue is compared between a host that received the treated cell relative to a host that received an untreated control cell. An increase in the biological function or vascularity of the neural tissue that received the treated cell identifies the agent as useful in the methods of the invention.

In another embodiment, an agent is locally administered to a neural tissue of interest in combination (e.g., prior to, during, or following) implantation of a cell of the invention. The biological function or vascularity of the neural tissue contacted with the agent is compared to the biological function of in a control host that did not receive the agent. An increase in the biological function or vascularity of the neural tissue contacted with the combination identifies the agent as useful in the methods of the invention.

In practicing the methods of the invention, it may be desirable to employ a purified population of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells. A purified population of multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, and progenitor cells has about 50-55%, 55-60%, 60-65% and 65-70% purity. In other embodiments, the purity is about 70-75%, 75-80%, 80-85%; and in still other embodiments the purity is about 85-90%, 90-95%, and 95-100%. Agents useful in the methods of the invention can also be detected by identifying an increase in expression of a cytokine or other desirable marker. The level of expression can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the genetic markers; measuring the amount of protein encoded by the genetic markers; or measuring the activity of the protein encoded by the genetic markers. The level of mRNA corresponding to a marker can be determined both by in situ and by in vitro formats. The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers described herein. The level of mRNA in a sample can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker being analyzed.

Assays for Angiogenesis, Proliferation, and Apoptosis The invention provides methods for increasing angiogenesis in a neural tissue of interest, increasing Schwann cell proliferation, increasing endothelial cell proliferation, increasing neural conductance, increasing pain responsiveness, decreasing apoptosis, or is otherwise useful for the treatment of a diabetic neuropathy. Methods for measuring an increase in angiogenesis are also known in the art and are described herein. In general, angiogenesis can be assayed by measuring the number of non- branching blood vessel segments (number of segments per unit area), the functional vascular density (total length of perfused blood vessel per unit area), the vessel diameter, or the vessel volume density (total of calculated blood vessel volume based on length and diameter of each segment per unit area). Angiogenesis can also be quantitated using endothelial cell markers. For example, angiogenesis can be assayed in a neural tissue using immunohistochemical staining with antibodies prepared against a specific endothelial cell marker isolectin B4 (Vector Laboratories). Capillary density is evaluated morphometrically by histological examination of randomly selected fields of tissue sections. Capillaries are recognized as tubular structures positive for isolectin. Such methods are described, for example, by Iwakura et al., Circulation 2003; 108: 3115-21.

Methods of assaying cell growth and proliferation are known in the art. See, for example, Kittler et al. (Nature. 432 (7020): 1036-40, 2004) and Miyamoto et al. (Nature 416(6883):865-9, 2002). Assays for cell proliferation generally involve the measurement of DNA synthesis during cell replication. In one embodiment, DNA synthesis is detected using labeled DNA precursors, such as ([3H]-Thymidine or 5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals) and then the incorporation of these precursors into genomic DNA during the S phase of the cell cycle (replication) is detected (Ruefii-Brasse et al., Science 302(5650):1581-4, 2003; Gu et al., Science 302 (5644):445-9, 2003). Assays for measuring cell survival are known in the art, and are described, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol.62, 338- 43, 1984); Lundin et al., (Meth. Enzymol.133, 27-42, 1986); Petty et al. (Comparison of J. Biolum. Chemilum.lO, 29-34, 1995); and Cree et al. (Anticancer Drugs 6: 398-404, 1995). Cell viability can be assayed using a variety of methods, including MTT (3-(4,5- dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop, Bioorg. & Med. Chem.

Lett.l: 611, 1991; Cory et al., Cancer Comm. 3, 207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays for cell viability are also available commercially. These assays include but are not limited to CELLTITER-GLO® Luminescent Cell Viability Assay (Promega), which uses luciferase technology to detect ATP and quantify the health or numbe of cells in culture, and the CellTiter-Glo® Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH) cytotoxicity assay (Promega).

Combination Therapies

Compositions and methods of the invention may be used in combination with any conventional therapy for neuropathy known in the art or in combination with any therapy known to increase angiogenesis. In one embodiment, a multipotent stem cell may be used in combination with any pro-angiogenic therapy known in the art.

EXAMPLES Example 1: Diabetic neuropathy is characterized by decreased vascularity and can bt reversed by VEGF gene

Experimentally-induced diabetic neuropathy was characterized by destruction of vasa nervorum Figures 1A-1B. This destruction was reversed by administration of an angiogenic growth factor. Diabetes was induced by intraperiotoneal injection of streptozotocin. Rats were included in the study protocol 12 weeks after induction of diabetes, at which time rats develop peripheral neuropathy. Age- and weight-matched rats were used as non-diabetic control animals. Diabetic rats were randomly assigned to three groups; one receiving saline and the other two groups receiving local intramuscular gene transfer of phVEGF-1 or- 2. Four weeks later, nerve vascularity, nerve blood flow and nerve conduction velocities were measured. In saline injected rats, nerve perfusion, as measured by laser Doppler imaging, and nerve vascularity (vasa nervorum), as detected by a locally administered fluorescent lectin (an endothelial specific ligand) analogue (BS-I lectin) (Figures 2A and 2B) was markedly attenuated, consistent with a profound reduction in the number of vessels observed. In parallel, a peripheral neuropathy developed characterized by slowing of motor (MCV) and sensory nerve conduction velocities (SCV). In contrast, vascularity and blood flow in the nerves of diabetic animals treated with VEGFs was similar to those of nondiabetic controls; constitutive overexpression of both transgenes resulted in restoration of large and small fiber peripheral nerve function demonstrated by increase of MCV and SCV to the level of nondiabetic controls.

Example 2: Sonic hedgehog induces functional recovery in DPN

In this study, the embryonic morphogen sonic hedgehog (SHIi) (PoIa et al., Nature Medicine. 2001 ;7:706-711), which has been shown to augment neovascularization of ischemic tissue, induced arteriogenesis in vasa nervorum and improved nerve function in DPN. Human SHh proteins were used to construct SHh-rat IgG fusion proteins to increase the half-life as described (Calcutt et al., J. Clin. Invest. February 15, 2003 2003;l 11(4):507- 514). Subcutaneous injection of SHh fusion proteins (1.0mg/kg) or saline was started 12 weeks after the induction of diabetes three times per week for four weeks. As shown in Figure, SHh treatment replenished vascular supply to sciatic nerves and also restored nerve function as measured by MCV and SCV. Without wishing to be bound by theory, this therapeutic effect of SHh is considered to be mediated through increase in expression of multiple angiogenic and neurotrophic cytokines. In fact, semiquantitative RT-PCR analysis revealed that SHh treatment results in a significant increase in the expression of mRNA of both angiogenic cytokines (VEGF-I, Angiopoietin-land -2 (Ang-1 and -2)) and neurotrophic factors (brain-derived neurotrophic factor (BDNF), IGF-I), which are downregulated in diabetic nerves (Figure 4).

Example 3: Statin improves DPN through restoration of vasa nervorum and direci neurotrophic effects (Ii et al., Circulation. July 5, 2005 2005;112(l):93-102).

In this study, rosuvastatin, a new HMG-CoA reductase inhibitor, restored nerve vascularity, including vessel size, and nerve function to the levels of nondiabetics in a db/db mouse model of diabetes. The effect of rosuvastatin on vasa nervorum is likely to be mediated via an NO-dependent pathway. Coadministration of a nitric oxide synthase inhibitor with rosuvastatin attenuated the beneficial effects of rosuvastatin on nerve function and limited the recovery of vasa nervorum and nerve function. Rosuvastatin also showed a direct neurotrophic effect. Neuronal nitric oxide synthase expression in sciatic nerves, which was reduced in diabetic mice, was preserved by rosuvastatin treatment. In vitro, rosuvastatin inhibited downregulation of neuronal nitric oxide synthase expression induced by hyperglycemic conditions in cultured Schwann cells. Collectively, restoration of vasa nervorum accompanied by functional recovery in diabetic animal models with the use of 3 distinct angiogenic agents; VEGFs, SHh, and rosuvastatin was identified. These data indicati the importance of angiogenesis and vascular integrity in the development and reversal of DPN.

Example 4: Diabetic neuropathy is characterized by decreased expression of multiple angiogenic and neurotrophic cytokines.

Diabetic complications including DPN is characterized by defective angiogenesis and reduced expression of VEGF. The angiogenic process involves multiple pro- and anti- angiogenic factors and their corresponding receptors including VEGFs, basic fibroblast growth, factor (bFGF), hepatocyte growth factor (HGF), placental growth factor (PLGF), Ang-land -2 (Ang-2), platelet-derived growth factor (PDGF)-BB, transforming growth facto (TGF)-βl, eNOS and VEGF receptor (VEGFR)-1, 2,3. Also factors like VEGF, bFGF and IGF-I possess both angiogenic and neurotrophic effects. Therefore, to further investigate the effect of diabetes on angiogenic and neurotrophic factors in DPN, the mRNA expression of the following representative factors in sciatic nerves: IGF-I, VEGF, bFGF, Ang-1, eNOS, NGF, BDNF and SHh was examined. At twelve weeks after induction of diabetes in rats, sciatic nerves were harvested and analysed using real-time reverse transcriptase polymerase chain reaction (RT-PCR). The expression levels relative to that of GAPDH, which was used as a control, were compared between diabetic and non-diabetic control rats. In diabetic nerves, mRNA levels of all the examined factors were significantly lower than those in non- diabetic nerves. In non-diabetic nerves, VEGF and IGF-I were the predominant cytokines (Figure 5). The advantage of quantitative (q)RT-PCR over semiquantitative RT-PCR or Northern analysis is well demonstrated in Figure 7, which clearly discloses the relative expression level of each cytokine. Real-time (Quantitative) RT-PCR (qRT-PCR) was carried out using the ABI 7500 Real-Time PCR system (Applied biosy stems, CA). Briefly, Taqman primer/probe sets for various cytokines and housekeeping genes were designed using the Primer Express Software. Total RNA from rat samples was extracted and was reverse transcribed with the Taqman Multiscribe RT Kit. Real-time PCR was performed in duplicate with cDNA using Taqman Universal Master Mix. Multiplex Taqman assays were performed using dyes with distinct emission wavelength. Target probes and housekeeping genes are labeled with Fam and VIC, respectively. This system measures the fluorochrome emission at each cycle of PCR which ii directly proportional to the amount of PCR product. The threshold cycle (Ct) of each sample during the log-linear phase, which is inversely proportional to starting copy number, was determined. Relative gene expression was calculated using the ΔCt method with normalization to GAPDH. AU the samples were run at the same session to avoid confounding factors.

Example 5: Diabetes is characterized by a decreased number of circulating EPCs (Yooi et al., Circulation. Apr 26 2005;lll(16):2073-2085).

To determine the vasculogenic abnormality which may contribute to the potential impairment of neovascularization in diabetes, the number of circulating EPCs in diabetic rats was evaluated during a period of one year using the standard EPC culture method (Yoon et al., Circulation. Apr 26 2005;111 (16):2073-2085). EPC counts revealed a progressive decrement in accordance with the duration of diabetes, becoming significantly lower beginning at 4 months (Figure 6). EPC culture assay was performed as described previously (Mallat et al., Circ Res. Sep 6 2002;91(5):441-448; Mallat et al., CzVc Res. Sep 6 2002;91(5):441-448). Briefly, mononuclear cells isolated from 500μl of PB or BM (5x10 /well) was cultured in 5% FBS/EBM-2 (Clonetics) medium with supplements

(SingleQuot Kit; Clonetics) on rat vitronectin (Sigma) with 0.1% gelatin coated 4-well glass chamber slides. After 4 days in culture, cells were incubated with Dil-acLDL (Biomedical Technologies) for 1 hour followed by FITC-BS-I lectin staining. The dual-stained cells, considered EPCs, were counted in ten randomly selected high-power fields (HPF) under fluorescent microscopy. Example 6. Paracrine effects mediate therapeutic effects of EPCs

Recent studies have demonstrated that adult stem cells may exert their therapeutic effects in part through paracrine action. A series of experiments investigating whether paracrine mechanisms may explain the therapeutic effects of EPCs in repairing myocardial injury following myocardial infarction was performed. Myocardial infarction was induced in C57B1/6J mice aged 8-10 weeks as described previously (Lee et al., Circulation, 2004;l 10 (Suppl III): 138; Weis et al., J. CHn. Invest. March 15, 2004 2004; 113(6): 885-894; Michael et al., American Journal of Physiology. 1995;269:H2147-H2154). Immediately following the induction of myocardial infarction, the mice were randomly assigned to one of 3 treatment

5 5 groups (n = 5, each group) and received 5 x 10 mouse EPC, 5 x 10 mouse endothelial cells (Endo) or PBS via direct injection into the periinfarct area. Two weeks after treatment, rats were sacrificed and cardiac samples were obtained. qRT-PCR analysis demonstrated that mRNA expression of various angiogenic and cytoprotective factors such as VEGF, bFGF, Ang-1, Ang-2, VEGFR-2, eNOS, HGF, PLGF, SDF-I , PDGF and IGF-I were significantly upregulated in the EPC transplanted group compared to the control groups (Figure 7). Western blot analysis demonstrated increased phosphorylation of Akt. Akt is one of the key mediators for cell survival and vascular homeostasis. Collectively, these findings indicated that the upregulation of multiple paracrine factors can, in part, contribute to the angiogenic and protective effect of EPCs on injured myocardium.

Example 7. Mononuclear cell transplantation restored vasa nervorum and improve nerve conduction velocities in DPN.

In the following experiments, diabetic neuropathy was induced by administration of streptozotocin in 8 week-old, female Fisher 344 (F334) syngeneic rats. Twelve weeks later, the rats were randomly assigned to one of the following treatment groups (n=9, each group):

6

3.3 xlO MNCs or the same volume of saline. As a non-diabetic control, age- and sex- matched normal rats which received saline were used. Mononuclear cells were obtained from 20 week-old, normal male Fisher rats and were pre-labeled with red fluorescent dye, CM-DiI (1.5μg/ml, Molecular Probes) to track the engrafted cells in histologic sections Kawamoto et al., Circulation. 2001;103(5):634-637; Yoon et al., Circulation. Jun 29 2004; 109(25):3154- 3157). Under general anesthesia, cells or solutions in a total volume of 500 μl were directly injected into the muscle of both limbs at five sites along the course of the sciatic nerve, from the sciatic notch to the popliteal area using a 27 gauge needle. Motor nerve conduction velocities (MCV) and sensory nerve conduction velocities (SCV) were measured bilaterally and averaged in all rats at baseline, and at two and four weeks after treatment. To determine the extent and magnitude of mononuclear cell engraftment, sciatic nerves were excised after local infusion of FITC-labeled BS-I lectin. A schematic diagram showing the experimental design is provided at Figure 8.

Under fluorescent microscopy, robust engraftment of transplanted mononuclear cells into the sciatic nerves was observed (Figure 9A). The number of vasa nervorum detected by a locally administered fluorescent BS-I lectin was markedly increased in the endothelial progenitor cell transplanted rats compared to saline control. In cross sections of these nerves, a portion of the transplanted mononuclear cells co-localized with the BS-I lectiή-positive cells, indicative of mononuclear cell differentiation into endothelial cells (Figure 9B). In addition, the number of capillaries per section was significantly increased in the mononuclear cell transplanted rats compared to the saline injected controls (71 ± 6 vs 38 ± 4 per section, P < 0.01). These results suggest that locally transplanted mononuclear cells increased vascular supply by augmenting neovascularization.

Before treatment, both MCV and SCV in diabetic rats were significantly decreased compared to those in nondiabetic rats. Four weeks later, MNC transplanted rats demonstrated significantly increased MCV and SCV compared with those injected with saline (n=9, each group). Taken together, these findings indicate that in a rat model of DPN, local transplantation of MNCs can increase neural vascularity and result in restoration of peripheral nerve function.

Example 8: EPC transplant restores motor nerve conduction velocity in vivo Twelve weeks after the induction of diabetes with streptozotocin in C57B1 mice (vs normal mice; 43±5 vs 66±3 m/s, P<0.05), the mice were randomly assigned to EPC or saline injection groups (n=25, each group) and received either IxIO⁶EPCs or saline around the sciatic nerves. In the EPC group, motor nerve conduction velocities (MCV) were recovered back to normal levels within eight weeks after treatment (EPC vs Saline, 64±5 vs 44 ± 3 m/s, P<0.05) (Figures 1OA and 10B). Sensory conduction velocity also showed significant recovery. Example 9: Endothelial progenitor cell or mesenchymal stem cell transplant rescued pain responsiveness in mice with diabetes

The Tail Flick assay is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, a radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. Figure 11 shows the results of a tail flick assay that compares the pain response of mice having induced diabetes that received endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), or no transplant (DM-saline) with normal control mice (NonDM-saline) and control mice that received an endothelial progenitor cell transplant (NonDM-EPC). As shown in Figure 11, endothelial progenitor cell or mesenchymal stem cell transplant partially restored the pain responsiveness of mice with diabetes.

Example 10: Blood flow increased following endothelial progenitor cell transplantation Microvascular circulation of sciatic nerve was assayed using laser Doppler perfusion imaging (LDPI) in normal control mice (NonDM-S), in diabetic control mice treated with saline (DM-saline), and in diabetic mice that received endothelial cell (DM-EPC) or mesenchymal stem cell (DM-MSC) transplants. Blood flow in the sciatic nerve as measured by laser Doppler perfusion imaging was markedly increased in the mice that received EPC or mesenchymal stem cell transplants (Figures 12 A and 12B).

Example 11: Capillary density increased following endothelial progenitor cell transplantation

Vascularity was measured in normal control mice, in diabetic control mice injected with saline, and in diabetic mice that received endothelial progenitor cell transplants. Mice were perfused in vivo with FITC-conjugated BS-I lectin. Samples taken from nondiabetic saline- injected control mice showed a normal pattern of vascularity. Samples taken from a diabetic mouse, 4 weeks after saline injection, showed a marked reduction in the vascularity of the sciatic nerve. At 8 weeks post-treatment vascularity in the endothelial progenitor cell was significantly higher in the EPC group than in the saline group (29±4 vs 20±4 /HPF, PO.05) (Figures 13 and Figure 14). Example 12: Engrafted endothelial progenitor cells colocalize with endothelial markers

Sciatic nerve engraftment of endothelial progenitor cells was assayed eight weeks following transplantation. Figure 15 provides fluorescence images of whole-mounted rat sciatic nerves following in vivo perfusion with FITC-conjugated BS-I lectin and robust engraftment of Dil-labeled endothelial progenitor cells (fluorescence colocalizing with lectin). A nerve sample from a saline injected diabetic rat shows markedly decreased vascular networks. Robust engraftment of EPCs was observed in sciatic nerves for at least 8 weeks following transplantation. Engrafted EPCs were colocalized with endothelial markers. These results likely indicate the transdifferentiation of EPCs into endothelial cells (vasculogenesis) in the vasa nervorum (Figure 16).

Example 14: Endothelial progenitor cell transplantation increased Schwann cell proliferation

The disorder is marked pathologically by degeneration of Schwann cells and myelinated neuronal fibers as well as loss of a population of the neurons located in the dorsal root ganglia. Transplantation of endothelial progenitor cells markedly increased Schwann cell proliferation at four weeks following transplantation (Figure 17).

Example 13: Endothelial progenitor cell transplantation increased proliferation and decreased apoptosis of endothelial cells

A higher number of BrdU positive endothelial cells and a lower number of TUNEL positive apoptotic endothelial cells were observed in the sciatic nerves of EPC group (Figure 18). Brdu staining co-localized with isolection B4 (ILB4), a marker for endothelial cells. EPC implantation also decreased sciatic nerve apoptosis as shown by TUNEL assay (Figure 19).

Example 14: EPC implantation increased angiogenic and neurogenic cytokines

EPC implantation also increased mRNA expression levels of the following cytokines, which were assayed using real-time RT-PCR on sciatic nerves: VEGF (2.4 fold), FGF-2 (1.4), BDNF (4.2), Shh(2.0), Gli(2.6) and SDF-Ia(1.9) (all PO.05). This analysis indicated that levels of these cytokines were significantly increased in the EPC group compared to the saline group. The protein levels were well correlated with mRNA expression levels (Figure 20). Without wishing to be bound by theory, it is likely that local transplantation of BM- derived EPCs reversed experimental diabetic peripheral neuropathy by augmenting neovascularization and increasing angiogenic and neurotrophic factors in peripheral nerves. Thus, EPC transplantation provides an therapeutic method for treating diabetic neuropathy.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application, and publication was specifically and individually indicated to be incorporated by reference. In particular, U.S. Patent applications Nos. 60/728,509, 11/412,416, 11/339,808, and International Application No. US06/10981 and US04/36298, are hereby incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method for increasing angiogenesis in a neural tissue of a subject in need thereof, the method comprising administering a cell having the potential to differentiate into an endothelial cell to a subject, thereby increasing angiogenesis in a neural tissue of the subject.

2. The method of claim 1 , wherein the subject has or has a propensity to develop diabetes.

3. The method of claim 1 , wherein the subject has or has a propensity to develop a neuropathy.

4. The method of claim 3 , wherein the neuropathy is a peripheral neuropathy, toxic neuropathy, diabetic dementia, or autonomic neuropathy.

5. The method of claim 1 , wherein the cell is an endothelial progenitor cell, a mesenchymal stem cell, or a mononuclear cell.

6τ The method of claim 1 , wherein said administration increases neural tissue expression of a polypeptide is a therapeutic polypeptide.

7. The method of claim 1 , wherein the therapeutic polypeptide is selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF-2, VEGF165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I), Hypoxia inducible factor (HIF-I) and nitric oxide synthase.

8. The method of claim 1 , wherein the method reduces apoptosis in the neural tissue.

9. The method of claim 1 , wherein the method increases proliferation in the neural tissue.

10. The method of claim 2, wherein the method increases Schwann cell or endothelial cell proliferation.

11. The method of claim 1 , wherein the cell is integrated into the miciovasculature of the neural tissue.

12. The method of claim 1, wherein the cell is an endothelial progenitor cell, mononuclear cell, mesenchymal stem cell, or a progenitor or progeny cell thereof.

13. A method for preventing or ameliorating a neuropathy in a subject in need thereof, the method comprising: (a) administering to the subject a cell having the potential to differentiate into an endothelial cell; and

(b) increasing angiogenesis in a neural tissue of the subject, thereby ameliorating a neuropathy in the subject.

14. The method of claim 13, wherein the subject has or has a propensity to develop a diabetic neuropathy.

15. The method of claim 13, wherein the cell is an endothelial progenitor cell, a mesenchymal stem cell, or a mononuclear cell.

16. The method of claim 10,, wherein said administration increases therapeutic polypeptide expression in the neural tissue.

17. The method of claim 13 , wherein the therapeutic polypeptide is a neurotrophic factor, angiogenic factor, or cytokine.

18. The method of claim 17, wherein the therapeutic polypeptide is selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF-2, VEGF165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I) Hypoxia inducible factor (HIF-I) and nitric oxide synthase.

19. The method of claim 1 , wherein the cell is locally administered.

20. The method of claim 1 , wherein the cell is integrated into the neural tissue.

21. The method of any one of claims 1 to 13 , wherein the cell is isolated and expanded in vitro to obtain a cell population enriched in bone marrow-derived stem or progenitor cells prior to being administered to the host subject.

22. The method of any one of claims 1 to 13, wherein the cell is genetically modified.

23. The method of any one of claims 1 to 13, wherein the cell is an endothelial progenitor cell (EPC).

24. The method of any one of claims 1 to 13, wherein the cell is isolated from bone marrow of a donor subject.

25. The method of any one of claims 1 to 13, wherein the EPC is isolated from peripheral blood of a donor subject.

26. The method of any one of claims 1 to 13, wherein the donor and the subject receiving the cell are the same individual.

27. The method of any one of claims 1 to 13, wherein the cell is a human multipotent stem cell having reduced levels of a marker selected from the group consisting of: CD90, CDl 17, CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD133, MHC class I receptor and MHC class II receptor.

28. The method of claim 27, wherein the cell expresses reduced levels of at least two, three, four, or more markers.

29. The method claim 28, wherein the cell expresses reduced levels of all markers

30. The method claim 29, wherein marker expression is undetectable in a standarc cell marker detection assay.

31. The method of any one of claims 1 to 13, further comprising administering to the host subject a therapeutic polypeptide or a nucleic acid encoding a therapeutic polypeptide.

32. The method of any one of claims 1 to 13, wherein the cell is locally administered.

33. A method for increasing the level of a therapeutic polypeptide in a neural tissue of a subject in need thereof, the method comprising locally administering a cell having the potential to differentiate into an endothelial cell to a subject, thereby increasing the level of a therapeutic polypeptide in a neural tissue of the subject.

34. The method of claim 33 , wherein the therapeutic polypeptide is expressed in a support cell of the neural tissue.

35. The method of claim 29, wherein the therapeutic polypeptide supports endothelial or neuronal survival, function, or proliferation.

36. The method of claim 35, wherein the therapeutic polypeptide increases angiogenesis or vascularity in a neural tissue.

37. The method of claim 29, wherein the therapeutic polypeptide is selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growtl factor, VEGF-2, VEGF165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I) Hypoxia inducible factor (HIF-I) and nitric oxide synthase.

38. A method for preventing or ameliorating diabetic neuropathy in a subject in need thereof, the method comprising locally administering to a neural tissue of the subject a cell having the potential to differentiate into an endothelial cell, thereby ameliorating diabetic peripheral neuropathy in the subject.

39. The method of claim 38, wherein the method increases therapeutic polypeptide production in the neural tissue.

40. The method of claim 38, wherein the method results in engraftment of the stem cell in the microvasculature of a neural tissue of the subject.

41. The method of claim 38, wherein the local administration involves injecting the cell into a muscle comprising the neural tissue.

42. A method for preventing or ameliorating neuropathy in a subject in need thereof, the method comprising administering to the subject a genetically modified cell having the potential to differentiate into an endothelial cell, thereby preventing or ameliorating diabetic peripheral neuropathy in the subject.

43. The method of claim 42, wherein the cell comprises an expression vector comprising a nucleic acid sequence encoding a therapeutic polypeptide that supports endothelial or neuronal cell survival, proliferation, or function.

44. The method of claim 43, wherein the therapeutic polypeptide is selected from the group consisting of acidic and basic fibroblast growth factors, vascular endothelial growth factor, VEGF-2, VEGF165, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor A, B, E, tumor necrosis factor α, hepatocyte growth factor, insulin like growth factor 1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF, Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1, angiopoietin-2, stromal cell derived factor (SDF-I) Hypoxia inducible factor (HIF-I) and nitric oxide synthase.

45. The method of claim 42, wherein the cell is derived from bone marrow, peripheral blood, or umbilical cord blood.

46. The method of claim 42, wherein the cell is selected from the group consisting of a bone marrow derived stem cell, an endothelial progenitor cell, a hematopoietic stem cell, a mesenchymal stem cell, and a mononuclear cell.

47. The method of claim 42, wherein the cell is a human multipotent stem cell having a reduced level of a marker selected from the group consisting of CD90, CDl 17,

CD34, CDl 13, FLK-I, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CDl 17, CD133, MHC class I receptor and MHC class II receptor.

48. The method of claim 47, wherein the cell expresses reduced levels of at least two, three, four, or more markers.

49. The method of claim 47, wherein the cell expresses reduced levels of all markers.

50. The method of claim 42, wherein the reduced marker expression is undetectable in a standard cell marker detection assay.

51. A packaged pharmaceutical comprising a therapeutically effective amount of i cell having the potential to differentiate into an endothelial cell, and instructions for use in treating a subject having a neuropathy.

52. The packaged pharmaceutical of claim 51 , further comprising a therapeutic polypeptide.

53. A packaged pharmaceutical comprising a therapeutically effective amount of a cell having the potential to differentiate into an endothelial cell, and instructions for use in treating or preventing a diabetic neuropathy in a subject.

54. The packaged pharmaceutical of claim 51 or 53, wherein the cell is genetically modified.

55. A method for identifying an agent useful for the treatment of neuropathy, the method comprising

(a) contacting a cell having the potential to differentiate into an endothelial cell with an agent;

(b) providing the cell to a host; and

(c ) measuring an increase in angiogenesis in a neural tissue of the host, wherein an increase in angiogenesis relative to a reference identifies the agent as useful for the treatment of neuropathy.

56. A method for identifying an agent useful for the treatment of neuropathy, the method comprising (a) contacting a cell having the potential to differentiate into an endothelial cell with an with at least one inhibitory nucleic acid molecule;

(b) providing the cell to a host; and

(c ) identifying a decrease in angiogenesis in a host tissue relative to the tissue of a corresponding control host, thereby identifying the target of the inhibitory nucleic acid molecule as an agent useful for the treatment of neuropathy.

57. The method of claim 56, wherein the inhibitory nucleic acid is identified usinj proteomics, bioinformatics, or genomics.

58. The method of claim 55 or 56, further comprising measuring Schwann cell proliferation, endothelial cell proliferation, neural conductance, or pain responsiveness in the host, wherein measuring an increase relative to a reference identifies the agent as useful for the treatment of neuropathy.

59. The method of claim 55 or 56, further comprising measuring apoptosis, wherein measuring a decrease in apoptosis identifies the agent as useful for the treatment of neuropathy.