GB2542776A - Treatment of neurological conditions by activation of stem cells - Google Patents

Abstract

A method of treating a neurological condition comprises administering to the subject valproic acid, sodium valproate, of divalproex sodium, where the free valproic acid concentration is 5.0 to 10.0 µgm/ml within cerebral spinal fluid. The free valproic acid may be used at 2.5 to 15 µgm/ml in combination with lithium carbonate at a concentration of from 0.05 to 0.25 mEq/ml; or the free valproic acid may be used at 2.5 to 15 µgm/ml in combination with lithium carbonate at 0.1 to 0.4 mEq/ml; or the free valproic acid may be used at 5.0 to 10.0 µgm/ml in combination with lithium at 0.2 to 0.4 mEq/ml. The combination therapy may comprise exposing stem cells of a subject, in which expansion has been induced, to the combination of lithium carbonate and valproic acid, and subsequently returning the stem cells to the subject; or may comprise returning stem cells in which expansion has been induced back to a subject followed by administering a combination of lithium carbonate and valproic acid. A method for assessing the clinical status of a subject having traumatic brain injury is also claimed.

Description

TREATMENT OF NEUROLOGICAL CONDITIONS BY ACTIVATION OF NEURAL STEM CELLS

The present disclosure is related to treatment of neurological conditions through the activation of endogenous stem cells within the brain and, more specifically, but not exclusively to specific dosages of lithium and valproic acid that induce proliferation, migration, and epigenetic reprogramming of stem cells.

Stem cell therapy is a common procedure for the treatment of blood disorders including leukemia, lymphoma and auto-immune conditions using transplantation of hematopoietic stem cells (HSCs). Other types of adult stem cells are now being transplanted to treat other conditions including skeletal muscular disorders, cardiovascular diseases, cardiomyopathy, etc. While activation of endogenous stem cells has been used to increase proliferation and differentiation of hematopoietic stem cells for several years, such activation of other adult stem cells including mesenchymal stem cells (MSCs), satellite cells, and neural stem cells (NSCs) has not yet reached routine clinical practice. A detailed study published in 2013 by scientists at the National Institutes of Health (NIH) showed reduction in lesion volume, diminution of blood brain barrier (BBB) disruption, reduced neuronal hippocampal degeneration and enhanced recovery of motor coordination in a mouse model of traumatic brain injury (TBI) by combined treatment with lithium and valproic acid, sodium valproate, and sodium divalproex(Li-VPA). Yu, F„ et al., J. Neurosurg. 119: 766-773,2013.

Similarly, combined Li-VPA had the following effects in an animal model of amyotrophic lateral sclerosis (ALS; SOD-1 -G93A mutant mice): delayed onset of symptoms, prolonged life-span and reduced neurological deficits while either agent alone produced smaller effects. Feng, H.L., et al., Neurosci 155: 567-572,2008.

Lithium itself also exerts neuroprotective effects and increases neurogenesis; it up-regulates neurotropic and growth factors, including brain-derived neurotropic factor (BDNF), modulates inflammatory molecules while increasing neuroprotective factors such as heat shock protein 70 (Hsp70), stimulates angiogenesis and diminishes pro-apoptotic factors. The data reported supports clinical trials in humans for treatment of traumatic brain injury (TBI) with lithium. Leeds, P.R., et al., ASC Chem Neurosci, Epub ahead of print, 2014, Apr 11. TBI is caused by a bump, blow, or jolt to the head via penetration of the brain or closed head injury. Some 5.3 million Americans (i.e., 2% of the United States population) currently live with disabilities resulting from TBI.

Clinical trials in Alzheimer’s disease using low-dosage Li treatment (300 pg daily for 15 months) resulted in prevention of cognitive loss. Nunes, M.A., et al.,Curr. Alzheimer’s Res 10:104-107, 2013.

Also, serum BDNF levels were significantly increased in a placebo-controlled clinical trial and cognitive impairment was diminished by 0.5 to 0.8 mM Li treatment of attention deficit (AD) patients. Leyhe, T. et al., J. Alzheimer’ Dis 16:649-656,2009.

In a clinical trial of ALS, it was found that 0.4 to 0.8 mM Li prolonged survival in patients as well as in the G93A animal model of ALS. Fornai, F, et al., PNASD 105: 2052-2057, 2008.

The pre-clinical results support the effectiveness of Li-VPA treatment of animal models of TBI and ALS. Li also has beneficial effects on neuroprotection, mitigation of inflammation and apoptosis and up-regulation of neurotrophic factors as well as angiogenesis. Clinical trials of lithium alone show potential therapeutic benefit in AD and possibly ALS.

The present disclosure teaches new methods and provides compositions related to the activation of endogenous neural stem cells within the brain to regenerate damaged tissues through various biological processes including inhibition of apoptosis, inflammation, oxidative stress and the induction of angiogenesis in addition to differentiation of NSCs to glial, various neuronal cells and endothelial cells.

The present disclosure arose from the finding that lower dosages of Li and VPA than those previously used result in proliferation, migration and re-programming of human stem cells. We disclose herein the combination of specific Li and VPA dosages that induce stem cell activation.

Cell-based assays are used to evaluate drug dosages and other purposes as well. Stem cells are maintained in physiological status and drugs are introduced to the cell culture medium to determine effects on proliferation, migration and epigenetic reprogramming. This approach is extended to patient-specific NSCs removed by biopsy of nasal epithelium tissue, enzymatic extraction of cells and expansion of neural stem cells in culture, thereby providing a sample of the patient's neural stem cells for several uses. For example, following treatment with stem cell activating agents, the stem cells may then be transplanted into the patient for various uses such as treatment of spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease and otherconditions amenable to therapy by stem cell transplantation. Also, we disclose analysis of multiple biomarkers in biological fluids to determine the status of the disease and its response to treatment through stem cell activation. These biomarkers are specific to processes including nerve cell damage or injury, neurogenesis, neuroprotection, apoptosis, angiogenesis, inflammation, oxidative stress, etc. As such, measurementof specific biomarkers allows readout of the status of specific biological processes such as neuroprotection, apoptosis, inflammation, neurogenesis, and angiogenesis. When combined with the stem cell activation treatment, the biomarker analysis allows determination of stem cell activation and its effects on specific biological processes. Together with clinical assessments, biological process-specific biomarkerprofiling allows in-depth analysis of biological responses to NSC activation and its clinical manifestation. Biological fluid analysis also includes pharmacodynamic endpoints of therapies such as measurement of histone acetylation, GSK3-p inhibition together with therapeutic drug monitoring (TDM).

The methods and compositions of the present disclosure are intended for treatment of various neurological conditions, including but not limited to, traumatic brain injury, Alzheimer’s disease, Parkinson’s disease, concussion, impaired cognitive function, dementia, memory loss, ALS, spinal cord injury, Huntington’s chorea, stroke, meningitis, and autism spectrum disorders. Similar strategies of stem cell activation are amenable to broader application than neurological conditions including treatment of auto-immune, skeletal muscular disorders, etc.

In accordance with one aspect of the present disclosure, there is provided a method of treating a neurological condition in a subject in need thereof, comprising administering to the subject a combined therapy comprising lithium (Li) and valproic acid (VPA), wherein the Li concentration is from about 0.1 to 0.4 mM. In the preferred embodiment, the Li concentration is from about 0.2 to 0.4 mM and the VPA concentration is from about 2.5 to 15 pgm/ml.

The neurological condition can be Alzheimer’s disease, traumatic brain injury, a concussion, Parkinson’s disease, amyotrophic lateral sclerosis, dementia, Huntington’s chorea, autism spectrum disorders, memory loss, spinal cord injury, or impaired cognitive function. Also, included are neuropsychiatric conditions, including butnot limited to, schizophrenia. A complete understanding may be obtained by reference to the accompanying drawing, when considered in conjunction with the subsequent detailed description, in which:

Figure 1 is a microphotograph depicting cell migration using fluorescent readout and cell tracker green as a fluorescent marker of human CB-MSCs;

Figure 2 is a line graph representation illustrating percent closure as a functionof time during live-cell data acquisition at different dosages of Substance P;

Figure 3 is a line graph representation illustrating migration of various human cell lines exposed to Substance P;

Figure 4 is a line graph representation illustrating less than 1 μΜ curcumin induced migration of human CB-MSCs and 1 to 10 μΜ curcumin blocked migration due to apparent toxicity;

Figure 5 is a line graph representation illustrating migration of CB-MSCs induced by exposure to lithium alone (green diamonds), VPA alone (black squares) and VPA in the presence of 200 μΜ lithium (red diamonds);

Figure 6 is a line graph representation illustrating migration of CB-MSCs, NSCs and colorectal CAFs by induced by increasing VPA concentration in 200 μΜ lithium;

Figure 7 is a line graph representation illustrating migration of human MSCs exposed to a combination of lithium and VPA with and without inhibition of MMP9and CXCR4;

Figure 8 is a line graph representation illustrating proliferation of different cell lines induced by increasing concentrations of FGF-b (FGF-2) after a 5 dayexposure, FGF being a fibroblast growth factor;

Figure 9 is a line graph representation illustrating proliferation of CB-MSCs induced by lithium, VPA and VPA in 200 μΜ lithium after a 5 dayexposure;

Figure 10 is a line graph representation illustrating proliferation of CB-MSCsand NSCs as a function of increasing VPA in 200 μΜ lithium after a 5 dayexposure;

Figure 11 is a bar graph representation illustrating PCR used to measure target genes known to be subject to epigenetic regulation by HDAC inhibitors including VPA: Oct 3/4, a pluripotency gene, Sirtl, age-related gene and FGF-21, whose expression is related to VPA-Li synergy; and

Figure 12 is a bar graph representation illustrating cytokine levels in cell culture media exposed to CB-MSCs for 24 days and determined by microarrayanalysis.

Disclosed herein are methods for activating stem cells, including methods for inducing stem cell proliferation, migration and epigenetic reprogramming. Aneurological condition can be treated in a subject in need thereof, the steps comprising administering to the subject a combined therapy comprising lithium (Li) and valproic acid (VPA), and related agents. The neurological condition can be Alzheimer’s disease, traumatic brain injury, a concussion, Parkinson’s disease, amyotrophic lateral sclerosis, dementia, Huntington’s chorea, meningitis, autism spectrum disorders, memory loss, spinal cord injury, or impaired cognitive function.

Adult stem cells reside in various regions throughout the body of warm-blooded animals including bone marrow where both hematopoietic and mesenchymal stem cells reside, satellite cells within muscles, various MSCs within other tissues, e.g., teeth pulp, endocrine glands, arteries (as pericytes).

Within the nervous system, NSCs are concentrated within thesubventricular zone (SVZ) of the lateral ventricles, the dentate gyrus of the hippocampus, within the olfactory epithelium and diffusely within the frontal cortex. The rostral migratory stream (RMS) is a CNS structure of continuous flow of neural progenitor cells, neural stem cells and transiently activated neural progenitor cells (TaP cells) whereby a migratory stream of cells provide potential regeneration of the adult brain through cellular activationat the level of the SVZ in the lateral ventricles. The RMS is a critical component of brain development and also maintains activity within the adult brain. Activation of stem cells in the SVZ through increased proliferation, migration and epigenetic reprogramming is a primary embodiment of the present invention. While cells migrating through the RMS are ultimately destined for the olfactory bulb, there are circumstances that promote migration of NSCs away from the RMS into other regions of the brain. Injury is known to induce migration of NSC and related progenitors to sites of injury and inflammation through chemical signaling involving chemokines and their chemokine receptors including the Stromal-derived factor 1a (SDF1a also known as CXCI12) ligand and its receptor, CXCR4 and related signaling systems. Injury includes sites of injury and inflammation characterized by primary pro-inflammatory cytokines including IL-1 and TNF, etc. that trigger secondary inflammatory chemokines including CXCL12 whether due to TBI, cell death from concussion, cellular degeneration due to neurodegenerative diseases, local regions of inflammation due to infection including meningitis, cell death induced by reduced blood flow/hemorrhage, etc. Other chemokines and factors are involved as well in the migratory process that ultimately results in the movement of neural stem cells into regions of cerebral injury. Additional factors are also known to promote ectopic migration to surrounding brain regions including changes in extracellular matrix proteins including neural cell adhesion molecule (NCAM) and tenascin-R. Sun, W., et al. Anat Cell Biol 43:269-279,2010.

While hematopoietic stem cells (HSCs) are relatively active and responsible for production of 200 billion red blood cells daily, other niche regions of adult stem cells are typically quiescent and are activated by a specific stimulation, such as response to injury. Stem cell activation, as referred to herein, involves three separate, although not necessarily independent, biological processes: proliferation, migration and epigenetic reprogramming. Proliferation increases the number of stem cells through cell division, while migration is characterized by movement to specific targets mediated through chemokine/chemokine receptor systems within stem cells and the local environment. This migration of stem cells allows for various regenerative and repair processes to occur at sites remote from the actual stem cell niches within the body. Epigenetic reprogramming involves increased expression of specific genes involved with pluripotency, e.g., Oct 3/4 and longevity (Sirtuin family including SIRT-1) and certain members of the FGF family of proteins, including FGF-21. Epigenetic reprogramming is mediated through HDAC inhibition resulting in modulation of DNA methylation patterns yielding altered gene expression including increased or decreased expression of specific genes. Proliferation is induced by several molecular mechanisms that yield increased numbers of cells through the process of cell division.

We disclose herein specific concentrations of Li and VPA that are effective in the activation of stem cells. Prior art shows improved recovery from TBI, stroke and ALS by the combined administration of Li-VPA. The effects of these agents were investigated on cultured human stem cells and other primary cells and results are described in detail within the examples provided herein (Examples 1-6, below). The results obtained from validated, cell-based assays provide additional evidence for the activation of stem cells by Li-VPA since either alone orin combination, Li-VPA result in dose-dependent proliferation and migration of both human MSCs and human NSCs. However, results showed the unexpected finding that significantly lower dosages of Li and VPA than previously used or therapeutic dosages were effective in the induction of stem cell activation. Hence, in the presence of maximally effective 200 μΜ lithium, VPA was effective at lower dosages than by itself in inducing stem cell migration (Example 4) and it was less effective than by itself in the induction of stem cell proliferation (Example 5). The EC50 for VPA was slightly less than 40 μΜ in the presence of 200 μΜ Li.

In clinical use of the treatment of bipolar disorder, lithium is usuallyadministered at 0.4 to 1.2 mEq/ml using blood serum lithium measurements to control the lithium concentration to ensure administration of sub-toxic levels. The data obtained shows that lithium is capable of stem cell activation at dosages less than needed for thetreatment of bipolar disorder. The EC50 for Li alone in the stem cell migration assay was 0.08 mM; 0.10 mM lithium induced 89% of maximal closure and 0.125 mM lithium induced maximal migration (See Example 4, Figure 5). In a cell proliferation assay, Li showed similar dosage effects. The EC50 was 0.08 mM; 0.10 mM induced 90% of the maximum proliferation and proliferation was maximum at 0.15 mM lithium and higher concentrations (See Example 5, Figure 9).

Results thus support a lower target dosage of lithium for adult stem cell activation than that used in the treatment of bipolar disorder. One hundred micromolar (0.10mM) lithium was an effective concentration for stem cell migration and proliferation in thecell-based assays reported here. The blood brain barrier is unlikely to create concentration differences between CSF and serum and studies of lithium distribution indicate passive transfer to CSF from serum and accumulation of brain lithium following chronic administration. Hillert, M, et al, Neurosci Lett 521: 62-66, 2012; Wraae, 0., Br. J. Pharmacol. 64: 273-279, 1978. Clinical usage of lower dosage lithium may increase compliance and also increase safety through diminished risk associated with toxic or near-toxic levels of lithium.

The lithium dosage for activation of neural stem cells according to the present invention is optimal at 100 to 500 μΜ, more preferable at 100 to 300 μΜ and most preferable at 100 to 250 μΜ. This dosage refers to CSF concentrations measurable through serum determinations or by direct measurement of CSF lithium concentration.

The dosage of lithium is optimal at 0.2 mM, while the VPA concentration is optimal at 10 mg/kg (microgram/ml). These concentrations refer to free molecular forms of these drugs at a cellular level. Various formulations and delivery routes are embodied within the present invention as well with each route of drug delivery resulting in specific dosage at the level of neural stem cells within the brain. The dosages proposed herein are generally less than therapeutic levels for approved indications. Flence one of the advantages of the present proposal is reduction of drug toxicity, improved tolerance and compliance. Other activating agents are also disclosed herein, including specific agents that provide potent and selective inhibition of GSIO-β (glycogen synthetase 3-β) and HD AC-1 (histone deacetylase I).

In the presence of 0.20 mM lithium, results show enhancement of the effectiveness of VPA compared to VPA in the absence of lithium with regard to stem cell migration (ECso= 38.45 μΜ alone and 32.96 μΜ in the presence of 200 μΜ lithium; Example 4, Figure 5) while VPA was less effective with lithium in inducing stem cell proliferation than by itself (Example 5, Figure 9). In either case, VPA in the presence of lithium resulted in an ECsoof about 40 μΜ that is equivalent to 5.8 μg/ml. VPA circulates as both free VPA and bound VPA; the latter is predominantly bound to albumin. Free VPA in serum is about 20% of total VPA although this varies with serum albumin levels. The exchange of VPA across the blood-brain barrier involves both active uptake and efflux from the CSF with efflux exceeding uptake by about 2-fold, possibly due to active transport of VPA through the carboxylic acid efflux system. Loscher, W, et al., Epilepsia 29:311-316, 1988; Gibbs JP, et al, Epilepsy Res. 58:53-66, 2004; Bachmeier CJ, Miller DW, Pharm Res. 22:113-21, 2005. Thus, free VPA in CSF was measured to be about one-half of the levels of free VPA in serum in children being treated for epilepsy with chronic VPA administration. It is thus apparent that free VPA levels in serum should range from 10 to 20 pg/ml in clinical trials in order to achieve optimal activation of NSCs through stimulation of migration and proliferation. It is generally agreed that free VPA is responsible for its pharmacological effects. Also, chronic administration at 10 to 20 pg/ml free VPA in serum is expected to result in epigenetic effects in the brain since increased expression of Oct 3/4, Sirtl and FGF-21 at 31.25 μΜ VPA are shown and these epigenetic effects appear to be a critical component of stem cell activation (Example 6, Figure 11).

Preferably, the concentration of VPA in the present invention is 2.5 to 15 pg/ml, more preferably 5 to 10 pg/ml, and most preferably 4 to 8 pg/ml as free VPA in CSF. Given that VPA circulates bound to albumen and possibly other serum proteins and that the blood-brain barrier results in efflux of VPA into serum, the levels of free VPAin serum are likely to be higher than those in CSF. Reference is preferably made to concentrations of free VPA within the CSF as this reflects the dosage level of exposure to CNS cells. Also, VPA and/or lithium may be administered in a variety of different routes of drug delivery, including orally, by use ofa nasal spray, intra-thecal injection, eye drops, dermal delivery systems, etc. The preferred concentrations of VPA and lithium are to be achieved irrespectively of the administration route of each drug and whether or not lithium or VPA are administered in combination, as by specific compounding, or as individual medications either alone or in combination.

More preferably, the Li is 0.1 to 0.3 mM and the VPA is 5 to 15 pg/ml. These concentrations refer to those in the immediate environment of the in-vivo stem cells which is not necessarily the same as the concentrations in serum. There are several factors that determine the local concentration of a drug substance as is well-known to those skilled in the art. For example, VPA is bound by albumin in serum and is subject to complex transfer across the brain-brain barrier due to interactions with specific transporting molecules. Thus, one particular embodiment centers on local concentration of Li and VPA within the various NSC niches within the brain. Also disclosed herein are methods to determine effective dosage through pharmacodynamic endpoints together with biomarker profiling of specific biological processes related to apoptosis, neuroprotection, neurogenesis, and angiogenesis, as well as inflammation and oxidative stress. Neuro-imaging of stem cell activation within specific brain regions such as the SVZ of the lateral ventricles by, for example and without limitation, imaging of labeled thymidine uptake, also allows direct determination of stem cell activation.

Another embodiment refers to concomitant treatments to both activate neural stem cells and to enhance the migration beyond the RMS to other brain regions. Neural stem cells are derived primarily from cell division in the SVZ of the lateral ventricle and then migrate through the RMS eventually differentiating into neurons within the olfactory bulb. Injury, cell degeneration, infection, etc. result in collateral migration to these sites from the RMS. This signaling is mediated through various factors elaborated at the injured site that promote tropic movement of NSCs away from the RMS. The RMS is composed of complex network of cells, vasculature and extracellular matrix (ECM) molecules that usually limit cell movement to the RMS itself. Specific ECM molecules especially NCAM and Tenascin R play significant roles in the migration of NSCs within the RMS. NCAM is particularly important in controlling RMS cellular migration and it interacts with itself or other molecules to regulate cellular migration within the RMS. The addition of homopolymers of sialic acid (polysialic acid or polySia-PSA) is a highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). Two enzymes, the polysialyltransferases, ST8Sial I (ST8Sia I) and ST8Sial V (ST8Sia V) are responsible for the biosynthesis of polySia. Since the removal of PSA from NCAM is known to allow diffuse migration of NSC and related cells from the RMS to surrounding CNS regions (Battista and Tutishauer, J. Neurosci 30:3995-4003, 2010) the present invention includes concomitant methods to activate Neural stem cells within the brain and particularly in the SVZ of the lateral ventricles together with methods to modify and limit the restraints from ectopic migration by interference with the process of PSA addition to NCAM. For example and without limitation, this may include use of synthetic precursors including N-butyl mannosamine, small inhibiting RNA to ST8Sia I, ST8Sia V, peptide mimetics that block addition of PSA to NCAM, and other enzymatic inhibitors and negative regulators of expression that result in blocking the addition of PSA to NCAM.

In another embodiment, we include compositions yielding desired levels of GSIO-β and HDAC-I inhibition. Thus, a single formulation of Li and VPA is provided wherein the composition yields a concentration of Li ranging from 0.1 to 0.4 mM and VPA at 2.5 to 15 pg/ml. This may be formulated as a single medication comprising Li-carbonate and Na-valproate, divalproex sodium, or valproic acidor equivalent compounds as the active pharmaceutical ingredients while using appropriate excipients and additives to achieve the desired local concentrations at NSC regions of the brain. Dosage scheduling is based on pharmacokinetic parameters and adjusted to assure steady state levels. Pharmacokinetic properties manipulated bytime-release formulations of drug substances well-known to those skilled in the art are also included. A further embodiment includes compositions comprising substances in addition to Li and VPA that have potent and selective ability to inhibit GSK3-p and HDAC-I. These compositions include, but are not limited to, curcumin as a GSK3-p inhibitor and Romidepsin (FK-228) as a potent and selective HDAC-I inhibitor.

Bioavailability of pharmacological agents is an important component of therapy and thus the liposomal formulations of curcumin are necessary to maintain adequate bioavailability. The compositions of the present invention thus includevarious compounding processes well-known to those skilled in the art to alter PK properties and combine appropriate dosages of GSK3-p inhibitor and HDAC-I inhibitors into single medications. Also, various administration routes may be utilized within the present invention to optimize drug delivery to CNS, Thus for example, but without limitation, activating drugs and NCAM modulating substances may be compounded together or alone into nasal sprays, eye drops, appropriate formulations for intrathecal delivery, dermal patches, etc. These formulations may involve the appropriate use of nano technology, liposomes; emulsions, ointments, etc. as are well-known to those skilled in the art.

Another embodiment involves the use of cell-based assays to provide optimal therapy to patients, including all warm-blooded animals. Cell-based assays include stem cells that the therapy is designed to activate in asuitable culture system to allow maintenance of appropriate environment of cell culture media, temperature, relative humidity, oxygen and carbon dioxide level. In one embodiment the stem cells may be allogeneic and represent specific classes of adult stem cells such as hematopoietic, mesenchymal, neural, muscle, etc. Use of such cultures is ideal for studies of basic issues such as dosage and comparative studies. Anotherembodiment involves use of patient-specific stem cells, expansion of these cells and use of the cells in cell-based assays of a specific element. For example, human neural stem cells may be derived by biopsy of nasal epithelia tissues. Girard, SD, et al., J. Vis Exp 54: e2762, 2011. Enzymatic digestion according to methods well-known in the art may be used to disperse and purify cells followed by cell culture using established methods of NSC expansion resulting in patient-specific NSC cultures. Preferably, these cultures are maintained on laminin or fibronectin-coated (or equivalent) tissue culture plates or flasks in a medium containing Y27632 at 10 to 40 μΜ final concentration and also containing DYRK inhibitor -ID-8 at 0.5-10 μΜ final concentration.

These cultures provide numerous advantages, including allowing the determination of personalized dosage regimes, and the direct study of cellular responses including proliferation, migration and epigenetic reprogramming. In addition the cell culture media may be collected from said cultures and analyzed for biomarker content to determine baseline secretion levels and those resulting from stem cell activation using Li and VPA and other activating agents as well. These cell-based assays are also amenable to discovery and characterization of novel activating agents as well. It should be noted that stem cell activation does not necessary involve a chemical process but may also occur through the use of appropriate energy input into stem cells, such low-power laser activation, exposure to electrical/magnetic fields or light at specific intensities and frequencies.

Furthermore, the patient-specific NSC cultures may be used for genetic analysis of the cells and their responses. Genetic abnormalities may be detected by standard methods of genomic profiling. Also, the expression of FGF-21 is a particularly sensitive indicator of the synergistic effects of Li and VPA since its expression can be accelerated several fold by Li-VPA. Leng, Y, et al., Mol. Psychiatry, Epub ahead of print, 2014, Jan 28.

Gene expression profiling also provides detailed analysis of the epigenetic reprogramming resulting from HDAC inhibition and the subsequent alterations in DNA methylation. A further embodiment includes use of patient-specific stem cells for treatment of various conditions by stem cell transplantation. Such stem cells may be provided to the patient in various conditions including quiescent cultures of viable cells or as activated cells such as those exposed to Li-VPA. It has been shown that such activation results in increased recovery of spinal cord injury when compared to non-activated cells. Such cells, either activated or not, may be used therapeutically through transplantation to treat other conditions amenable to stem cell therapy, including for example, macular degeneration, stroke, Alzheimer’s disease, Parkinson’s disease, etc. Also, a further extension of this embodiment includes use of differentiating agents to drive patient-specific stem cell into particular lineages. Thus, for example, patient NSCs maybe differentiated into cholinergic or dopaminergic neurons for use in autologous stem cell therapy of Alzheimer’s or Parkinson’s disease, respectively. Other differentiated cell lineages and therapies are readily apparent to those skilled in the art and are not intended to be limited from this embodiment.

We also contemplate molecular analytical procedures to assess the extent of stem cell activation and its effects on specific biological responses. Since the mechanism of the biological effects of Li is primarily through inhibition of the enzyme GSiO-β, assays of beta catenin are used to quantify the degree of GSK3-p inhibition in patients. Similarly, VPA is a well-known histone deacetylase inhibitor and histone acetylation is a measure of HDAC-I inhibition. These analyses are used together with standard TDM analyses of lithium and especially, free VPA in serum to gain a complete pharmacodynamic profile of the Li-VPAtherapy. PGE2 is known to mediate immunosuppressive effects of stem cells and PGE2 synthetase expression is increased 9-fold in the rat sub-ventricular zone in response to VPA treatment. P. Laeng, et al, J. Neurochem. 91: 238-251,2004. Thus, the analysis of PGE2 levels is an indicator of stem cell activation. Similarly, substance P is powerful vasodilatory agent that is released as a consequence of injury to the brain and excessive cranial edema is a principal cause secondary injury in TBI. NSE (nerve-specific enolase) is a marker of nerve cells and its presence in serum, CSF, etc. is indicative of neural cell damage. S100-beta is similar to NSE but derived from astrocytes, thus providing another biomarker for extent of brain injury.

Ubiquitin hydroxy-terminal hydrolase L1 is specifically elevated in serum following blood-brain barrier disruption and its serum levels thus indicate the functional status of the blood-brain barrier. Donkin, J.J., et al., Neurotrauma 28: 217-224, 2011. Heat shock protein 70 is a potent neuroprotective agent (Li, Y., et al, Epilepsy Res. 103: 203-210, 2013) and its serum levels reflect the severity of TBI and elevated levels correlate with TBI-related fatalities. Kim, N, et al., Inflammopharmacology 20:177-185, 2012. Monocyte chemoattractant protein-1 (MCP-1): Promotes inflammatory responses and myelin degradation. Plasma MCP-1 contents are associated with the severity ofTBI and the index of comprised axonal fiber integrity in the frontal cortex. Noh, H., J.

Alzheimer’s Dis. 31(2):301-313, 2012. It signifies inflammation and also indicates extent of injury.

Neurogenesis is associated with increased levels of various biomarkers, including but not limited to, MMP9, CXCR4, SDF1 alpha, HBEGF, PGE2, CXCL5, BMP11, BDNF, NGF and NTF3. Measurement of these biomarkers provides quantitative assessment of the process of neurogenesis and its activation in response to stem cell activation. Angiogenesis is associated with increased levels of various biomarkers, including but not limited to, CXCL2, CXCL5, VEGF, EGF, FIBEGFand MDK. Measurement of these biomarkers provides quantitative assessment of the process of angiogenesis and its activation in response to stem cell activation. Neuroprotection is associated with increased levels of various biomarkers, including but not limited to, Hsp70, BDNF, GDNF, NGF, NTF3, GFAP, Melatonin, PDGF-AA. Measurement of these biomarkers provides quantitative assessment of the process of neuroprotection and its activation in response to stem cell activation. Apoptosis is associated with increased levels of various biomarkers, including but not limited to,

Fas, C-reactive protein, Survivin, Cytochrome C, BLC, caspase 9 and related molecules, Bcl2 and Bcl-XL. Measurement of these biomarkers provides quantitative assessment of the process of apoptosis and its activation in response to stem cell activation.

Oxidative stress and inflammation are associated with increased levels of various biomarkers, including but not limited to, IL-6, NFkb, IL-6, TNF-alpha, IL-17, andMCP-1. Measurement of these biomarkers provides quantitative assessment of the process of apoptosis and its activation in response to stem cell activation.

Analysis of stem cell activation according to the present invention also includes neuro-imaging methods to assess NSC status within patients. While manyimaging procedures provide information on multi-cellular structures, other methods well-known in the art allow imaging at a cellular level. Pyrimidines are selectively taken up by proliferating cells and a preferred, without limiting, method of NSC proliferation imaging of the present invention is by positron-emission tomography of F-labeled3’-deoxy-3’-fluorothymidine at known anatomical NSC niches including the SVZ of the lateral ventricles.

Examples A series of assays quantified various functional aspects of stem cell activation including proliferation, migration, epigenetic reprogramming and stem cell secretome analysis. These assays were validated through comparison to prior results. Results show that Li and VPA enhance proliferation and migration of MSCs and NSCs in a dose-dependent manner. Cell migration by VPA was inhibited by blockage ofCXCR4 and Li-induced cell migration was inhibited by blockage of MMMP-9, suggesting their involvement in the mechanism of stem cell activation. Additional molecular mechanisms are possible. The data suggest a role for epigenetic modulation in stem cell activation.

Example 1: Expansion of cell lines for use in cell-based assays: Native human cord blood-derived MSCs (Vitro Biopharma Cat. No. SC00A1), human pancreatic fibroblasts (Vitro Biopharma, Cat. No. SC00A5), colorectal cancer-associated fibroblasts (Vitro Biopharma, Cat. No. CAF05), and pancreatic stellate cancer-associated fibroblasts (Vitro Biopharma, Cat. No. CAF08) were plated at 7500 cells/cm2 and grown to 90% confluency in T-25 tissue culture (TC) flasks (BD Falcon, Cat. No. 353108) in MSC-Gro™ low serum, complete medium (Vitro Biopharma Cat. No. SC00B1). Neural stem cells (Vitro Biopharma, Cat. No. SC00A1-NSC), were similarly cultured except that neural MSC-Gro™ medium (Vitro Biopharma, Cat. No. NSCB1) and laminin-coatedT-25 flasks (Corning BioCoat, Cat. No. 354533) were used. Cultures were maintained in a humidified chamber equilibrated with 5% CO2, 1% O2, balance N2at 37SC. Cells were detached using Accutase (Innovative Cell Technologies Inc., Cat No. AT-104) and collected by centrifugation (450 x g) for 7 minute. Following aspiration, the cell pellet was resuspended in 1ml_ PBS and cells were counted using a Beckerman-Coulter Z2 particle counter (range 10 μιτι-30 pm).

Example 2: Stem Cell Migration Assay: In order to investigate the effects of drugs on human stem cell migration, a stem cell-based assay of migration was developed and validated. Figure 1 shows the basic components of the assay and shows the effects of substance P, a well-known inducing agent of cell migration. Figure 1 is a microphotograph depicting cell migration using fluorescent readout and cell tracker green as a fluorescent marker of human CB-MSCs. These cell images show fluorescent human MSCs (green) at the beginning of the assay (left panel) of the Control vs. Activating Agent (Substance P at 3.7 nM)) and 24 hours later (right panel). MSCs migrated to the cell-free center of the well and also filled open areas in other regions of the culture as a result of Substance P exposure (lower right panel) but did not similarly migrate in its absence (upper right panel). The EC50was determined as a measure of effectiveness of activating agents. Initial kinetic data allowed determination ofthe optimal assay parameters for further experiments. An initial analysis of the cell free zone using Image J software was used to screen concentrations of activating agents for dose-response determination in further experiments. It was determined that MSCs plated at 25,000/well, incubated for 24 hours and then exposed to appropriate activator concentrations gave optimal results.

Figure 1 shows the results of testing the effect of 3.7 nM Substance P on the migration of human CB-MSCs. In the absence of Substance P (upper panels), no migration was detected into the cell-free zone created by culturing cells in the presence of an occluding plug that prevented cell attachment in the center of the well during24 hours of culture and this was a consistently observed result under control conditions, i.e., no activator. On the other hand, 3.7 nM Substance P-induced MSC migration into the cell-free center of the well and increased fluorescence within the original, cell containing region of the well (Figure 1, lower two panels). Analysis of fluorescence within the cell-free region at the center of the well was used as a quantitative measure of cell migration as described below.

Figure 2 is a line graph representation illustrating percent closure as a function of time during live-cell data acquisition at different dosages of Substance P. This Figure 2 shows the dose-response of Substance P-induced migration of CB-MSCs. Per cent closure is shown under control conditions (no Substance P) and with increasing concentrations of Substance P as a function of time during the 24-hour period of live cell analysis. No migration is seen without Substance P, while migration increased in a dose-dependent manner by exposure to Substance P from 0 to 18.5 nM. Migration exhibited saturation at higher Substance P concentrations.

Figure 3 is a line graph representation illustrating migration of various humancell lines exposed to Substance P. This Figure 3 shows the dose response relationship for Substance P-induced migration of CB-MSCs, a primary human pancreatic cell line and NSCs as well. The data shows the dose-response curve of CB-MSCs (Red diamonds), human primary pancreatic fibroblasts (blue triangles), and human NSCs (blacksquares) together with EC50 values. Per cent closure was determined at 24 hours. By fitting the data to a sigmoidal curve, EC50 values of 2.48, 2.5 and 2.35 nM were calculated for CB-MSCs, human pancreatic fibroblasts, and NSCs, respectively. The EC50 obtained for human fibroblasts, i.e., 2.5 nM, compares well with a prior study of human fibroblast migration induced by Substance P of 2.2 nM using a suspension culture system to measure cell migration. Human fibroblast migration was mediated through theNK-1 receptor, since NK-1 receptor agonists mimicked Substance P and NK-1 receptor antagonists blocked Substance P induction of fibroblast migration. Fibroblast migration induced by Substance P is an important response to injury in addition to the induction of MSC migration. Parenti, A., et al., Naunyn Schmiedeberg’s Arch Pharmacol 353:475-481, 1996. Since the ECsofor Substance P is comparable to prior results, these results provide validation support for the cell migration assay.

The cell migration assays described above were set up as follows: One million cells/cell line were resuspended in 10 ml_ MSC-Gro™ serum free, quiescent medium (Vitro Biopharma Cat. No. SC00B17) containing 5 pg/mL mitomycin C (Sigma, Cat. No. M4287) to inhibit proliferation and incubated for 2 hours at room temperature with end- to-end agitation at 7 RPM. In some experiments, curcumin was used at 1 μΜ to block proliferation. Cells were centrifuged (450 x g) for 7 minutes, washed with PBS and then resuspended in 1 ml_ MSC-Gro™ low serum, complete medium (Vitro Biopharma Cat. No. SC00B1) and plated at 25,000 cells/well in black 96 well, TC-coated cell culture plates, (ThermoScientific, Cat. No. 165305) containing cell seed-stoppers (Platypus, Cat. No. CMAUFL4) to form a cell free zone at the centre of the well and incubated in 5%C02, 1% 02, 94% N2at 37QC in a humidified chamber for 24hrs. Plates used for culture of NSCs, were first treated with 10 pg/mL fibronectin (Sigma, Cat.No. F0556) for 2 hours at 37SC. Following washout with PBS (3x), cell seed stoppers were inserted, NSCs were plated at 25,000/well and incubated in 5%C02,1%02, 94%N2at 37eC in a humidified chamber for 48 hrs. For studies of the effects of CXCR4 and MMP-9 inhibition, following 24 hours of cell culture, appropriate wells were dosed with 15μΜ GM6001 and 20 μΜ AMD3100 for 6 hours.

Cells were washed once with PBS then incubated in serum free, MSC-Gro™ (Vitro Biopharma Cat. No. SC00B17) containing 5 μΜ Cell Tracker Green CMFDA (Molecular Probes, Cat. No. C7025) at 37SC for 30 minutes. The wells were then washed with serum free, MSC-Gro™ (Vitro Biopharma Cat. No. C00B17)and incubated for 30 minutes at 37SC. Cells were washed once with PBS and replaced with MSC-Gro™ serum free, quiescent medium (Vitro Biopharma Cat. No. SC00B17) containing different concentrations of activating agents. Substance P was from Tocris Bioscience, (Cat. No. 1156) and curcumin from Santa Cruz Biotechnology (Cat. No. SC- 200509A), VPA from Reagents Direct (Catalog Number 25-B43) and lithium chloride from Sigma Chemical Co. (Catalog number L4408). A TopSeal (PerkinElmer, Cat. No. 6050195) covered the plate for live-cell imaging in a BioTek Cytation3 Imaging Reader. Kinetic data was acquired every 2hrs for 24hrs using a GFP filter and bright field data acquisition. The gas phase throughout the acquisition of kinetic data was 5% 02j5% C02with the balance nitrogen maintained by a BioTek C02/02gas controller. Images were saved as TIFF files to calculate percent closure using imaging data.

Example 3: Effect of curcumin on human MSCs/NSCs migration: Because curcumin had been previously shown to enhance rat NSC proliferation and enhanced recovery of spinal cord injury by use of NSCs pre-treated with curcumin (Ormond, DR, et al, PLoS ONE 9: e88916, 2014), the effects of curcumin were tested onMSC migration and the results are shown in Figure 4, which is a line graph representation illustrating less than 1 μΜ curcumin induced migration of human CB-MSCs and 1 to 10 μΜ curcumin blocked migration due to apparent toxicity. Percent closure was determined after a 36-hour run period. At concentrations less than 1 μΜ, curcumin induced migration with an apparent EC50 of 250 nM and at concentrations greater than 1 μΜ, curcumin blocked migration by apparent toxicity indicated by reduced cell number with an estimated LD50 of 3 μΜ. These results also compare with prior studies (Ormond, DR, et al, PLoS ONE 9: e88916, 2014) providing further validation support for the cell migration assay. In addition to inducing proliferation of NSCs, curcumin also induces migration of MSCs and is toxic to MSCs at concentrations greater than 1 μΜ. Curcumin is thus emerging as a natural substance that serves as an activator of adult stem cells including MSCs and NSCs through increased stem cell proliferation and migration.

Example 4: Effects of lithium and VPA on migration of human stem cells: Figure 5 is a line graph representation illustrating migration of CB-MSCs induced by exposure to lithium alone (green diamonds), VPA alone (black squares) and VPA in the presence of 200 μΜ lithium (red diamonds). Percent closure is plotted as a function of dose and the data was modeled by sigmoidal curve fitting to calculate EC50 values. This Figure 5 shows lithium-induced MSC migration with a calculated ECsoof 79.12 μΜ and maximal migration at 200 μΜ lithium. VPA also induced CB-MSC migration with an ECsoof 38.45 μΜ with maximum closure at 100 μΜ. Since maximal lithium-induced closure occurred at 200 μΜ, the closure induced by increasing VPA concentrations in 200 μΜ lithium was then investigated. The results showed a lower ECsothan that observed with VPA only, i.e., 32.96 μΜ suggesting a synergistic effect of Li-VPA on CB-MSC migration.

The effect of Li-VPA on cell migration of different cell lines was determined; the results are shown in Figure 6, which is a line graph representation illustrating migration of CB-MSCs, NSCs and colorectal CAFs by induced by increasing VPA concentration in 200 μΜ lithium. Per cent closure is plotted as a function of VPA in medium containing 200 μΜ lithium. Percent closure is plotted as a function of dose and the data was modeled by sigmoidal curve fitting to calculate EC50 values. Migration of CB-MSCs (green squares), NSCs (blue diamonds) and colorectal CAFs (red triangles) are shown together with calculated EC50 values. While increasing VPA in 200 μΜ lithium showed similar kinetics between CB-MSCs and NSCs, ECsowas 36.02 and 35.19 μΜ, colorectal CAFs did not migrate to the same extent as MSCs and NSCs and similar data was obtained for other CAFs (data not shown). Thus, while MSCs and NSCs are robustly induced to migrate by Li-VPA, CAFs do not similarly migrate.

The molecular mechanisms of the effect of lithium and VPA were then investigated on stem cell migration. Since a prior report suggested that VPA up-regulated CXCR4, a critical chemokine receptor involved with cellular mobility, and that lithium up-regulated MMP-9 (Tsai, LK, et al., Stroke 42(10): 2932-2939, 2011), the effects of known inhibitors of CXCR4 and MMP-9 on the migration of CB-MSCs were determined and the results are shown in Figure 7, which is a line graph representation illustrating migration of human MSCs exposed to a combination of lithium and VPA with and without inhibition of MMP9 and CXCR4. The data shows the dose-response curve of CB-MSCs treated with Li and VPA alone (black squares), treated with Li and VPA and with inhibition of CXCR4 by AMD3100 (red diamonds), treated with Li and VPA and inhibition of MMP9 by GM6001 (green triangles), and treating with Li and VPA while inhibiting both CXCR4 and MMP9 (blue diamonds). Percent closure is plotted as a function of dose. The results indicate that the CXCR-4 inhibitor, AMD 3100, blocked the VPA-induced CB-MSC migration and that GM 6001, a competitive inhibitor of MMP-9, blocked lithium induced CB-MSCs. Also, in the presence of both AMD 3100 and GM6001, CB-MSC migration induced by Li-VPA was also blocked. These results suggest that CXCR4 and MMP-9 are molecular components of the VPA and lithium-induced migration of CB-MSCs thus confirming and extending prior results of Tsai, LK, et al., Stroke 42:2932-2939, 2011.

Example 5: Effects of lithium and VPA on stem cell proliferation: The effects of lithium and VPA on stem cell proliferation were then investigated, using an assay based on Presto Blue, a fluorescent marker of cellular reduction. Figure 8 is a line graph representation illustrating proliferation of different cell lines induced byincreasing concentrations of FGF-b (FGF-2) after a 5 day exposure. Relative fluorescent units were measured using a FITC filter on a Modulus Microplate Reader and plotted as a function of FGF-2. The data was modeled by sigmoidal curve fitting to calculate EC50 values. This Figure 8 shows the effect of FGF-2 on the proliferation of different cell lines including CAFs, primary fibroblasts and CB-MSCs. Proliferation of these mesenchymal cells was stimulated by recombinant human FGF-2 with EC50 values in the range of 3 to 6 ng/ml with maximal proliferative responses at about 10 ng/ml. Since these results are comparable to previous studies (Lee, TH, et al, Biochem Cell Biol. 26:1-8, 2015), these data provide validation support for the cell-based proliferation assay.

The proliferative effects of lithium and VPA were compared, either alone or in combination on CB-MSC proliferation, and the results are shown in Figure 9, which is a line graph representation illustrating proliferation of CB-MSCs induced by lithium, VPA and VPA in 200 μΜ lithium after a 5 day exposure. Relative fluorescent units were measured using a FITC filter on a Modulus Microplate Reader and plotted as a function of FGF-2. The data was modeled by sigmoidal curve fitting to calculate EC50 values. The EC50 value was 76.7 μΜ for lithium and this was comparable to the lithium effect on MSC migration (Figure 5). The ECsofor VPA was 47.71 μΜ and in the presence of 200 μΜ lithium, the ECsofor VPA was 63.31 μΜ. While MSC migration showed an apparent synergy with combined Li-VPA this does not appear to be the case for MSC proliferation, since larger VPA dosages were needed for equivalent MSC proliferation in the presence of 200 μΜ lithium.

The proliferative responses of CB-MSCs and NSCs were compared to Li-VPA and the results are shown in Figure 10, which is a line graph representation illustrating proliferation of CB-MSCs and NSCs as a function of increasing VPA in 200 μΜ lithium after a 5 day exposure. Relative fluorescent units were measured using a FITC filter on a Modulus Microplate Reader and plotted as a function of FGF-2. The data was modeled by sigmoidal curve fitting to calculate EC50 values. While both NSCs andMSCs exhibited comparable EC50, 36.78 and 39.02 μΜ, respectively, the extent of proliferation is reduced in NSCs compared to MSCs and this may reflect intrinsic proliferative capacity. Also, since the cell migration results yielded similar results, both proliferation and migration of MSCs and NSCs may be activated at similar reduced dosage when lithium and VPA are administered in combination. Thus in the presence of 200 μΜ lithium, VPA is 50% effective in inducing cell proliferation and migration at 35 to 39 μΜ (mean 37 μΜ).

Example 6: Effects of VPA and lithium on stem cell gene expression: In Figure 11, results of gene expression analysis are shown following the exposure of MSCs to VPA. Figure 11 is a bar graph representation illustrating PCR used to measure target genes known to be subject to epigenetic regulation by FIDAC inhibitors. The graph shows the result of human MSCs treated with VPA alone or in combination with lithium. The expression of Oct 3/4, a well-known pluripotency gene, was increased about20-fold compared to untreated human MSCs. The expression of SIRT-1 was highly elevated compared to untreated MSC by ~300-fold without differences between treatment with either VPA or lithium-VPA. The expression of FGF-21 was also elevated by 3 to 5-fold and its expression was higher in MSCs treated with Li-VPA, although this increase with not significant. All gene expression was normalized to untreated MSCs.

Beta actin was measured as a house-keeping gene. The graph shows the result of gene expression analysis of human MSCs treated with VPA only or VPA+ lithium. Gene expression was quantified by determining the amount of gene-specific DNA/total cDNA. Data is mean +/- SD of 4 replicates. These results thus show increased expressionof Oct 3/4, SIRT-1 and FGF-21 as a result of exposure to VPA or Li-VPA using 200 μΜ lithium and 31.25 μΜ VPA.

Native human cord blood-derived MSCs (Vitro Biopharma Cat. No. SC00A1) were expanded from cryopreservation in a T-25 TC-coated flasks (BD Falcon, Cat. No. 353108) in MSC-Gro™ low serum, complete medium (Vitro Biopharma Cat. No. SC00B1). Cells were sub-cultured and counted on a Beckerman-Coulter Z2particle counter (range 10 μιη-30 pm). Cells were plated at 10,000/cm2 a TC-coated Greiner Bio-One T75 flask and maintained in MSC-Gro™ serum free, complete medium (Vitro Biopharma Cat. No. SC00B3) in a reduced 02environment (1%02, 5%C02, 94%N2) at 37SC in a humidified chamber. The MSCs were treated continuously for up to 2weeks. Cultures were fed every three days. Cells were harvested using Accutase(Innovative Cell Technologies Inc., Cat No AT-104) and centrifuged (450 x g) for 7 minute. Cell supernatant was aspirated off and cells were resuspended in 1ml_ PBS and counted on a Beckerman-Coulter Z2 particle counter (range 10 pm-30pm).

Total RNA was extracted using RNeasy Mini Kit (Qiagen Cat. No. 74104). RNA was quantified using an absorbance measurement at 260nm. RNA was converted to cDNA using Quantitect Reverse Transcription Kit (Qiagen Cat. No.205310) in a thermocycler. RNA was incubated in the gDNA elimination reaction for 2 minutes at 42SC then incubated in the reverse-transcription master mix for 15 minutes at 42SC.

Immediately after, it was incubated at 95SC for 3 minutes for inactivation. cDNAwas sent to an outside lab (CU-Anschutz Metabolic Laboratory) for q-PCR to detect relative or absolute gene expression levels. cDNA was diluted 1:5 and iTaq Universal Supermix fluorescent probe (BioRad Cat. No. 172-5120) used to detect the threshold cycle (Ct) during PCR. Dilution factors and cDNA concentrations were calculated into recorded values then normalized to untreated hMSCs (Vitro Biopharma Cat. No.SCOOAl).

Example 7: Secretion of cytokines from MSCs: Preliminary experiments were performed concerning the composition of cytokines within the soluble factors secreted from stem cells and the results are shown in Figure 12, which is a bar graph representation illustrating cytokine levels in cell culture media exposed to CB-MSCs for 24 days and determined by microarray analysis. Conditioned media was analyzed using an inflammation microarray, Th17 microarray and a bone metabolism array. Results show an increase in inflammatory cytokines as well as adhesion factors. With a substantial increase in the content of MIP-3a, ICAM-1, IVCAM-1 and VE-Cadherin within the conditioned medium, this suggests the well-known immunosuppressive role of MSCs.

Native human cord blood-derived MSCs (Vitro Biopharma Cat. No. SC00A1) were plated at 1,000/well in a tissue cultured 6-well plate (BD Falcon, Cat Number 353046) in MSC-Gro™ low serum, complete medium (Vitro Biopharma Cat. No. SC00B1). Cells were continuously grown for a period of 30 days. Conditioned media was collected at day 3, 6, 12, 18, and 24. Multiple microarrays were run using conditioned media for cytokine secretion determination after a continuous 24 dayculture period. An inflammation microarray (Ray Biotech, Cat. No. QAH-INF-3), bone metabolism microarray (Ray Biotech, Cat. No. QAH-BMA-1) and aTh17 microarray (Cat. No. QH-TH17-1) were used to analyze the conditioned media. A laser scanner (Molecular Probes, Genepix 4000B) was used to measure the fluorescent signals of each microarray.

Claims (17)

Claims

1. A method of treating a neurological condition in a subject in need thereof, comprising administering to the subject a combined therapy comprising lithium carbonate and valproic acid, sodium valproate, or divalproex sodium, wherein the lithium concentration is from about 0.2 to 0.4 mEq/ml and the free valproicacid concentration in cerebral spinal fluid is about 5.0 to 10.0pgm/ml.

2. The method of treating a neurological condition of claim 1, whereinthe neurological condition is at least one of the group: Alzheimer’s disease, traumatic brain injury, a concussion, Parkinson’s disease, amyotrophic lateral sclerosis,dementia, Huntington’s chorea, meningitis, stroke, autism spectrum disorders, memory loss, spinal cord injury, and impaired cognitive function.

3. A method of treating a neurological condition in a subject in need thereof, comprising obtaining stem cells from the subject and culturing the stem cells to induce expansion, followed by exposure to lithium carbonate at a concentration betweenabout 0.05 to 0.25 mEq/ml and to VPA at a concentration of between about 2.5 to 15pgm/ml and subsequent transplantation of said stem cells back into the subject.

4. The method of treating a neurological condition of claim 3, wherein the stem cells from the subject are obtained by biopsy from the subject's nasal epithelia.

5. The method of treating a neurological condition of claim 3, wherein the neurological condition is at least one of the group: spinal cord injury and macular degeneration.

6. A method for assessing the clinical status of a subject having traumatic brain injury, comprising measuring S-100-beta, nerve-specific enolase, MCP-1,SDF1a, MMP-9, CXCR4, IL6, CXCL5, heat shock protein 70 and prostaglandin E2, Fas,CRP and BDNF in the subject.

7. A method of treating a neurological condition in a subject in need thereof, comprising: a) obtaining stem cells from the subject and culturing the stem cells to induce expansion, b) transplanting the expanded stem cells back into the subject; and c) subsequently administering to the subject a combined therapy comprising lithium carbonate at a concentration of from about 0.1 to 0.4mEq/ml and free VPA at concentration of from about 2.5 to 15pgm/ml.

8. The method of treating a neurological condition of claim 7, wherein the stem cells from the subject are obtained by biopsy from the subject's nasal epithelia.

9. The method of claim 7, wherein the stem cells from the subject are mesenchymal stem cells obtained from the subject.

10. A method of treating a neurological condition in a subject in need thereof, comprising administering to the subject valproic acid, sodium valproate, or divalproex sodium wherein the free valproic acid concentration is about 5.0 to 10.0 pgm/ml within cerebral spinal fluid.

11. A method of treating a neurological condition in a subject in need thereof, comprising administering to the subject valproic acid, sodium valproate, or divalproex sodium wherein the free valproic acid concentration in cerebral spinal fluid is about5.0 to 10.0 pgm/ml together with methods that result in the dissociation of polysialicacid from neural cell adhesion molecule.

12. The method according to claim 11 wherein the method of dissociation of polysialic acid from neural cell adhesion molecule involves administration of atleast one of: a mimetic peptide, small inhibiting RNA specific to ST8Sia I and ST8Sia V,and N-butylmannosamine.

13. A combination therapy for treatment of a neurological condition, comprising lithium carbonate and valproic acid, sodium valproate, or divalproex sodium, whereinthe lithium concentration is from about 0.2 to 0.4 mEq/ml and the free valproicacid concentration in cerebral spinal fluid is about 5.0 to 10.0pgm/ml.

14. A combination therapy for treatment of a neurological condition in a subject from whom stem cells had been obtained, cultured to induce expansion, and then returned to the subject, the combination therapy comprising lithium carbonate at a concentration of from about 0.1 to 0.4 mEq/ml and free valproic acid at a concentration of from about 2.5 to 15pgm/ml.

15. A combination therapy according to Claim 14, wherein the free valproic acid is provided by at least one of valproic acid, sodium valproate and divalproex sodium.

16. A compound selected from valproic acid, sodium valproate and divalproex sodium for use in the treatment of a neurological condition in a concentration of free valproic acid in cerebral spinal fluid of about 5.0 to 10.0 pgm/ml in combination with an agent adapted to result in dissociation of polysialic acid from neural cell adhesion molecule.

17. A combination therapy comprising a compound according to Claim 15 and at least one of a mimetic peptide, small inhibiting RNA specific to ST8Sia I and ST8Sia V, and N-butylmannosamine.