WO2019053729A1 - An enriched artificial cerebrospinal fluid compositions methods for preparation of same and uses thereof - Google Patents

Abstract

The present disclosure provides an enriched artificial cerebrospinal fluid (EA-CSF) composition comprising: an electrolyte containing solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and at least one neuroprotective factor. Also provided by the present disclosure are methods for producing the EA-CSF as well as methods of using the same for treating diseases, specifically, neurological diseases.

Description

AN ENRICHED ARTIFICIAL CEREBROSPINAL FLUID COMPOSITIONS METHODS FOR PREPARATION OF SAME AND USES THEREOF

TECHNOLOGICAL FIELD

The invention relates to enriched artificial cerebrospinal fluids, methods of preparation and uses thereof.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

• Kristof RA, Clusmann H, Koehler W, Fink KB and Schramm J: Treatment of accidental high dose intraventricular mezlocillin application by cerebrospinal fluid exchange. J Neurol Neurosurg Psychiatry 64: 379-81, 1998.

Geng L, Ma F, Liu Y, Mu Y, Zou Z. Massive Cerebrospinal Fluid Replacement Reduces Delayed Cerebral Vasospasm After Embolization of Aneurysmal Subarachnoid Hemorrhage. Med Sci Monit. 2016 Jul 10;22:24Q4-8.

Menendez Gonzalez M. Mechanical Dilution of Beta-amyloid Peptide and Phosphorylated Tau Protein in Alzheimer's Disease: Too Simple to be True? Cureus. 2017 Feb 28;9(2):el062. doi: 10.7759/cureus. l062.

• International Patent Application Publication No. WO 16/059162

International Patent Application Publication No. WO 2015/056258

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Degenerative diseases of the central nervous system (CNS), affecting cognitive and motor functions, are the most common causes of chronic disability in adult life. Although impressive advances in understanding the mechanisms of neurodegenerative diseases were accomplished, no effective drug is yet available.

One strategy for the treatment of neurological diseases may be to "change maximally the CNS milieu from a pathogenic to a healthy milieu" . One route for such a change of CNS milieu is by exchanging the fluid which is in direct contact with the whole CNS, i.e. the cerebrospinal fluid (CSF). Optimal functioning of CSF depends on homeostatic balances (production, composition, flow and resorption), and their disruption can directly underline CNS disorders, such as in Alzheimer's disease, in which deficit in CSF turnover is implicated. Changes in the composition of the CSF are evident in many neurodegenerative diseases, including disease-specific proteins and others (like growth factors and neurotrophic agents). Its immediate contact with the brain and its ability to swiftly distribute signals across vast distances in the CNS - makes the CSF a suitable route for therapeutic intervention.

Kristof RA et al. describe that CSF exchange performed on a patient with serial epileptic seizures results in the patient recovery of clinical status.

Geng L, et ai. describe that massive cerebrospinal fluid replacement after embolization surgery for aneurysmal subarachnoid hemorrhage (SAH) can significantly reduce Delayed cerebral vasospasm (DCVS) occurrence and effectively improve the outcomes.

Menendez Gonzalez M. describes the rationale of mechanical dilution of cerebrospinal fluid (CSF) as a therapeutic approach in Alzheimer's Disease and present models of implantable systems allowing mechanical dilution of CSF by means of CSF replacement and CSF filtration (liquorpheresis).

WO 16/059162 describes the use of CSF, more particularly external CSF or CSF- like compositions for the treatment and prevention of different diseases by administration of CSF to the intrathecal space or the cerebral ventricles of patients to increase intracranial pressure and/or CSF flow.

WO 2015/056258 describes methods of inducing transdifferentiation of mesenchymal stem, cells (MSC) into a population of cells comprising terminally differentiated neurons, astrocytes and oligodendrocytes, using several steps and comprising culture in a media comprising human derived cerebrospinal fluid (CSF). SUMMARY OF THE INVENTION

In a first aspect, the present disclosure provides an enriched artificial cerebrospinal fluid (herein "enriched aCSF", "EA-CSF" or in sort "eCSF") composition which, based on the experimental data presented herein, is considered to be an artificial neuroprotective CSF composition. More specifically, the EA-CSF composition disclosed herein comprises an electrolyte containing solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and at least one neuroprotective factor. Such EA-CSF was found to have a beneficial therapeutic effect in animal models of acute (short tenn) and chronic (chronic) ne rodegenerative diseases as further described below.

In a second aspect, the present disclosure provides a method of preparing an EA-CSF composition. More specifically, the method comprises culturing MSCs in a culture medium comprising an electrolyte solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium, ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and in due course, removing cells from the culture medium (collecting medium or cell supernatants of the cells, containing the aCSF with the cell secretions) to obtain the EA-CSF composition.

In a third aspect, the present disclosure provides a method of preparing an EA-CSF composition, the method comprises mixing an electrolyte solution comprising at least one electrolyte selected from the group consisting of sodium, ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion with umbilical cord plasma.

In yet a fourth aspect, the present disclosure provides a method of treating a disease in a subject in need. More specifically, the method comprises administering to the subject an EA-CSF composition comprising an electrolyte solution comprising at least one ion selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and at least one neuroprotective factor. BRIEF DESCRI PTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figure 1 A-1B: Schematic representation of the Study design

Fig, 1A: is a schematic illustration of a method of producing an enriched artificial CSF ("enriched aCSF") according to which MSCs driven from bone marrow were grown in artificial CSF (aCSF) and the supernatant was collected and then used in in vitro assays.

Fig, IB: is a schematic illustration of a method of producing the enriched aCSF and use thereof for CSF exchange therapy in EAE-mice, including infusion of the enriched aCSF into one ventricle ("in"), and withdrawal of endogenous CSF from the other ventricle ("out") of the mice.

Figures 2A-2B: MSCs are viable when growing in aCSF

Fig, 2A is a is a graph showing cell viability using the MTT assay when using various aCSF systems.

Fig, 2B is a graph showing the Optical density (OD) of MSCs grown in aCSF and in Nutristem™ medium (Biological Industries, Israel) for 7 days for comparing the viability of the MSCs grown for 1-5 days in artificial CSF to that of the ceils grown with Nutristem™.

Figure 3A-3B: Enriched aCSF increases cell viability of PC12 neuronal cell line after 5 days of culture.

Fig. 3A: is a graph showing optical density of PC12 cells grown in either aCSF, or in 10K or 100K MSC secretion enriched aCSF, after 2 day s of culture;

Fig, 3B: is a graph showing optical density of PC 12 cells grown in either aCSF, or in 10K or 100K MSC secretion enriched aCSF, after 5 days of culture . Figure 4A-4B: Enriched aCSF increases cell viability of SH-SY5Y and PC12 neuronal cell lines.

Fig. 4A: is a graph showing optical density of SH-SY5Ycells grown in either aCSF, or in 10K or 25K MSC secretion (secretions derived from 5 days growing) enriched aCSF. Fig, 4B: is a graph showing optical density of PC I 2 cells grown in aCSF, or in 10K or 25 K MSC secretion (secretions derived from 5 days growing) enriched aCSF.

Figure SA-SB: Enriched aCSF increases cell viability of neuronal cell lines exposed to H2O2 and Αβ neurotoxins.

Fig, 5A: is a graph showing optical density of PC 12 cells grown in aCSF only or cells grown in either aCSF or in 10K or 25K MSC secretion enriched aCSF (secretions derived from 5 days grown 10K-25 7 ml MSCs) and exposed to H2O2

Fig. SB: is a graph showing optical density of SH-SY5Y cells grown in aCSF only, or cells in either aCSF only, or in 10K or 25K MSC secretion (secretions derived from 5 days growing) enriched aCSF, and exposed to Αβ

Figure 6: Optical density of PC 12 cells grown in aCSF only, or in aCSF enriched with 15% umbilical cord plasma.

Figure 7: Suppression of spleen lymph node cell (LNC) proliferation by Enriched aCSt.

Figure 7 is a graph showing cell proliferation where LNCs excised from mice were grown with aCSF enriched with the MSCs secretions (secretions derived from 5 days growing) or with non-enriched aCSF, and assayed for their proliferation in response to the concanavalin mitogen. As positive control conditioned medium (DMEM) grown on MSC ceils was used to demonstrate suppression of LNC proliferation

Figure 8A-8D: Enriched aCSF (eCSF) (in/out) exchange therapy showed a short- term amelioration of the EAE clinical symptoms.

Fig. 8A: is a graph showing the Average clinical score along time of EAE control mice and mice treated with (in/out) enriched aCSF ("eCSF").

Fig, 8B: is a graph showing the Average cumulative score of EAE control mice and in mice treated with (in/out) eCSF. Fig, 8C: is a graph showing the Average maximal score of EAE control mice and in mice treated with (in/out) eCSF.

Fig. 8D: is a graph of the Kaplan Meier analysis showing the proportion of disease free mice relative to the untreated mice. Log-Rank Test: p=^:0.028

Figure 9A-9D: Prolonged amelioration of the EAE clinical symptoms during a prolonged CSF exchange therapy.

Fig, 9A: is a graph showing the Average clinical score along time of EAE control mice, mice treated with (in/out) eCSF, mice treated with (in) eCSF and mice treated with (in/out) aCSF. Start treatment (5 days/wk)

Fig, 9B: is a graph showing the Average cumulative score of EAE control mice, mice treated with (in) eCSF, (in/out) aCSF and (in out) eCSF.

Fig, 9C: Graph showing the Average maximal score of EAE control mice, mice treated with (in) eCSF, (in/out) aCSF and (in/out) enriched aCSF,

Fig, 9D: is a graph of the Kaplan Meier analysis revealing significant less EAE disease- free mice among the different groups.

Figure 10A-10J: Enriched aCSF reduced xonal damage and demyeUnation in EAE- mice.

Fig. 10A: is a graph showing the bieischowsky score of EAE control mice and mice treated with (in/out) eCSF.

Fig, 10B: is a graph showing the bieischowsky score of EAE control mice and mice treated with (in) eCSF.

Fig, 10C: is a graph showing the bieischowsky score of EAE control mice and mice treated with (in/out) aCSF.

Fig, 10B: is a picture of histological section of brain of EAE control mice with bieischowsky staining

Fig, 10E: is a picture of histological section of brain of mice treated with enriched aCSF with bieischowsky staining

Fig. 10F: is a graph showing Luxol Fast Blue score of EAE control mice and mice treated with (in/out) enriched aCSF (eCSF). Fig, 10G: is a graph showing Luxol Fast Blue score of EAE control mice and mice treated with (in) enriched aCSF (eCSF).

Fig. 10H: is a graph showing Luxol Fast Blue score of EAE control mice and mice treated with (in/out) aCSF.

Fig, 101: is a picture of histological section of brain of EAE control mice with LFB staining

Fig, 10 J: is a picture of histological section of brain of mice treated with enriched aCSF with LFB staining

Figure 11A-11C: Enriched aCSF reduced cognitive deficits in a prolonged therapy in AO-mice.

Fig, 11 A: is a schematic representation of the design of AD-mice treated with (in/out) Enriched aCSF exchange therapy at days 2-4 (a cluster of 3 days therapy) following disease induction by amyloid-beta (Αβ) ICV, and further treated repeatedly at days 7-17 for 2 times/ week. Cognitive tests were conducted during these 7-17 days of therapy. Fig, 11B: is a graph showing the Y-maze results ("correct trios") of non-AD mice, AD mice, AD mice treated with enriched aCSF (AD-eCSF) and AD mice treated with aCSF (AD-aCSF).

Fig, 11C: is a graph showing the Radial arm water maze results (seconds to reach platform) obtained with non-AD mice, AD mice, AD mice treated with enriched aCSF (AD-eCSF) and AD mice treated with aCSF (AD-aCSF).

Figure 12A-12C: Short-term therapy with Enriched aCSF reduces cognitive deficits in AD-mice.

Fig, 12A: is a schematic representation of the design of AD-mice were treated with (in/out) enriched aCSF exchange therapy at days 2-4 (a cluster of 3 days therapy only) following disease induction. Cognitive tests were conducted at days 9-14 following disease induction.

Fig. 12B: is a graph showing the Radial arm water maze results obtained with non-AD mice, AD mice and AD mice treated with enriched aCSF (AD-eCSF). Fig, 12C: is a graph showing die Y maze results obtained with non-AD mice, AD mice and AD mice treated with enriched aCSF (AD-eCSF).

Figure 13: is a graph showing the T maze results obtained with AD-Tg mice, AD-Tg mice treated with CSF exchange therapy using the umbilical cord plasma (diluted in artificial CSF) and Wild Type mice

Figure 14: is a graph showing the Y maze results obtained with Wild Type mice, Tau- Tg mice and Tau-Tg mice treated with CSF exchange therapy (in/out) using umbilical cord plasma

Figure 15: is a graph showing the results of the Open field habituation test (delta distance of day2~dayl) obtained with Wild Type mice, Tau-Tg mice and Tau-Tg mice treated with CSF exchange therapy using umbilical cord plasma

Figure 16A-16B: Performance in open field between CSF exchange (in/out) treated ALS Tg mice and control ALS Tg mice.

Fig. 16A: is a graph showing the Distance traveled by treated animals (enriched aCSF) in comparison with untreated animals (control) along age (days).

Fig, 16B: is a graph showing the time spent in the center zone by enriched aCSF treated animals (eCSF) in comparison with untreated animals (control) along age (days).

Figure 17A-17F: are graphs of levels of neuroprotective factors secreted from hMSC, neuralized MSC and terminally differentiated MSC (diff MSC) as obtained from a Ready-to-Use Elisa Kit (results presented in Optical Density Measurement (OD405).

Fig, 17A is a graph showing levels of BDNF; Fig. 17B is a graph of levels of NGF; Fig. 17C is a graph showing levels of GDNF; Fig. 17D is a graph showing levels of NT~4; Fig. 17E is a graph showing levels of NT-3; Fig. 17F is a graph showing levels of CNTF, DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based on the development of an enriched artificial CSF that was found, inter alia, to improve clinical parameters of acceptable animal models for neurodegenerative diseases and disorders. Neurodegenerative disorders are characterized by progressive loss of neuronal function due to death of those cells in tlie brain. Many ne urodegenerative disorders have similar pathological characteristics, even though they may express plethora of phenotypes. Most studies failed to provide evidence of the pathological triggers leading to tlie onset of the disorders. Therefore, many of the neurodegenerative diseases are still incurable. One approach in treating neurodegenerative disorders involve "washing out" of toxic elements secreted from cells by exchanging the "damaged^" CSF, to thereby delay neuronal death of tlie brain and disease progression. CSF replacement can be achie ved by using artificial CSF that is suitable for supporting the viability of neurons.

Artificial CSF (aCSF) are known in the art and are typically used as a vehicle for administration of test material in animal models to the CNS.

The term "artificial CSF' (aCSF or A-CSF) as used herein refers to a buffer solution that closely matches the electroh'te concentrations of cerebrospinal fluid and is used experimentally to immerse isolated brains, brain slices, or exposed brain regions to supply oxygen, maintain osmolarity, and to buffer pH at biological levels.

Artificial CSF can be obtained commercially, or prepared according to known protocols. Some exemplary aCSF include the following composition (which have been used in the following examples, see Fig. 2B):

Composition aCSFl NaCl 125mM, KC3 2.5nM, MgCl₂,6H₂0 I mM, NaH₂P0₄ 1.25mM,

CaCl₂.2H₂0 2mM, NaHC0₃ 25mM, Glucose 25mM

aCSF2 NaCl 7-l m.M. KC1 1.5nM, MgC1.6H₂0 0.4m M. NaH₂PG₄ 1.125mM,

CaCl₂.2H₂Q 1 ,4mM, Na₂HP0₄ 1.5niM

aCSF3 NaCl !25mM, KC1 2.5nM, MgCl₂.6H₂0 ImM, NaH₂PQ₄ 1.25mM,

CaCl₂.2H₂() 2mM, NaHC0₃ 25mM, Glucose 25mM, MgCl₂ ImM aCSF5 NaCl 125mM, KC1 2.5nM, MgCl₂.6H₂() ImM, NaH₂P0₄ 1.25mM,

CaCi .21 1 .0 2mM, NaHCQ₃ 25mM, Glucose 25mM, D-glucose 25mM aCSF5 NaCl 125mM, KC1 2.5nM, MgCl₂.6H₂0 ImM, aH₂P0₄ 2.5mM,

CaCb.2H₂0 2mM, NaHCOs 25mM, Glucose 25mM The present disclosure is based on the development of an enriched aCSF, i.e. CSF that at minimum comprises electrolytes, as in various known artificial CSF compositions, and is supplemented by secretions from cells possessing neuroprotective properties, thus enriching the electrolyte solution (specifically, aCSF) with a protective and supportive environment to the neurons.

This novel approach may be beneficial for many neurodegenerative indications, as it offers the elimination or dilution of "damaged" CSF together with its enrichment with protective and supportive components to the neurons, regardless of the underlying mechanisms or types of neuronal toxins secreted.

Therefore, in a first aspect, the present disclosure provides an enriched artificial cerebrospinal fluid ("enriched aCSF", "AE-CSF" or in short "eCSF") composition. More specifically, the EA-CSF composition comprises an electrolyte solution and at least one, but typically more than one, neuroprotective factor.

An electrolyte solution is a solution that generally contains ions, atoms or molecules that have lost or gained electrons, and is electrically conductive.

In accordance with the present disclosure, the electrolyte solution comprises at least one electrolyte selected from sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and/or carbonate ion.

In some embodiments, the electrohle solution comprises at least 2, or at least three, or at least 4 or at least 5 or at least 6 of all the above listed electrolytes.

Additional molecules or ions that can form part of the electrolyte solution according to the present disclosure, include, without being limited thereto, a saccharide. In some embodiments, the saccharide can be a glucose, including D-glucose.

In some embodiments, each ion of the EA-CSF disclosed herein, if present in the electrolyte solution, is at a concentration that correlates with its concentration within human cerebrospinal fluid. In the context of this embodiment, when referring to a correlating concentration it is to be understood as having a similar but not necessarily identical concentration as in the human CSF, the similarly should be sufficient to provide an overall functional aCSF, i.e. an aCSF that provides the supportive environment for neurons. In some embodiments, each electrolyte, when present in the composition, is at a concentration as follows :

Sodium ion: at least 50mM, at times between 50 mM and 250 mM, at times, between 70mM and 200mM, at times, between lOOmM and 150mM; in some embodiments, at a concentration of 125nM ±20%.

Potassium ion: at least InM, at times, between 1 nM and 4.5 tiM, at times, between 1.5nM and 3nM, at times between 2nM and 3nM, in some embodiments, at a concentration of 2.5nM±20%.

Calcium ion: at least InM, at times, between 1 nM and 2.5 mM, at times between 500nM and 2.5mM, at times between ImM and 2.5mM, in some embodiments, at a concentration of 2mM±20%.

Magnesium wn: at least lOOnM, at times, between 0.3 mM and 2 mM, at times, between ImM and 1.8mM, at times between ImM and 1.5mM; in some embodiments, at a concentration of lmM±20%.

Phosphor ion: at least lOOnM, at times, between 0.5 mM and 3 mM, at times, between ImM and 2.5mM, at times, between ImM and 1.5mM; in some embodiments, at a concentration of 1.25mM±20%.

Chloride ion: at least lOnm and at times between 50 mM and 200 mM.

Carbonate ion: at least ImM, at times between 10 mM and 50 mM, at times between 20mM and 30mM; in some embodiments, at a concentration of 25mM±20%.

In some other embodiments, the EA-CSF composition disclosed herein comprises one or more, at times, two, three, four, five, four or even ail sever of the salts selected from the group consisting of NaCl, KC1, MgCh, Na3¾P04, CaC , \a>! I O : and NaHCOj, each combination of salts constituting a separate embodiment of the present disclosure.

In some embodiments, the EA-CSF comprises the following compositions (the indicated concentration may fluctuate by ±10%): NaCl 125mM, KC1 2.5nM, MgCl₂.6H₂0 ImM, NaH₂PQ₄ 1.25mM, CaCl₂.2H₂0 2mM, NaHC0₃ 25mM, Glucose The electrolyte solution can be manufactured by mixing the electrolytes at the desired concentrations.

In some embodiments, the human CSF is used as a source for the electrolytes/electrolyte solution, as further described below. Thus, in the context of the present disclosure, when referring to EA-CSF it is to be understood as encompassing also a composition obtained from human CSF that has, however, been artificially supplemented with one or more neuroprotective factors.

As used herein, the tenn "neuroprotective factor" relates to any biological factor enabling the relative preservation of neuronal structure and/or function and/or preventing or slowing disease progression and secondary injuries by halting or at least slowing the loss of neurons. In the case of an ongoing insult (a neurodegenerative insult) a relative preservation of neuronal integrity implies a reduction in the rate of neuronal loss over time.

In some embodiments, the neuroprotective factor comprises one or a combination of neurotrophic factors, anti-inflammatory cytokines and anti-oxidative agents.

In some embodiments, the neuroprotective factor comprises a neurotrophic factor.

In the context of the present disclosure, the term "neurotrophic factor" or "NTFs" relates to a family of biomolecules that support the growth, survival, and differentiation of both developing and mature neurons. In the mature nervous system, they promote neuronal survival, induce synaptic plasticity, and modulate the formation of long-term memories. Some neurotrophic factors also promote the initial growth and development of neurons in the central nervous system and peripheral nervous system, and they are known to be capable of re-growing damaged neurons in test tubes and animal models.

Most neurotrophic factors belong to one of three families: (1) neurotrophins,

(2) glial cell-line derived neurotrophic factor family ligands (GFLs), and

(3) neuropoietic cytokines. Any neuroprotective factor out of these families can be used in the-EA-CSF disclosed herein. In some embodiments, the neuroprotective factor comprises at least one neurotrophic factor.

In some embodiments, the neurotrophic factor is selected the group consisting of Ciliary neurotrophic factor (CNTF), Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophm-4 (NT-4).

In some embodiments, the EA-CSF comprises a combination of neurotrophic factors.

In some embodiments, the EA-CSF comprises a combination of two, three, four, five, or even six neurotrophic factors selected the group consisting of BDNF, GDNF, NGF, NT-3 and NT-4, each possible combination constituting a separate embodiment of the present disclosure.

In some embodiments, the EA-CSF comprises at least BDNF.

In some embodiments, the EA-CSF disclosed herein comprises a concentration of at least O. lpg/ml at times between 1 pg/ml and 2,000 pg/ml, at times between Ipg/mi to l,000pg/ml, further at times 1 pg/ml to lOOpg/ml, for each neurotrophic factor present in the composition. In some embodiments, and without being limited thereto, the concentration is 22±4.83pg/ml, as may be obtained in secretions of 600,000MSC.

In some embodiments, the neuroprotective factor comprises an anti -inflammatory cytokine .

In the context of the present disclosure, the term "anti-inflammatory cytokine" refers to any immunoregulatory molecule that is involved and may control pro-inflammatory cytokine responses.

In some embodiments, the anti-inflammatory cytokine is selected from the group consisting of Transforming Growth Factor beta (TGF-β), Interleukin 4 (IL-4) and Jnterleukin 10 (IL-10).

In some embodiments, the EA-CSF comprises a combination of anti -inflammatory cytokines. In some embodiments, the EA-CSF comprises anti-inflammatory cytokines at a concentration of at least O. lpg/ml, at times, at least Ipg/mi, at times between O. lpg/ml and 2,000pg/ml, at times between I pg/ml and l,000pg/ml.

In some embodiments, the EA-CSF comprises at least TGF-β.

In some other embodiments, the EA-CSF disclosed herein comprises the antiinflammatory cytokine(s) TGF-β at a concentration of at least Ipg/rnl and at times between 100 and l,000pg/ml, and in some further embodiments, at a concentration of 535.25±49.16 pg/ml as may be obtained in secretions of 600,000MSC.

In some embodiments, the neuroprotective factor comprises an anti-oxidative agent.

In the context of the present disclosure, the term "anti-oxidative agent" refers to compounds that inhibit oxidation i.e. a chemical reaction producing free radicals which would lead to cells' oxidative damage.

Without being limited thereto, aCSF enriched by secretions of 600,000 MSG cells was tested for the anti-oxidant capacity (Sigma kit), showing about 0.2 ± 0.05 nmol/microliter Troiox (vitamin E analogue) equivalents of non-enzymatic anti-oxidant capacity (attributed to compounds such as: ascorbic acid (vitamin C), glutathione, uric acids, carotenes, a-tocopherol (vitamin E), and ubiquinol (coenzyme Q).

In some embodiments, the anti-oxidative agent is selected from the group consisting of ascorbic acid (vitamin C), glutathione, uric acids, carotenes, a-tocopherol (vitamin E), and ubiquinol (coenzyme Q) and any combination of same. Each antioxidant constituting a separate embodiment.

In some embodiments, the EA-CSF composition disclosed herein comprises a combination of one or more neurotrophic factors, one or more anti-inflammatory cytokines and/or one or more anti-oxidative agents. As such, in the context of the present disclosure, the neuroprotective factor within the EA-CSF is to be understood as encompassing either a single factor, or a cocktail of two or more neuroprotective factors including factors of different types and different concentrations.

In some embodiments, the EA-CSF composition comprises a combination of at least BDNF, TGF-β and a mixture of anti-oxidant compounds including at least one of ascorbic acid (vitamin C), glutathione, uric acids, carotenes, a-tocopherol (vitamin E), and ubiquinol (coenzyme Q).

The EA-CSF disclosed herein also encompasses any neuroprotective factor present or derived from umbilical cord plasma. Thus, an alternative for artificially adding or obtaining neuroprotective factor from secretion of MSC, the present disclosure also provides an EA-CSF composition comprising the electrolyte solution as defined herein and umbilical cord plasma.

Umbilical cord blood plasma (UCB-PL) contains various cytokines, growth factors, and immune modulatory factors that regulate the proliferation and function of immune cells and adult stem cells. As such, the UCB-PL is rich with neuroprotective factors suitable for use in the EA-CSF of the present disclosure.

Without being limited thereto, factors detected in umbilical cord plasma (without dilution with aCSF) include BDNF (>1000 pg/ml), TGF-β (30,000 -35,000pg/ml, such as 31,585 pg/ml), IL-10 (5-15pg/ml, such as 9.54 pg/ml) and total anti-oxidant capacity (>20nmol/microliter) .

Hie EA-CSF composition disclosed herein was found to be advantageous in treating, both in vitro an in vivo, of neural cells. Specifically, it lias been found that the EA-CSF has a beneficial effect, over artificial CSF (i .e. that is lacking the neuroprotective factors) in increasing cell viability (e.g. in PC 12 cells and human neuroblastoma ceils (SH-SY5Y) ceils), even after exposure to H2O2 and Αβ neurotoxins, in suppressing Con-A induced lymphocyte proliferation: and in in vivo animal models of neurodegenerative diseases, e.g. in showing a short term as well as prolonged amelioration of EAE clinical symptoms.

Thus, it has been proposed that the EA-CSF disclosed herein is suitable for use in treating diseases, particular, neurological diseases.

The following non-limiting Example 3 shows that the EA-SF disclosed herein has a beneficial effect in animal models of Multiple Sclerosis, Alzheimer disease and Amyotrophic Lateral Sclerosis.

In some embodiments, the neurological disease is a neurodegenerative diseases. In some embodiments, the neurological disease is Multiple Sclerosis (MS). In some embodiments, the neurological disease is Alzheimer's disease (AD).

In some embodiments, the neurological disease is tenopathies (Frontotemporal dementia).

In some embodiments, the neurological disease is Amyotrophic lateral sclerosis. The EA-CSF disclosed herein can be administered to the subject in need thereof m various ways. In one embodiment, the administration is by Intracerebroventricular (TCV) delivery/infusion.

In some embodiments, the administration comprise intrathecal administration.

In some embodiments, the administration of EA-CSF is by CSF exchange technology (in/out).

Methods of using the EA-CSF for treating a disease is further described hereinbelow.

The EA-CSF can be obtained by combining an artificial CSF, namely an electrolyte solution as defined herein, with a neuroprotective factor (single or cocktail).

In some embodiments, the method comprises, at minimum the following procedures. Firstly, culturing mesenchymal stem cells (MSCs) in a culture medium comprising an electrolyte solution as defined herein . During the culture period, the MSCs secret various molecules into the medium and after a period of time, the secretions together with the electroKtes are then separated from the cells. Separation can be achieved by centrifugation, filtration or the like.

In some embodiments, the electrolyte solution is an artificially produced solution, specifically, an artificial CSF.

In some other embodiments, the culturing is in human CSF (that inherently contains the electrolyte solution as defined herein).

Once separated, the cell free medium constitutes the EA-CSF composition.

In another embodiment, the EA-CSF can be obtained by combining, e.g. mixing, the electrolyte solution, as defined herein, with umbilical cord plasma. It has been found, as also disclosed herein, that the EA-CSF composed of umbilical cord plasma ameliorates cognitive impairments in a Tg mouse model for tenopathy/ Alzheimer' s disease .

In some aspects of the present disclosure, as will be further discussed below, treatm ent of neurological disease is by administering umbilical cord plasma without any dilution with aCSF.

When prepared by cuiturmg with MSC, it is preferable that the culturing is for at least 48 hours, preferably, for at least and preferably more than 72 hours, more preferably for a time period of at least 4 days, and most preferably for at least 5 days.

Culturing can be conducted under commonly acceptable culturing conditions, including incubator conditions that maintain optimal temperature (e.g. approximately 37°C), humidity (typically >95%) and other conditions such as carbon dioxide (CO2, typically 5%) and oxygen content of the atmosphere inside the incubator.

In some embodiments, the time of culture is for at least 5 days.

The source of MSC according to the present disclosure is not limited and may be derived from any appropriate biological source, for example from bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and peripheral blood. The source may be human or non-human. In one embodiment, the MSC are human MSC.

In some embodiments, the MSC are obtained from bone marrow.

The MSCs may be obtained from the BM by conventional methods such as aspiration or biopsy or any other method for providing MSCs. In BM aspiration a semi- liquid is obtained which may be further diluted with peripheral blood.

In some embodiments, the BM is harvested and the BM sample is treated to select the mesenchymal cells (also referred to as stromal ceils). Selecting for BM mesenchymal/stromal cells may be in a number of ways. For example, stromal cells may be disaggregated and cultured inside a plastic container and then separated by their survival in specific media and adherence to the plastic.

The BM sample obtained from a subject may be stored under appropriate conditions prior to use for example the sample can be stored in liquid nitrogen after the separation of BM in mononuclear cells using, for example, Sepax separation method or may be used immediately after removal.

In some embodiments the BM may be obtained from a healthy donor. Alternatively, the BM may be obtained from a subject diagnosed with a disease, including, without being limited thereto, neurodegenerative diseases or inflammatory disorders, including autoimmune disorders.

The MSC may be identified by using markers by FACS analysis for identification of the cells.

In some embodiments, the MSC are not obtained from an adipose tissue.

In the context of the present disclosure, MSCs can include any one or combination of undifferentiated MSCs, Neuralized MSCs (NMSCs) and terminally differentiated MSCs.

In the context of the present disclosure, when referring to undifferentiated MSC it is to be understood to encompass MSC characterized by being negative for CD34 and CD45 and positive for CD73, CD 105 and CD90. The term negative is used to note that no intensity or an intensity that is like the control intensity is observed in the FACS analysis. The term positive is used to note that a higher intensity than control is observed in the FACS analysis.

When referring to neuralized MSC or NMSC it is to be understood to encompass non-adherent (free-floating) spherical clusters of stem cells and progeny therefrom, that have a neurosphere-like structures characterized by the non-limiting markers, Nestin and PS-NCAM, which are characteristic antigens for neurospheres; the NMSC are further characterized by the capability to induce a dose -dependent suppression of lymphocytes proliferation. As such, while having a neurosphere structure, they are distinguished from classical neurospheres generated from adult neural stem cells (at least for the reason that they are produced from a different cell source).

The NMSC formation can thus be characterized by low expression or lack of (negative) MSC marker selected from the group consisting of CD90, and CD 105 (both being typical for the undifferentiated MSC) and positive expression of a neurosphere marker selected from the group consisting of nestin and PSN CAM. The NMSC can differentiate into cells of an ectoderm lineage including terminally differentiated astrocyte, neurons and oligodendrocyte. Differentiation induced by human CSF (with which it is cultured) resulted in the formation of Microtubule-associated protein 2 (MAP2)-and Class III β-tubulin (βΙΠ-tubulin or β- tubulin 111), positive cells with neuronal morphology, Glial fibrillary acidic protein (GFAP)-positive cells with astrocyte morphology, and 2',3'-Cyclic-nucleotide 3'- phosphodiesterase (CNPase) -positive cells with oligodendrocyte morphology.

The terminally differentiated MSC can thus be characterized expression of a marker selected from the group consisting of Microtubule-associated protein 2 (MAP2), Tubulin-beta-III, Glial fibrillary acidic protein (GFAP) and Cyclic-nucleotide 3^!-phosphodiesterase (CNPase).

Methods for obtaining NMSCs and terminally differentiated MSC are disclosed in WO 2015/056258, the content of which is incorporated herein by reference in its entirety.

In some embodiments, the MSCs are bone marrow MSCs,

In some other embodiments, the MSCs are human MSCs (hMSCs).

Advantageously, the EA-CSF can be used by conventional and safe CSF injection or replacement procedures. Therefore, and in accordance with another aspect, the present disclosure provides a method of treating a disease in a subject in need. More specifically, the method comprises administering to the subject an enriched artificial CSF (EA-CSF) composition as disclosed herein.

As briefly described herein above, the EA-CSF can be used for treating neurological diseases.

The term "neurological disease" or "neurological disorder" refers to any disorder of the nervous system. Structural, biochemical or electrical abnormalities in the brain, spinal cord or other nerves can result in a range of symptoms. Examples of symptoms include paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness. There are more than 600 disorders that afflict the nervous svstem. In some embodiments, the neurological disease may be one of but not limited to Multiple Sclerosis, Alzheimer's Disease (AD), tauopathies, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Parkinson's Disease (PD), and other dementias, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Huntington's Disease (HD), Prion Diseases, Frontotemporal dementia, Dementia with Lewy bodies, Progressive supranuclear palsy, Corticobasal degeneration, Multiple system atrophy. Hereditary spastic paraparesis, Spinocerebellar atrophies, Amyloidoses, Motor neuron diseases (MND), Spinocerebellar ataxia (SCA), stroke and Spinal muscular atrophy (SMA).

In one embodiment, the neurological disease is selected from the group consisting of Multiple Sclerosis, Alzheimer's disease, tauopathies and Amyotrophic lateral sclerosis.

In one embodiment, the neurological disease is Multiple Sclerosis. Multiple Sclerosis, also known as disseminated sclerosis or encephalomyelitis disseminata, refers to an inflammatory disease in which myelin sheaths around axons of the brain and spinal cord are damaged, leading to loss of myelin and scarring

In one embodiment, the neurological disease is Alzheimer s disease. Alzheimer's disease, also referred to simply as Alzheimer's, is a chronic neurodegenerative disease characterized by the difficulty in remembering recent events (short-term memory loss). The disease process is associated with plaques and tangles in the brain.

In one embodiment, the neurological disease is tauopathies. Tauopathy belongs to a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in neurofibrillary or gliofibrillary tangles in the human brain. Tangles are formed by hyperphosphorylation of a microtubule-associated protein known as tau, causing the protein to dissociate from microtubules and form aggregates in an insoluble form. Tauopathies may be one of but not limited to Alzheimer's disease, Primary age- related tauopathy (PART), Chronic traumatic encephalopathy (CTE), Progressive supranuclear palsy, Corticobasal degeneration, Frontotemporal dementia, Parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-dementia complex of Guam), Gangiioglioma, Gangiiocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, encephalopathy, tuberous sclerosis, Pantothenate kinase-associated neurodegeneration and lipofuscinosis.

In one embodiment, the neurological disease is Amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis (ALS), also known as motor neurone disease (MND), or Lou Gehrig's disease, is a specific disease which causes the death of neurons controlling voluntary muscles. Some also use the term motor neuron disease for a group of conditions of which ALS is the most common. ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscles decreasing in size.

The EA-CSF may be administered by any means known in the art.

In one embodiment, the administration of the EA-CSF is by injection.

In some embodiments, the injection comprises intrathecal administration or toracerebro ventricular (ICV) delivery/infusion.

As appreciated, intrathecal administration is a route of administration for drugs via an injection into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). The drug needs to be given this way to avoid being stopped by the blood brain barrier. An intrathecal pump is a medical device used to deliver medications via an intrathecal catheter, directly into the (intrathecal) space between the spinal cord and the protective sheath surrounding the spinal cord.

Further, as appreciated, intracerebro ventricular (ICV) administration is an invasive injection technique of substances directly into the cerebrospinal fluid in cerebral ventricles in order to bypass the blood brain barrier.

In some embodiments, the EA-CSF is administered by CSF-exchange techniques. "CSF exchange" involves the replacement of the CSF of a subject by artificial CSF, and in the context of the present disclosure, by the enriched artificial CSF (EA-CSF) disclosed herein.

Thus, in accordance with some embodiments of the present disclosure, there is provided a "CSF exchange therapy" for neurologic diseases of the CNS.

As described in the following non-limiting Examples, the effect of CSF exchange with the enriched artificial CSF disclosed herein was tested and found beneficiary in acute (short-term) models for CNS degeneration: EAE for MS-model, ICV injected Αβ as an acute model for AD, Tg model for AD, and Tg mouse model for ALS (all these models were tested with MSC secretions EA-CSF). In addition, the following non-limiting examples provide results for Tau-tg mice (model for tauopathy/Alzheimer) tested with umbilical cord plasma EA-CSF.

The non-limiting Examples can also support the use of the EA-CSF for treating stroke and/or psychiatric conditions.

In the context of the present disclosure, when referring to treating it is to be understood as encompassing the administration of an amount of the EA-CSF that is effective to ameliorate undesired symptoms associated with a disease or disorder, preferably of the nervous system, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the medical condition, to slow down the deterioration of symptoms associated with the condition, to slow down the irreversible damage caused by the chronic stage of the condition, to lessen the severity or cure a condition, to improve survival rate or more rapid reco very form such a condition.

A further aspect disclosed herein concerns umbilical cord plasma (UCP) for treatment of neurological diseases as defined hereinabove. Administration of the UCP can be by any method known in the art, including the methods listed above with respect to administration of the EA-CSF.

Thus, in the context of the present disclosure also provided is a method of treatment of neurological diseases comprising administering to a subject in need of said treatment an amount of UCP, the amount being sufficient for treating the disease.

As used in the specification and claims, the forms "a", "an" and "the" include singular as well as plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells and the term "cells" includes one ceil as well as more than one ceil.

As used herein, the term "or" means one or a combination of two or more of the listed choices.

Further, as used herein, the term "comprising" is intended to mean that the methods and culture systems includes the recited elements, but does not exclude others. Similarly, "consisting essentially of is used to define methods and systems that include the recited elements but exclude other elements that may have an essential significance on the functionality of the culture systems of the inventions. For example, a culture system consisting essentially of a basic medium and medium supplements will not include or will include only insignificant amounts (amounts that will have an insignificant effect on the propagation of cells in the culture system) of other substances that have an effect on ceils in a culture. Also, a system consisting essentially of the elements as defined herein would not exclude trace contaminants. "Consisting of shall mean excluding more than trace amounts of other elements. Embodiments defined by- each of these transition terms are within the scope of this in vention.

Further, all numerical values, e.g., concentration or dose or ranges thereof, are approximations which are varied (+) or (-) by up to 20%, at times by up to 10%, from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

N ON-LIMITING EXAMPLES

Experimental procedures

Cell culture studies

Materials

Selecting ofaCSF composition allowing high viability of neuronal cell lines

For determining the optimal aCSF, viability of SY -SY5Y and PC12 cells grown overnight (ON) in different aCSFs, relative to optimal cell medium was tested. Specifically, 2()xl0⁴ cells/ml (in volume of 0.5ml) were grown in different aCSF (See below) in comparison with optimal medium (RPM1 for SY-SY5Ycells, and DMEM for PC 12 cells). After ON incubation viability was tested by MTT assay. aCSF #1 was selected for further studies since it showed high cell viability in both neuronal cell lines.

aCSFl (basic): NaCi 125mM, KC1 2.5nM, MgCl₂.6H20 lrnM, NalfcPC.25mM, CaCl₂.2H₂0 2mM, NaHCC 25mM, Glucose 25mM

aCSFl (Alzet) (calculated from site) ; NaCl 74mM, KC1 1.5nM, MgC1.6H₂0.4mM, NaH₂P0₄ 1.125mM, CaCl₂.2H₂0 l ,4mM, Na₂HP0₄ 1.5mM

aCSF3: basic + MgCl₂ ImM

aCSF4: basic + D-glucose 25mM aCSF5: basic + NaH₂P04 1.25mM

Unless otherwise stated, the artificial CSF used in the following contained NaCl 7.31gr/L, KCi 0.186 gr/L, MgCl 0.2033 gr/L, NaH₂P0₄ 0.15gr/L, CaCh 0.294 gr/L, aHCOj 2.1025 gr/L, glucose (D) 4.505 gr/L and was prepared in our iab using reagents from companies like Sigma,

Growing ofhMSC

The MSCs were obtained from the lab of Prof. Karussis (Hadassah Medical Center, Jerusalem, Israel). The preparation included:

Bone marrow aspiration

Fresh bone marrow aspirated according to the routine medical center procedure from the patient's iliac-crest under local anesthesia and sedation by an anesthetist. This procedure was performed in aseptic conditions at the department of bone marrow transplantation of the Hadassah Medical Center. The bone marrow aspiration procedure is preceded by documentation reporting negative test results for HBV, HCV and HIV Bone marrow (-100 mi) was aspirated using aspiration needles into heparin containing sterile bags (Macopharma, USA). The aspirated patients' bone marrow, which is the source of the MSC, was transferred immediately to the GMP -facility. The patient's aspirated bone marrow sample is labeled by the physician or by the attending Technical assistant.

Separation of Mononuclear cells (MNC) from whole bone marrow

BM aspirates w¾re transferred from the heparin containing bone marrow aspiration bags into sterile 50ml conical tubes (Corning, USA) using two spike tubing sets (Macopharma, USA) and diluted 1 : 1 (v:v) in Hank's Balanced Salt Solution (HBSS, Sigma-Aldrich), and MNC were separated from total bone marrow cells by Ficoll density gradient (1.073 gr/ml) centrifugation (GE Healthcare, USA). Diluted BM were transferred in barrier-containing 50ml tubes (LEUCOSEP™, Greiner-bio one, Germany) prefilled with 15ml of Ficoll and centrifuged for 10 minutes, lOOOxg , 24°C. The MNC layer was removed using sterile pasture pipette (Greiner-bio one, Germany) and transferred in 50ml sterile tubes and diluted with 30ml CTS™ DPBS. Cells were centrifuged twice 10 minutes, 1000 rpm, 24°C. Cell were re-seeded in "complete culture media" containing Nutristem™ XF Basal Media supplemented with Nutristem™ Supplement media for further processing.

Propagation of MSC

MNC were counted using hematocytometer and cell viability was evaluated using trypan-blue dye staining (Sigma-Aldrich, Israel). MNC were washed and re- suspended with Nutristem XF™ complete media and seeded on Cell-Stack™ (636 cm², Coming, USA) pre-coated with Attachment Solution XF™. The seeding density was 100,000 cells/ cm² and incubated in a 37°C/5% CO2 humidified incubator for 48 hours. The cell culture is examined under the microscope. At this stage, non-adherent, mononuclear cell will be floating in the culture supernatant and plastic -adherent MSC will have attached to the flask surface. The culture supernatant containing the nonadherent mononuclear cells is removed, and the adherent cells are gently washed with 100 ml DPBS. The step from MNC seeding to hMSC harvesting is presented as Passage 0 (P0). The P0 cells are incubated in a 37°C/5% C02 humidified incubator and growth medium is replaced twice a week, with fresh complete NutriStem™ XF growth medium as until the culture is 80-90% confluent but for no more than 12 days. Cells are subcultured at regular intervals, when the culture reaches 80- 90% confluence. Each subculture cycle is counted as a new passage. The cultures may be sub -cultivated up to Passage 3. For sub-culturing MSC, the culture supernatant is removed from the flask and a CTS™ TrypLE™ Select solution is added to each flask. The flask is incubated for 8 min at 37°C and culture medium then added to each CellStack™ to inactivate the enzymatic action. The detached cell suspension were transferred into centrifuge tubes, washed, re-suspended in growth medium, counted and reseeded at a density of 5,000 cells/cm in 120ml of NutriStem™ XF growth medium in new CellStack™. The cultures are then incubated in a 37°C, 5% CO2 humidified incubator for further culturing.

Characterization of isolated human MSC

Isolated hMSC cells of MS patients were characterized by flow cytometry analysis (FACS) of surface antigen expression . The International Society for Cellular Therapy (ISCT) recommended MSC should be characterized by a set of minimum criteria including the presence/absence of the below specific surface markers. MSC of MS patients were tested for the positive markers CD 105, CD73 and CD90 on the cell surface (>95% positive) and to lack expression (<2% positive) of CD34, CD45, CD79alpha, CD 19, and HLA-DR. List of the antibodies used are presented in Table 2. The stained cells were read and analyzed with CYTOMICS FC500 (Beckman Coulter, Germany).

Testing viability of the MSCs grown in aCSF

The viability of the MSCs, as a source of secreting materials for enriching aCSF, was tested: MSCs at a concentration of 25K/mi were grown for 1-7 days in aCSF, and their viability was compared to that of MSCs grown in an optimal culture conditions with Nutristem™ (Biological Industries, Israel), a special chemically-defined medium (a DMEM based buffer which is serum-free and xeno-free medium). Cell viability was tested using the MTT assay (Ginsburg , Phytotherapy research 2011). Phytother Res. 2011 May;25(5):740-3. doi: i0, 1002/ptr.3459. Epub 2011 Mar 17. The herbal preparation Padma® 28 protects against neurotoxicity in PC 12 cells. Ginsburg II, Rozenstein-Tsalkovich L, Koren E, Rosenmann H).

Preparation of aCSF enriched with secretions of MSCs

MSCs were grown in the aCSF and the supernatant were collected and frozen at -20°C to be used as "MSC secretions-enriched aCSF" ("enriched aCSF") for the various in vitro and in vivo assays. The preparation of the enriched aCSF and design of the study in EAE-mice model is schematically represented in Figs. 1A-1B.

Testing the viability of neuronal cell lines treated with the MSC secretions "enriched aCSF"

To find the optimal conditions for achieving effective "enriched aCSF" (MSCs concentration and the minimal culture time), I 0K-100K/ml MSCs were grown in aCSF for 2 and 5 days, and the supernatant constituting the "enriched aCSF" was collected and tested for its effect of the viability of PCI2 cells following an overnight (ON) treatment. This was further followed by using "enriched aCSF" from 10K-25 K/ml MSCs growing in aCSF for 5 days, and testing its effect on the viability of PC 12 cells as well as SH-SY5Y cells. The effect of the "enriched aCSF" was also tested in the neuronal cell lines under exposure to neurotoxins as follows: treating the cells for ON with "enriched aCSF", further followed by addition of 0.5mM H2O2 (Sigma) for 4h, or addition of 4μΜ Αβ 25-35 (Sigma) (pre -aggregated at 37°c for 10 min) for ON. MTT assay was performed.

Testing the viability of neuronal cell lines treated with umbilical cord plasma.

PC12 cells were treated for ON with aCSF enriched with 15% umbilical cord plasma. MTT assay was performed.

Measurement of secreted neurotrophic factors, anti-inflammatory cytokines and antioxidant capacity in the MSC secretions enriched-aCSF

MSCs (6Q0K/ml) were grown in aCSF in a 24-wells plate (Nunc, Denmark) for 5 days. Supernatants were collected. The levels of the neurotrophic factors: brain- derived neurotrophic factor (BDNF), and for the levels of the anti-inflammatory cytokines: transforming growth factor beta (TGF-β) was determined by ELISA (ELISA kits, R&D Systems), as well as the total anti-oxidant capacity (Sigma). The absorbance at specific nm was recorded.

Measurement of secreted neurotrophic factors, anti-inflammatory cytokines and anti- oxidant capacity in the umbilical cord plasma

The presence of the neurotrophic factors, anti-inflammatory cytokines and antioxidant capacity was also measured in the umbilical cord (purchased from the umbilical cord databank (https://www.mdais.org).

Testing the proliferation of spleen lymphocytes treated with the "enriched aCSF"

Draining lymph nodes were excised from C57BL mice and cultured as single- cell suspensions. Lymph node cell (LNC) proliferation in response to the concanavalin mitogen was assayed.

In vitro proliferation of mouse splenocytes

Splenocytes were excised from C57BL mice and cultured as single-cell suspensions. Splenocytes proliferation was assayed in vitro by ^JH-thymidine incorporation. All cultures were carried out in triplicate in 96-well, flat-bottom, microtiter plates. The assay was carried out by seeding 4 x ! 0⁵ cells/well in 0.2 mL of RPMI medium (Sigma, Rehovot, Israel) supplemented with 2.5% PCS, ImM L- glutamine, and antibiotics. Basal ³H-thymidine incorporation was determined in response to Concanavalin A (ConA, l^tg/rriL), The cultures were incubated for 48 hours m a humidified atmosphere of 5% carbon dioxide at 37°C and then pulsed for 16 hours with ¾~thymidme ( I Ci/well). Cells were harvested on fiberglass filters using a multiharvester and the radioactivity was counted.

Animal Studies

C57BL6 mice [purchased from Harlan/Envigo, Israel]

Insertion of the CSF exchange device

About 9-week -old female C57BL6 mice went through a standard stereotaxic surgical procedure for the insertion of the CSF exchange therapy device, as follows: briefly, the animals were anesthetized (ketamine 3mg/kg cepetore 0.03mg/kg i.p) and placed in a Kopf stereotaxic frame, and an incision was made to expose the skull. Small holes were drilled above the lateral ventricles, and the 2.5mm guide cannula (RWD- 900-0062-060) was cemented with dental acrylic cement mixed with cyanoacrylate glue to secure the cannula to the skull. Cannula dummies (RWD-900-0062-131 2) were inserted into the guide cannulas to keep them patent {means: in place). Stereotaxic coordinates used for ICV in mm from the bregma: -0.2 mm anteroposterior, ±1 mm mediolateral, dorsoventral -2.3mm according to the atlas of Slotnick and Leonard.

Induction of EAE

One week following the insertion of the device, chronic EAE was induced by immunizing the mice with an emulsion (0.2ml) containing 300 ag of purified myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide in phosphate-buffered saline (PBS) and an equal volume of complete Freund adjuvant containing 5 mg of H37Ra (Difco Laboratories, Detroit, Michigan). In addition, 300 ng of Bordetella pertussis toxin in 0.2 mL of PBS were injected intraperitoneally the same day and 48 hours later. Animals with EAE were scored daily for neurological symptoms according to the EAE clinical severity scale: 0 = asymptomatic; 1 = partial loss of tail tonicity; 2 = tail paralysis; 3 = hind limb weakness; 4 = hind limb paralysis; 5 = 4-limb paralysis; 6 = death (Kassis 2008). [Arch Neurol. 2008 Jun;65(6):753-61. doi: lO. lOOl/archneur.65.6.753. Neuroprotection and immunomoduiation with mesenchymal stem ceils in chronic experimental autoimmune encephalomyelitis. Kassis II, Grigoriadis N, Gowda-Kurkalli B, Mizrachi-Kol R, Ben-Hur T, Slavin S, Abramsky O, Karussis D.]

CSF exchange therapy

For CSF exchange therapy, mice were moved to individual therapy cages. The dummy cannula was removed and replaced by the injector cannula secured with fixing screw (Fixing Screw-Connecting Double Injector and Guide, RWD Life Science Inc, China, RWD-900-0062-521). In order to minimize stress and enable free spatial movement, all infusion ("in") and withdrawal ("out") tubing were connected to a low- torque dual channel swivel (375/D/22LT, Instech Laboratories, Inc, USA), mount to counter-balanced lever arm (MCLA, Instech Laboratories, Inc).

At day 6 following the EAE induction the CSF exchange therapy was started.

In the first part of the study the EAE -mice (n=6) went through a short-term CSF exchange ("in/out") therapy of 3 days (days 6-8 following EAE induction), as follows: infusion of MSC secretions enriched aCSF into one ventricle ("in"), and withdrawal of endogenous CSF from the other ventricle ("out"), at a rate of 5 μΐ/ r for 3 hours.

As control mice, the EAE-mice which were also inserted with the CSF exchange device but did not go the CSF exchange therapy were used (n=12).

Experiment #1 served as a proof of concept study, as well as for testing a protocol treatment in the EAE-mice.

This was followed by other studies in which the treatment was more prolonged

(performed at days 6-18 following disease induction, 5 times a week) at a rate of S j/hr for 3 hours. This included 3 experiments with different protocols of the CSF therapy as follows:

Experiment #2. EAE-mice received "in/out" CSF exchange therapy with MSC secretions enriched aCSF (n=5) vs non- treated EAE-controls (n^;=6);

Experiment #3. EAE-mice received only infusion of MSC secretions enriched aCSF into one ventricle ("in") without withdrawal of endogenous CSF (n=9), vs non- treated EAE -controls (;i 7).

Experiment #4. "in/out" CSF exchange therapy with aCSF only (non- enriched) (n^;=8) vs non- treated EAE-controls (n=13). Histological Examination

Mice were anesthetized with a lethal dose of pentobarbital and perfused via the ascending aorta with 4% paraformaldehyde, and brains were removed and preserved at-80°C. Serial 10 μΜ sections were made. The modified Bielschowsky staining was used to evaluate the axonal loss, using the following score: 0, normal axonal density; 1, focused mild to moderate axonal loss;2, scattered mild to moderate axonal loss; 3, focused severe axonal loss; and 4, scattered severe axonal loss. Luxol fast blue staining was used to grade the demyelination, as follows: 0, no demyeiination; 1, a few scattered naked axons; 2, small groups of naked axons; 3, large groups of naked axons; 4, confluent foci of demyelination and 5, widespread demyelination. Hematoxylin-eosin staining was performed for grading the inflammation, by counting the no. of perivascular mononuclear infiltrates and the no. of cells per infiltrate. Brain sections were evaluated under (20*) magnification of optical fields [Immunol Lett. 2013 Jul- Aug;154(l -2):70-6. doi: l Q.1010/j .milet.2013.06.002. Epub 2013 Aug 28. Mesenchymal stem cells (MSC) derived from mice with experimental autoimmune encephalomyelitis (EAE) suppress EAE and have similar biological properties with MSC from healthy donors. Kassis I, Petrou P, Halimi M, Kamssis D.] . Images were processed using Image -Pro Pius software.

Statistical analysis

The data were presented as mean ±SEM. Clinical analysis data were analyzed using one way or repeated ANOVA. Further analyses were performed using Tukey Post-hoc test. For survival analysis we used the Kaplan Meier analysis. The unpaired t test was used for the cell culture and histological studies analysis. Statistical analysis was performed using IBM SPSS Statistics V.23.

suits

Selection of the optimal aCSF composition

First, the effect of different aCSF on growth of the cells was examined. Figure 2A shows that aCSF#l is the optimal artificial CSF and was thus further used in the following experiments. Ve fying that MSC are viable when growing in aCSF

Next, the viability of the MSCs when being grown into artificial CSF was examined. Without being bound by theory, it was hypothesized that if MSCs cells grow in aCSF, they may secret beneficial/protecti ve compounds and if MSCs show distress in aCSF it may induce secretion of toxic elements. For this purpose, the viability of the MSCs grown for 1-7 days in aCSF was compared to that of the cells grown under the optimal conditions of a DMEM medium (considered as a rich medium). As can be seen in Figure 2B, the viability of the MSCs grown in aCSF was lower than that in the rich DMEM medium, but it reached about 75% of this optimal viability. This pointed that the MSCs did not show a severe distress under aCSF at least for 7 days.

MSCs grown in aCSF secrete beneficial factors

The ability of the MSCs grown in aCSF to secrete neurotrophic factors and antiinflammatory cytokines, and anti-oxidants was next examined. It was found that MSCs grown in the chosen aCSF (aCSF#l) for 5 days secreted at least BDNF, ΤΟΡβ and anti- oxidant compounds.

The levels detected in secretions of 600,000 MSC were as follows:

BDNF: 22 ±4.83 pg/ml

TGF-β: 535.25 ± 49, 16 pg/ml

Total anti-oxidant capacity: 0.2 ± 0.05 nmol/microliter

Umbilical cord plasma contains beneficial factors

BDNF, TGFp and lL-10, as well as anti -oxidants were detected in the umbilical cord plasma (maybe copy the levels detected).

The levels detected were as follows:

BDNF: >1000 pg/ml

TGF-β: 31585 pg/ml

IL-10: 9,54 pg/ml

Total anti-oxidant capacity: >20nmoi/microiiter In vitro neuroprotective studies

The MSG secretions enriched aCSF increases cell viability of neuronal cell lines.

The aCSF enriched with MSG secretions as described above was collected, and used for testing the protective effect in neuronal cell lines (as described above).

Specifically, PC 12 cells from rats were treated overnight (ON) with "enriched aCSF" containing secretions of 10K or 100K /ml MSCs which were grown for 2 or 5 days in aCSF. As seen in Figures 3A-3B, while secretions of 2 days growing 10K or 100K /ml MSCs in aCSF showed marginal increase in PC 12 cell viability, the enriched aCSF containing the secretions of 5 days growth of MSCs in the aCSF showed a significant increase in the PC12 cell viability relative to aCSF only treated cells. This increase was noticed in both MSCs lOK/ml and l OOK/ml MSCs cell concentrations, showing a comparable effect (increase of 30.8% (p=0.017 (2 tail) and 22.7% (p=0.0065) (2 tail), respectively, relative to the aCSF-only treated PC 12 cells), pointing that a higher concentration at this range is not necessarily more effective.

The use of lower concentration was further validated for this beneficial effect of the enriched aCSF of secretions of lOK/ml and also of 25K/ml in PC 12 and also in human neuroblastoma cells (SH-SY5Y) cells. As shown in Figures 4A-4B, in both cell lines, a significant increase in cell viability was noticed, with a dose effect in the SH- SY5Y cells (in SH-SY5Y: increase of 36.1% (p=0.008)(2 tail) and 88.8% (p 0.00013} (2 tail), respectively, relative to aCSF treated ceils, with a stronger effect of the secretions of 25K MSCs relative to 10K (p=0.0002)(2 tail); in PC I 2: increase of 57.17% (p 0.U007K2 tail) and 50.8% (p 0.00-0(2 tail), respectively, relative to aCSF treated cells, pointing that the enriched aCSF containing secretions of 10K-25K/ml MSCs grown for 5 days in aCSF is a beneficial/potent fluid, with some dose positive effect.

The MSC secretions enriched aCSF increases cell viability of neuronal cell lines exposed to I !.·()· and Αβ neurotoxins

Next, it was examined whether the MSC secretions enriched aCSF is also beneficial under the neurotoxic environment of H2O2 and Αβ,

The results presented in Figure 5A show that while H2O2 reduced robustly PC12 ceil viability relative to control cells, the enriched aCSF increased significantly the cell viability ( I nk. increase of 91.7% (p=0.021) ( 1 tail), 25K: 179.2% (v 0.00 1 > ( 1 tail), with a trend of stronger effect of the 25K MSCs secretions relative to 10K (p 0.07) (1 tail).

A beneficial effect was also demonstrated under Ap neurotoxicity in SH-SY5Y cells as seen in Figure SB, while ceil viability was reduced under Αβ, a significant increase in cell viability was noticed in the enriched aCSF treated mice ( 10K: 58.2% (p=0.000004) (1 tail), 25K: 36.5% (p=0.00004) (1 tail), with a comparable effect of the secretions of both MSCs concentrations.

The umbilical cord plasma enriched aCSF increases cell viability of the neuronal cell line

PC 12 ceils were treated overnight (ON) with "enriched aCSF" containing 15% of umbilical cord plasma or with aCSF only. As seen in Figure 6, there was a significant increase in cell viability of the cells treated with the enriched CSF relative to the aCSF only (non-enriched) (pO.0001). [We also detected that PC 12 cell grown in the DMED medium enriched with 15% umbilical cord plasma had a higher cell viability than the cells grown in the medium containing Fetal calf serum+ human serum (1.07 ± 0.05 vs 0.89 ± 0.04, respectively, p=0.006)] .

The enriched aCSF suppressed Con-A induced lymphocyte proliferation

Figure 7 shows that the proliferation assay in the presence of the mitogen con-A revealed a significant suppression of the proliferation of spleen lymphocytes treated with the MSC secretions enriched CSF (less CPM) relative to that of the lymphocytes treated with aCSF only (t-test p=0.0007). The positive control of spleen lymphocytes treated with conditioned medium (DMEM) grown on MSC cells showed a significant suppression relative to splenocytes only (non-treated) (t-test p=0.0004).

In vivo neuroprotective studies:

The CSF exchange procedure was established in mice i.e. surgical procedure of installation a guide cannula in each ventricle, a tether (anchor of halter) fixed to the skull. The cannulas (the injection and the removal cannulas) entered the brain through the guide cannulas. The mouse was connected to the micro-injection pump, using polyethylene tubes. The pump connected to one ventricle allowed the injection of aCSF, while ihe pump connected to the other ventricle allowed the CSF removal (withdrawal). The halter was directly connected to the swivel, avoiding the mouse to get tangled with piping, and allowing free roaming in the cage. The CSF exchange procedure is schematically represented in Figure IB.

After optimizing a safe CSF exchange protocol (daily exchange of 3 hours), its efficacy was tested in the following animal models: Experimental Autoimmune Encephalomyelitis (EAE) mouse model for Multiple Sclerosis (MS), ICV Αβ injected mouse model for Alzheimer's disease (AD) and Tg mouse model for Amyotrophic Lateral Sclerosis (ALS) as detailed below.

While in the cell line studies, the secretions of the lOK/ml and 25K/ml MSCs were used, in the animal studies, secretion of 25K/ml MSCs were employed, also grown for 5 days in aCSF.

CSF exchange in EAE mouse model for MS

A short-term MSC secretion enriched aCSF (in/out) exchange therapy showed a short term amelioration of the EAE clinical symptoms

The MOG-EAE-mice was used as a model for MS, an inflammatory disease with neurodegenerative components, with early/acute clinical manifestations of motor deficits, which can be monitored at a daily basis. EAE-mice treated with "in/out" enriched aCSF exchange therapy delivered at days 6-8 following EAE-induction showed a trend of a lower average clinical EAE-score at days 9-1 1 post EAE-induction (which are 3 days following the CSF exchange therapy) {repeated ANOVA f(l ,16)=3.73 p=1234567890-0.07, p=0.07]), with t-test for day 10 revealed a significant difference in the average clinical score between treated and untreated mice (0 vs 1.08 ± 0.30, respectively, p^:=0.012)} .As shown in Figure 8A, while the untreated mice developed the disease already at day 9, the treated mice developed it only at day 11 following the induction.

When comparing the average cumulative score as well as the average maximal score of days 0-10, significant lower scores were detected in the treated mice (t-test, p=0.017, p=0.012, respectively), with the average cumulative score significantly lower in the treated mice also at day 11 (3 days after end of therapy) (t-test p=0.035) (median cumulative value for days 0-10 being 0.75, and for days 0-11 being 1.5 in the treated mice, while score o in the control-EAE mice; median maximal score for days 0-10 being 0.5, while o in EAE -controls), as seen in Figures 8B-8C. However, from days 12-13 (which are 4-5 days after the end of therapy) the lines in the graph of the average score of the treated and untreated mice became merged, and no significant differences were noticed in the cumulative and maximal scores.

Using the Kaplan Meier analysis for calculating the proportion of the disease - free mice revealed significant less EAE disease-free mice among the treated mice relative to the untreated (Log-Rank Test: p=0.028), with the median value for the 50% chances to stay free of disease symptoms being at day 1 1 for the treated and at day 8 for the untreated mice, as shown in Figure 8D.

These results point that already after 3 days of CSF exchange therapy using the MSC secretion enriched aCSF, a beneficial effect of delaying disease onset and ameliorating the disease clinical symptoms was demonstrated, yet this effect lasted for only a few days.

Prolonged amelioration of the EAE clinical symptoms during a prolonged CSF exchange therapy

Next, EAE-mice were treated with a more prolonged CSF exchange therapy, which was delivered during days 6-22 after disease induction (performed 5 times a week for 2 weeks), in order to see whether the beneficial effect of therapy will be stronger and more prolonged. A number of 3 protocols of prolonged CSF exchange therapy was used: " in/out" enriched aCSF (eCSF), "in" eCSF, and in/out aCSF (aCSF only). Since the clinical symptoms of the control EAE -mice in these experiments, measured as average, cumulative, and maximal score as well as in the Kaplan Meier analysis - was similar (no significant difference in the one-way ANOVA analysis) the control EAE-rnice results were pooled, and w ere used for comparison of the efficacy of the different CSF exchange therapies. Repeated ANOVA analysis showed a significant difference between the different treatment groups [f(2, 24)=9.59, p<0.001)], with Tukey Post-hoc analysis showing significant differences between the control EAE-mice and each treatment group (p<0.001, p= 0.035 and p=0.001 for "in/out" eCSF, "in" eCSF, and "in/out" aCSF, respectively). Figure 9A shows that while the "in/out" eCSF mice showed a significant lower average score relative to control along all days of follow-up of disease symptoms (p=0.005, p=0.013, p<0.001, p=0.001, p=0.001, p=0.005 for days 11, 12, 14, 15, 17, 18 post induction), the "in" eCSF mice showed a significant lower average score (p<0.001, p=0.01, p=0.002) only at days 11, 12, 14, respectively; and the "in/out^"' aCSF mice showed a significant lower score (p<0.001, p=0.01, p=0.019) only at days 14, 15,17 (ith a similar trend (p=0.066) also at day 18)] . A lower average score of ^"'in/out" eCSF-mice was demonstrated relative to that of "in" eCSF-mice at days 15 and 17 (p=0.06 and p=O.03, respectively). These results point that the "in/out" eCSF therapy was the most effective one, affecting both age at onset (EAE-control mice develop the disease at day 10 while the treated mice at day 14 post induction) and the disease progression, while the "in" eCSF treatment was beneficial in delaying disease onset (EAE-control mice develop the disease at day 10 while the treated mice at day 12 post induction), and the "in/out" aCSF treatment was mostly effective in interfering with disease progression rather than in delaying its onset.

Further, when comparing the cumulative score a significant difference between the groups was noticed {one-way ANOVA [f (3 ,44)= 11.04 p<0.001]}, with further analysis with Tukey Post-hoc showing that the "in/out" eCSF-mice and the "in out" aCSF had lower scores compared to EAE-controls (p<0.001, p=0.008, respectively), with "in/out" enriched aCSF-mice having lower scores than the "in" enriched aCSF- mice (p^:=0.004), with comparable scores of the "in" eCSF-mice and the "in/out" aCSF- mice, as seen in Figure 9B.

Similar results were noticed when analyzing the maximal score: a significant difference between the groups was noticed {one-way ANOVA [f(3,48)=5.55, p=0.003] }, with further analysis with Tukey Post-hoc showing that the "in/out" eCSF- mice had lower scores compared to control and "in" eCSF-mice (p=0.002, p=0.02, respectively), with comparable scores of the "in" eCSF-mice and the "in/out" aCSF- mice, as shown in Figure 9C.

Using the Kaplan Meier analysis for calculating the proportion of the disease- free mice revealed significant less EAE disease- free mice among the different groups (Log-Rank Test: pO.001), with each pair of groups showing significant differences ("in/out" eCSF-mice vs EAE-control, "in" eCSF-mice and "in/out" aCSF-mice: p 0.000387. 0.00507 and 0.0041, respectively; "in" eCSF-mice vs EAE-control and ^"'in/out" aCSF-mice: p=0.0415 and 0.0141, respectively), but not "in/out" aCSF-mice vs EAE-control. The median value for the 50% chances to stay free of disease symptoms being 15, 13, 1 1 and 9 days for the , ("in/out" eCSF-mice, "in" eCSF-mice, "in/out" aCSF-mice and EAE-control, respectively, as seen in Figure 9D.

While the short-term treatment reached a decrease of about 1 unit of the average clinical score (maximal effect at day 10: 1.08 ± 0.30 vs 0 in control vs treated, respectively), the prolonged treatment reached a decrease of about 2 units (at day 18: 2.75 ± 0.22 vs 0.7 ±0.2 in control vs treated, respectively). The decrease in the average cumulative score was about 1.9 units (at days 0-11 : 2.375 ± 0.68 vs 0.416 ± 0.327 in control vs treated, respectively), while 11.2 units in the prolonged treatment (at days 0- 18: 15.096 ± 0.077 vs 3.8 ± 0.8 in control vs treated, respectively). The decrease in the average maximal score was about 1 unit (at days 0-10: 1.08 ± 0.30 vs 0 in control vs treated, respectively), while reaching 1.8 units in the prolonged treatment (at days 0-18: 3.11 ± 0.21 vs 1.3 ± 0.2 in control vs treated, respectively). As for delay of onset, the short-term therapy delayed the disease in 3 days (50% to stay free of disease at 8 days, while 11 days in control vs treated, respectively), while 6 days delay in the prolonged therapy (50% to stay free of disease at 9 days, while 15 days in control vs treated, respectively). This indicates a stronger and more prolonged beneficial effect of the prolonged CSF exchange therapy.

CSF exchange therapy reduced axonal damage and demyelination

Bielschowsky staining was performed for evaluating the effect of the CSF exchange therapy on the degree of axonal damage in the EAE mice. Figures 10A-10E show that a significant lower axonal damage was noticed in the "in/out" enriched aCSF treated mice relative to EAE-control mice (evaluated in the short-term treatment with a high number of brains for the histological analysis (1.88±0.46 (n=7) vs 2.57±0.22 ( n 1 i ). respectively, p=0.048, t-test)). A similar trend of reduced axonal damage was also detected in the prolonged treatment of "in" enriched aCSF-mice relative to the EAE-control mice (2.06±0.22 (n=9) vs 2.6H0.23 (n=7), respectively, p=0.09, t-test) while no difference in the prolonged treatment of "in/out" aCSF-treatment vs EAE- control ( 2.58 : 0.20 in 8} vs 2.28±0.32 {« ! ()). respectively, p 0..?.!!?. t-test). Further, Figures 10F-10J show that LFB staining of these research groups for the degree/extent of demyeimation following CSF exchange therapy - revealed a trend of less demyelination in the "in/out" enriched aCSF treated- mice relative to the EAE- control mice (2.58±0.22 vs 2.98±0.14, respectively, p=0.09, t-test), and moreover, a significant lower demyelination was noticed in the "in" enriched aCSF-mice relative to the EAE-control mice (2.69±0.19 vs 3.21±0.09, respectively, *p=0.02, t-test), and no difference in demyelination in the "in/out" aCSF-treatn ent vs EAE-control ( 2.56 : 0. 1 87 i n 8} vs 2.70±0.255 (n=10), respectively, p 0. 14. t-test).

CSF exchange in ICV A3 injected mouse model for AD

Reduced cognitive de ficits in a prolonged therapy with eCSF in AD-mice.

AD-mice were treated with (in/out) eCSF exchange therapy at days 2-4 (a cluster of 3 days therapy) following disease induction [by amyloid-beta (Αβ) ICV), and further treated repeatedly at days 7-17 for 2 times/ week. Cognitive tests were conducted during these 7-17 days of therapy; the design is presented in Figure 11A .

Figure 11B shows that a significant better performance following the eCSF therapy was demonstrated in the Y-maze {One way anova [f(2,2 i)=7.123, p=0.004] showed a significant difference among the groups, followed by Tuckey Post hoc analysis: while the performance of the AD-mice was significantly worse than the non- AD-mice (*p=0.02), the performance of eCSF treated mice was significantly better than that of the (u -treated) AD-mice (*p=0.001), and also better from the AD-mice treated with aCSF only (*p=0.03), and being similar to the performance of the non-AD-mice.

Figure 11 C shows a beneficial effect of the eCSF therapy was also demonstrated in the Radial arrn water maze. One way anova [f(3,37)=4.68847, p 0.00 1 showed a significant difference among the groups, followed by Tuckey Post hoc analysis, showing that while the performance of the AD-mice was significantly worse than the non-AD-mice (*p=0.025), the performance of eCSF treated was similar to the non-AD-mice (no statistical difference), while the aCSF treated was significantly worse than the non-AD- mice (*p=0.024). A trend of reduced cognitive deficits in the short-term therapy with eCSF in the AO- mice

AD-mice were treated with eCSF exchange therapy at days 2-4 (a cluster of 3 days therapy only) following disease induction. Cognitive tests were conducted at days 9-14 following disease induction: the design is presented in Figure 12A.

Figure 12B shows a trend of better performance in the eCSF treated mice was demonstrated in the Radial arm water maze {One way anova showed a significant difference among the groups [f(2,23)=4.688, p=0.021], followed by Post hoc analysis} : while the performance of the non-AD-mice was significantly better than that of the AD- mice, *p=0.02, it did not differ statistically from that of the eCSF treated mice (however it seem s that the performance of the eCSF was somewhere between the performance of the non-AD and AD- mice).

Some similar effect was noticed also in the Y-maze {Contrast analysis revealed a trend of difference among groups (p=0.06): with the performance of the non-AD-mice being better than that of the AD-mice, p=0.024, with no difference of the non-AD-mice from that of the eCSF treated mice as seen in Figure 12C.

CSF exchange in a Tg mouse model for Alzheimer [amyloid model: 5XFAD Tg; (Intraneuronal beta-amyloid aggregates, neurodegeneraiion, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R. J eurosci. 2006 Oct 4;26(40): 10129-40)]

AD-Tg mice were treated with (in/out) eCSF exchange therapy 2 times a week from 6 months of age (about age of development of cognitive deficits) for 3 weeks.

Figure 13 shows that a significant better performance following the eCSF therapy was demonstrated in the T-maze. While the Tau-tg mice showed a worse performance relative to the WT- mice (t-tesi, p=0.03), this impairment of the Tau-tg mice was ameliorated in the Tau-tg mice treated with the CSF exchange therapy using the umbilical cord plasma (diluted in artificial CSF) (t-test, p=0.04). The performance of the treated mice was comparable to that of the WT-mice. CSF exchange therapy using Umbilical cord plasma ameliorates cognitive impairments in a Tg mouse model for tauopathy '/Alzheimer 's disease

Tg mice model for tauo thy/alzheirne s disease [Tau-tg, expressing double mutant tau protein, generated by us (Exp Neurol. 2008 Jul:212(l): 71~84. doi: 10.1016/j .expneurol.2008.03.007. Epub 2008 Mar 21. A novel transgenic mouse expressing double mutant tau driven by its natural promoter exhibits tauopathy characteristics. Rosenmann HI, Grigoriadis N, Eldar-Levy H, A vital A, Rozenstein L, ^"fouloumi O, Behar L, Ben-Hur T, Avraham Y, Berry E, Segal M, Ginzburg ΐ, Abramsky O)] were treated with the CSF exchange therapy using human umbilical cord plasma (supplied by MAADA bank) diluted by us with artificial CSF (1 : 1 ). Cognitive analysis was performed. The results presented in Figure 14 show an amelioration of the cognitive impairment as presented in the Y- maze: while the Tau-tg mice showed a worse performance relative to the WT- mice (t-test, p=0.03), this impairment of the Tau-tg mice was ameliorated in the Tau-tg mice treated with the CSF exchange therapy using the umbilical cord plasma (diluted in artificial CSF) (t-test, p=0.04). The performance of the treated mice was comparable to that of the WT-mice.

This beneficial effect was also demonstrated in the Open field habituation test, the results of which are presented in Figure 15: the WT-mice showed a significant better performance relative to the Tau-tg (t-test, p-0,02), with a comparable performance to that of the Tau-tg treated mice.

In a further experiment, Tau-Tg mice are treated with the CSF exchange therapy using human umbilical cord plasma (supplied by MAADA bank). Cognitive analysis are performed. An amelioration of the cognitive impairment (Y-maze, T-rnaze and Open field habituation test) are expected in Tau-tg treated mice in comparison to untreated Tau-Tg mice.

CSF exchange in a Tg mouse model for ALS (SOD1-G93A)

Analyzing the motor performance of the mice on an accelerating rotating rod revealed no difference in the latency to fail when comparing treated animals to controls. No difference was also detected in the ALS clinical score.

Interestingly, as seen in Figure 16A, a significantly better performance was detected in the open field test: the CSF exchange -treated mice showed a higher distance traveled (p^:=0.02, repeated ANOVA) relative to the controls. Since open field performance measures not only locomotor activity, but also the degree of anxiety calculated by the percentage of time the animal spends in the center zone, this parameter was also calculated and it revealed a similar trend in which the treated animals spent more time in the central zone, as exhibited in Figure 16B. Without being bound by theory, this may suggest that the higher open field performance in the treated mice is attributed to an improvement in the degree of anxiety rather than to an improvement in locomotor activity.

Preparation of enriched CSF with secretions of various types of differentiated MSCs Figure 17 and Table 1 below show that differentiated neuronal MSC secret a higher level of neurotrophic factors than the non-differentiated (naive MSC , human MSC).

Table 1 : level of secretion from MSC or differentiated MSC

BDNF GDNF NGF NT-4 NT-3 CNTF

20- 90-

MSC 120pg/ml 18-40 pg/ml 10-30pg/ml 10-45pg/ml 20-60pg/ml 240pg/ml

400- 70- 90; 120- 150- 340- diffNMSC 2580pg/mi 420pg ml 550pg/ml 450pg/ml 650pg ml 1200pg/mi

Claims

CLAIMS:

1. An enriched artificial cerebrospinal fluid (EA-CSF) composition comprising: an electrolyte containing solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion;

and at least one neuroprotective factor.

2. The EA-CSF composition of claim 1, wherein each ion, when present in the electrolyte solution, is at a concentration that correlates with its concentration within human cerebrospinal fluid.

3. The EA-CSF composition of claim 1 or 2, comprising one or more salts selected from the group consisting of NaCl, KC1, MgCh, Nai Ι.·ΡΟ · . CaCk, Na2HP0₄ and NaHC0₃.

4. The EA-CSF composition of any one of claims 1 to 3, comprising at least one saccharide.

5. The EA-CSF composition of claim 4, wherein said at least one saccharide comprises or is glucose.

6. The EA-CSF of any one of claims 1 to 5, wherein said neuroprotective factor is selected from the group consisting of neurotrophic factor, anti-inflammatory cytokine and and -oxidative agent,

7. The EA-CSF composition of any one of claims 1 to 6, wherein said neuroprotective factor comprises at least one neurotrophic factor selected the group consisting of Brain-derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT-3) and NT4 Neurotrophin-4 (NT-4) .

8. The EA-CSF composition of claim 6 or 7, comprising a combination of two or more neurotrophic factor.

9. The EA-CSF composition of claim 6 to 8, comprising a combination neurotrophic factors EA-CSF, at least three of the neurotrophic factors selected from the group consisting of BDNF, GDN F, NGF, NT-3 and NT4.

10. The EA-CSF composition of any one of claims 6 io 9, wherein said anti -inflammatory cytokine comprises Transforming Growth Factor beta (TGF-β).

11. The EA-CSF composition of any one of claims 1 to 10, comprising a combination of anti -inflammatory cytokines.

12. The EA-CSF composition of any one of claims 6 to 11, wherein said antioxidant e agent is selected from the group consisting of ascorbic acid (vitamin C), glutathione, uric acids, carotenes, α-tocopherol (vitamin E), and ubiquinol (coenzyme Q).

13. The EA-CSF composition of any one of claims 1 to 12, wherein said at least one neuroprotective factor comprises a combination of neurotrophic factors, antiinflammatory cytokines and anti -oxidative agents.

14. The EA-CSF composition of any one of claims 1 to 13, comprising umbilical cord plasma.

15. The EA-CSF composition of any one of claims 1 to 14, for use in a method of treating a disease.

16. Tlie EA-CSF composition of claim 15, for use in treating a neurological disease.

17. The EA-CSF composition of claim 16, wherein said neurological disease is selected from the group consisting of Multiple Sclerosis, Alzheimer's disease, tauopathies and Amyotrophic lateral sclerosis.

18. The EA-CSF composition of any one of claims 1 to 17, in a form suitable for injection.

19. The EA-CSF composition of any one of claims 1 to 18, in a form, suitable for intrathecal administration or Intracerebroventricular (ICV) deliver}'.

20. The EA-CSF composition of any one of claims 1 to 19, in a form suitable for use in CSF exchange technology.

21. A method of preparing an enriched artificial CSF (EA-CSF) composition, the method comprising:

a) culturing mesenchymal stem cells (MSCs) in a culture medium comprising an electrolyte solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and

b) removing cells from the culture medium to obtain the EA-CSF composition.

22. The method of claim. 21, wherein, said culturing of MSCs in the culture medium, comprising the electrolyte solution is for at least two days.

23. The method of claim 21 or 22, wherein said MSCs are selected from the group consisting of undifferentiated MSCs, Neurahzed MSCs (NMSCs) and terminally differentiated MSCs.

24. The method of claim 23, wherein said NMSCs are characterized by expression of a neurosphere marker selected from the group consisting of nestin and Polysialylated-neural cell adhesion molecule (PSNCAM).

25. The method of claim 23, wherein said temiinaily differentiated MSCs are characterized by expression of a marker selected from the group consisting of Microtubuie-associated protein 2 (MAP2), Tubulin-beta-IIl, Glial fibrillary acidic protein (GFAP) and Cyclic-nucleotide 3'-phosphodiesterase (CNPase).

26. The method of any one of claims 21 to 25, wherein said MSCs are bone marrow MSCs.

27. The method of claim 26, wherein said MSCs are human MSCs (hMSCs).

28. The method of any one of claims 21 to 27, wherein said EA-CSF composition comprises at least one neuroprotective factor

29. The method of claim 28, wherein said neuroprotective factor is selected from the group consisting of neurotrophic factor, anti -inflammatory cytokine and anti -oxidative agent.

30. The method of any one of claim s 21 to 29, wherein each ion, when present in the electrolyte solution, is at a concentration that correlates with its concentration within human cerebrospinal fluid.

31. The method of any one of claims 21 to 30, wherein said electrolyte solution comprises one or more salts selected from the group consisting of NaCl, KCl, MgCh, Xal l -PC . CaCb, Na₂HP0₄ and NaHCC

32. The metliod of any one of claims 21 to 31, wherein said electrolyte solution comprises at least one saccharide.

33. The method of any one of claims 21 to 32, wherein said neurotrophic factor is selected the group consisting of Brain-derived neurotrophic factor (BDNF), Glial cell- derived neurotrophic factor (GDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT- 3) and NT4 Neurotrophin-4 (NT-4).

34. The metliod of any one of claims 21 to 33, wherein said electrolyte solution comprises a combination of two or more neurotrophic factors.

35. The method of any one of claims 21 to 34, wherein said electrolyte solution comprises a combination neuroprotective factors, at least three being selected from the group consisting of BDNF, GDNF, NGF, NT-3 and NT4.

36. The metliod of any one of claims 21 to 35, wherein said anti -inflammatory cytokine comprises Transforming Growth Factor beta (TGF-β).

37. The method of any one of claims 21 to 36, wherein said at least one neuroprotective factor comprises a combination of neurotrophic factors, antiinflammatory cytokines and anti -oxidative agents.

38. A method of preparing an EA-CSF composition comprising mixing an electrolyte solution comprising at least one electrolyte selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion with umbilical cord plasma.

39. The method of claim 38, wherein said electrolyte solution is as defined in any^¬ one of claims 2 to 5.

40. A method of treating a disease in a subject in need comprising administering to said subject an enriched artificial CSF (EA-CSF) composition comprising an electrolyte solution comprising at least one ion selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, phosphor ion, chloride ion and carbonate ion; and at least one neuroprotective factor.

41. The method of claim 40, wherein said EA-CSF composition is as defined in anyone of claims 1 to 20.

42. The method of claim 40 or 41, wherein said administering comprises injection.

43. The method of claim 41, wherein said injection comprises intrathecal administration or Intracerebroventricuiar (ICV) delivery.

44. The method of claim 41, wherein said injection comprises CSF exchange.

45. The method of any one of claims 40 to 44, for treating a neurological disease.

46. The method of claim 45, for treating a disease selected from the group consisting of Multiple Sclerosis, Alzheimer's disease, tauopathies and Amyotrophic lateral sclerosis.

47. A method of treating a neurological disease in a subject in need comprising administering to said subject an amount of umbilical cord plasma.