WO2017064688A1

WO2017064688A1 - Neurite regeneration therapy based on exosomes derived from menstrual stem cells

Info

Publication number: WO2017064688A1
Application number: PCT/IB2016/056234
Authority: WO
Inventors: Maroun Khoury; Ursula WYNEKEN; Maria Alejandra LOPEZ
Original assignee: Cells For Cells, S.P.A.; Universidad De Los Andes
Priority date: 2015-10-15
Filing date: 2016-10-17
Publication date: 2017-04-20

Abstract

The present invention offers an effective alternative method for treating neurological disorders, preferably those induced by a nerve injury or by a neurodegenerative process. The present invention is the first to show that a substantially pure population of exosomes derived from menstrual stem cells (MenSCs) have the capacity to increase the neuritic outgrowth of cortical and sensory neurons. The present invention shows that a substantially pure population of MenSCs-derived exosomes increases the length of the longest neurite and the ramification pattern of cortical neurons. MenSc and bone marrow stem cells (BMSCs) also promote the neuritic growth of neurons from the dorsal route ganglia. Overall, the invention offers a promising alternative method to treat neurological disorders involving the loss of nerve tissues. Since it is principally composed of exosomes produced by the stem cells present in menstrual fluid, the invention provides an ease access and repeated sampling in a non-invasive manner.

Description

Neurite regeneration therapy based on exosomes derived from Menstrual Stem Cells

Field of the invention

The present invention can be included in the field of new medical treatments, wherein specific organelles of specific cells are used for treating a given disease or disorder. In particular, a substantially pure population of exosomes from Menstrual Stem cells is used in the present invention to treat a loss of nerve tissue caused by a nerve injury or by a neurodegenerative disease.

Background of the invention Mesenchymal stem cells (MSCs) are self-renewing, multipotent progenitors that have the capacity to promote tissue-repair and neuroprotection (Kfoury and Scadden, 2015). MSCs can be harvested from bone marrow (BM), adipose tissue, skin, umbilical cord (UC), dental pulp and discard tissues such as menstrual fluids (Meng et al., 2007; Domini ci et al., 2009). In the nervous system, MSCs obtained from the BM improve behavioural deficits in a rat model of Parkinson's Disease and counteract depressive-like behavior by promoting neuronal growth and survival (Levy et al., 2008; Tfilin et al., 2010), while MSCs from UC enhance brain angiogenesis after stroke (Zhu et al., 2015) and MSCs from adipose tissue increase the number of lumbar motoneurons and motor performance in a mouse model of amyotrophic lateral sclerosis (Marconi et al., 2013). Researchers originally postulated that following their injection, MSCs home to injured tissues, thus replacing damaged cells. The administration of MSCs after hypoxic-ischemic brain injury improved recovery, however, with a low level of MSC engraftment in the ischemic zone (Chen et al., 2003; Bennet et al., 2012). Similarly, little MSC engraftment was quantified following cell treatment of lung and heart injuries (Chimenti et al., 2010; Katsha et al., 201 1), as well as in a rat model of Parkinson's Disease, supporting a paracrine mechanism of action through many secreted factors including MSC-derived extracellular vesicles, which contain a large array of biologically active molecules (Somoza et al., 2010; Lavoie and Rosu-Myles, 2013; Yu et al., 2014).

Microvesicles and exosomes are extracellular vesicles secreted by most cell types. Microvesicles (also named microparticles or ectosomes) display diameters ranging from 100 nm to 1000 nm. They are released to the extracellular environment directly by evagination from the plasma membrane (Heijnen et al, 1999). In contrast, the size of exosomes is restricted to diameters from 30 to 200 nm. They are contained in an endosomal compartment within multivesicular bodies which, once they fuse with the plasma membrane, release them into the extracellular space (Colombo et al., 2014; Yanez-Mo et al., 2015). Both vesicle types are enriched in distinctive sub-fractions after differential centrifugation steps of biological fluids and/or conditioned media of cell cultures (Thery et al., 2006). However, some confusion remains about the correct nomenclature regarding their distinctive subcellular origin and characteristic size distribution (Colombo et al., 2014; Kreimer et al., 2015). The fact that both extracellular vesicle types contain proteins, mRNA, microRNA and lipids, which can trigger cellular responses in target cells, supports their role as mediators of cell-to cell communication (Mittelbrunn and Sanchez- Madrid, 2012). Interestingly, MSC-derived extracellular vesicles have been shown to exert functions similar to those of their secreting MSCs: exosomes are able to reduce the size of myocardial infarctions (Lai et al., 2010a), and to improve neurovascular re-modelling after traumatic brain injury (Zhang et al., 2015b), while a extracellular vesicle fraction containing both exosomes and microvesicles were able to stimulate tubular cell proliferation and improve recovery from acute kidney injury (Bruno et al., 2009). It is well-known that each cell type secretes extracellular vesicles with a specific molecular signature (Villarroya-Beltri et al., 2014). Thus, extracellular vesicles secreted by MSCs of different origins might differ in their content and consequently, in their biological function on target cells. We have recently shown that MenSCs exert superior paracrine-mediated responses compared to bone marrow MSCs, by secreting vascular endothelial growth factor and basic fibroblast growth factor, and improving angiogenesis in endothelial cells (Alcayaga- Miranda et al, 2015). Additionally, they present several practical advantages in comparison to other presently known adult-derived stem cells. MenSCs are easy to obtain in a non-invasive and periodic manner and devoid of ethical dilemmas (Khoury et al., 2014). However, a systematic study of the growth-promoting effect of extracellular vesicles derived from different MSC sources on neurons is still lacking. Thus, for the first present invention we first focused on the effect of MenSCs on neuronal outgrowth comparing cell-cell contact with the total secretome and their extracellular vesicle fractions. Then, we compared the effect of exosome-enriched fractions harvested from MenSCs with stem cells from chorion (Ch-SCs), umbilical cord (UC-SCs) and bone marrow (BM-SCs). The differential advantages and benefits of MenSC-derived exosomes compared to other MSC sources in our experimental paradigm forward them to the frontline of promising candidates as novel treatments in neurological diseases. Brief description of the invention

Regenerative approaches in neurological diseases using MSCs involve injection of living cells. These therapies propose that upon transplantation into the brain, MSCs will promote endogenous neuronal growth and synaptic connections, reduce apoptosis and inflammation, primarily through paracrine mechanism by producing trophic factors. However, as already stated, the administration of MSCs after hypoxic-ischemic brain injury improved recovery, yet, with a low level of MSC engraftment in the ischemic zone.

Surprisingly, the present invention provides an alternative improved approach to treat a loss of nerve tissue caused by a nerve injury or by a neurodegenerative disease. In this sense, said approach involves the use of MSC derivatives, in particular exosomes derived from MSCs derived from menstrual fluid (MenSCs). The superior effect of MenSC-derived exosomes on the growth of cerebrocortical neurites with respect to exosomes derived from other MSC sources provides for a novel and alternative therapies for the treatment of diseases of the nervous system.

Brief description of the fieures

Fig 1. Figure 1 shows that MenSCs contact with neurons inhibits neurite outgrowth, while their secretome increases it.

A) Neurons were stained with antibodies against acetylated tubulin (green) and MAP2 (red) to visualize neurites. DAPI staining (blue) shows the cell nuclei. In the contact condition, the presence of nuclei surrounding the neuron indicate the presence of MenSCs. Scale bar=10 lm. (B) Quantitative analysis of neuronal morphology assessed by Scholl analysis. The following parameters were evaluated: longest neurite, maximal branch number and distance to maximal number of neurites. *P<0.05 compared to control, #P <0.05 compared to other treatments. Data are presented as mean±SEM (n=150 neurons per experimental group in 4 independent experiments).

Fig 2. Figure 2 shows that MenSCs secrete microvesicles and exosomes.

(A) Morphological characterization of MenSC microvesicles by electron microscopy. Scale bar=100 nm. (B) Morphological characterization of MenSC exosomes. Scale bar=100 nm. (C) Diameter distribution of microvesicles (black bars) and exosomes (gray bars) from MenSCs in negatively stained preparations. (D) Dot plot showing the purification process of microvesicles (MV) from the conditioned medium of MenSCs. Both the forward scatter area (FSC-A), associated to the relative size of the particles, and the side scatter area (SSC-A), associated to the complexity of the particles, were used for the analysis. Fraction 1 shows debris, cells and microvesicles after centrifuging the total supernatant once at 10,000 g. Fraction 2 shows the microvesicle pellet, obtained in sequential steps after removing cells and debris with centrifugations (300g and 2,000g) followed by a 10,000g centrifugation. The 10,000g supernatant (Fraction 3) reveals the poor detection capacity of smaller vesicles by flow cytometry. (E) Detection and characterization of microvesicles by flow cytometry show the presence of CD73, CD90, CD105, al integrin, CD63 and HLA-I in microvesicles(lower panel). MenSCs (upper panel) were used as positive controls and matching isotype antibodies were used as negative controls (light gray histograms). Histograms are representative of n=4 independent xperiments using microvesicles from 4 different MenSC donors. (F) Characterization of MenSC exosomes by western-blot. Purified exosomes (Exo, 20 μg of exosomes) are positive for the exosomal markers CD63, TSGlOl, Hsp70 and Hsp90. The non-exosomal protein Rab5 was not detected in exosomes. MenSC lysate (CL, 20 μg per lane) was used as a positive control.

Fig 3. Figure 3 shows the neurotrophic effect of MenSC secretome and extracellular vesicles. (A) Flow chart of the differential centrifugation-based protocols. The secretome fraction was isolated with protocol 1, while microvesicles (MVs) were isolated after the 10,000g centrifugation followed by exosome (Exo) isolation using protocol 2. Microvesicles (MV) and exosomes (Exo) were isolated together using protocol 3. (B) Cortical neurons were immunostained with acetylated tubulin (green) and MAP2 (red) antibodies to visualize neurites. Scale bar=10 μιτι. (C) Quantitative analysis of neuronal morphology. *P<0.05 compared to control, #P <0.05 compared to other treatments. Data are presented as mean ± SEM (n=150 neurons per group in 4 independent experiments).

Fig 4. Figure 4 shows the effect of exosomes from different sources of MSCs on neuronal growth.

(A) Characterization of exosome size, shape and presence of specific markers (CD63 and TSGlOl). Exosomes were purified from bone marrow (BM), chorion (Chor) and umbilical cord (UC)-derived MSCs. Scale bar=100 nm. (B) Cortical neurons were immunostained with acetylated tubulin (green) and MAP2 (red) antibodies to visualize neurites. Scale bar=10 μηι. (C) Quantitative analysis of cortical neurons morphology (data are mean±SEM). *P<0.05 compared to control, #P <0.05 compared to other treatments. Data are presented as mean±SEM (n=150 neurons per group in 4 independent experiments). (D) Axonal regeneration of DRGs after vehicle (Ctrl) or MSC exosomes treatment during 4 days. DRGs were stained for acetylated tubulin (Ac-Tub, green) and nuclei with DAPI (blue). Scale bar=500 μηι. (E) Quantification of the rate of axonal growth after treatment with vehicle (Ctrl) or exosomes from different MSC sources (mean ± SEM, n=5 DRG per treatment from three separate experiments; *P<0.05, linear regression).

Detailed description of the invention

Definitions

In the context of the present invention, the term "neurodegenerative disorder" is understood as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system.

In the context of the present invention, the term "nerve injury" is understood as an injury of a nervous tissue.

In the context of the present invention, the term "neurite" refers to any projection from the cell body of a neuron. This projection can be either an axon or a dendrite. The term is frequently used when speaking of immature or developing neurons, especially of cells in culture, because it can be difficult to tell axons from dendrites before differentiation is complete. - In the context of the present invention, the term "Menstrual stem cells (MenSCs)" is understood as stem cells isolated from the menstrual fluid of woman that are in fertile ages. These cells show spindle-shape morphology, and stem cell-like phenotypic markers: MenSCs express CD105, CD44, CD73, CD90 and HLA-ABC, but show negative expression for CD45, CD34, CD14 and HLA-DR. They also show mesodermal lineage differentiation under specific protocols of the laboratory. In the context of the present invention, the term "Bone marrow stem cells (BMSCs)" is understood as

In the context of the present invention, "exosomes" refer to small vesicles (30-200 nm) that originate when the inward budding of endosomal membrane forms Multivesicular bodies (MVBs). Exosomes are released into the extracellular space when the MVBs fuse with the plasma membrane. They contain a specific molecular cargo, which comprising proteins, DNAs, m NAs and miRNAs.

In the context of the present invention, "MenSCs-derived exosomes" refers to the exosomes produced by the MenSCs. They are isolated by a process of serial dilutions by centrifugation of the supernatant of the MenSCs culture, followed by two ultracentrifugations of the supernatant to save the pellet, which contains the exosomes.

In the context of the present invention, the term "isolated" indicates that the exosomes or exosome population is not within the environment where it originated. The exosome or the population of exosomes has been substantially separated from surrounding environment. In some embodiments, the exosomes or exosome population is substantially pure or enriched if it was separated from surrounding environment and if the sample contains at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, in some embodiments at least about 85% in some embodiments at least about 90%, and in some embodiments at least 95% of exosomes. In other words, the sample is substantially pure or enriched if it was separated from the surrounding environment and if the sample contains less than about 50%, less than 40%, less than 30%, preferably less than 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the exosomes. Such percentage values refer to percentage by weight or of a population of extracellular vesicles, wherein extracellular vesicles refer to membrane surrounded structures released by cells to the extracellular environment. They display diameters ranging from 20 nm to 5 μηι, depending on their subtype. There are released by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of extracellular vesicles includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. The term isolated also encompasses exosomes which have been removed from the environment from which they originated. The term also encompasses exosomes which have been removed from the environment where they originated, and subsequently re-inserted into an organism. The organism which contains the re-inserted exosomes may be the same organism from which the cells that produced the exosomes originated, or it may be a different organism, i.e. a different individual of the same species.

Description

Menstrual Stem Cells (MenSCs) are stem cells obtained from the menstrual fluid of woman that are in fertile ages. These cells show spindle-shape morphology, and stem cell-like phenotypic markers: MenSCs express CD105, CD44, CD73, CD90 and HLA-ABC, but show negative expression for CD45, CD34, CD14 and HLA-DR. They also show mesodermal lineage differentiation under specific protocols of the laboratory. In particular, it is noted that mesodermal lineage induction of the MenSCs showed positive specific staining for fat, bone and cartilage differentiation, and that MenSCs are obtained from menstrual fluid from fertile healthy woman donors aged 13 to 50 years old, preferably 19 to 45 years old, more preferably between 20 and 40 years old. It is still further noted that the MenSCs of the present invention are strictly non-embryonic derived stem cells.

This population of MenSCs out-performs the broadly studied bone marrow derived mesenchymal stem cells (BMSCs) in proliferation rate and support of hematopoietic stem cells (HSCs) expansion in vitro.

Exosomes are small vesicles of 30 to 200nm, preferably 30 to 150 nm, preferably 40 to 120 nm, more preferably between 50 and 100 nm in diameter that originate when the inward budding of endosomal membrane forms multivesicular bodies (MVBs) and are produced by almost all cell types and cancer cells. Exosomes are released into the extracellular space when the MVBs fuse with the plasma membrane. They are emerging as key mediators in intercellular communications through horizontal transfer of information via their molecular cargo, which includes proteins, DNAs, mRNAs and miRNAs that could trigger specific intracellular cascades that affect the gene expression of the recipient cells.

A substantially pure population of exosomes can be isolated from the supernatant (conditioned media) of the MenSCs culture following serial centrifugation steps, as described in the Examples. It is known that immunoblotting shows positive expression of HSP90, HSP70, TSG101 and CD63, which were enriched in comparison with the cell lysate, while the non- exosomal protein RAb5 was absent in the purified exosome fraction.

The present invention is based on the discovery that the substantially pure population of exosomes isolated from a culture of MenSCs have neurite-regenerative or outgrowth capacities and can be used to treat a neurological disorder involving a loss of nerve tissue caused by a nerve injury or by a neurodegenerative disease, such as, but not limited to, an ischemic stroke or a Head and Spinal Cord Trauma, Alzheimer^'s disease, Parkinson disease, glioblastoma multiforme, Epilepsy, Huntington's Disease, amyotrophic lateral sclerosis and Multiple Sclerosis. It is noted that neuritic outgrowth refers to the increase in the length and/or number of neurites from a completely or partially differentiated neuronal cell.

As it was previously reported (Paul R. Sanberg et al., Neurological disorders and the potential role for stem cells as a therapy, British Medical Bulletin 2012) the pathological characteristics of Parkinson's disease include the loss of the dopaminergic projection neurons of the substantia nigra pars compacta and the presence of a-synuclein-positive Lewy bodies, whereas Alzheimer's disease is characterized by the loss of neurons from the cortex and hippocampus and the presence of beta-amyloid plaques and tau-tangles. Multiple sclerosis involves the loss of the myelin sheath surrounding neurons and these are all progressive disorders. The more acute disorders such as stroke, traumatic brain injury or spinal cord injury involve the loss of cells in direct response to an insult such as ischemia or blunt trauma,though indirect cell loss with time also occurs. In epilepsy, cells fire abnormally which can result in seizures and changes in attention or behavior. The progressive neurodegenerative disorders also include diseases caused by a genetic mutation or deletion such as Huntington's disease, muscular spinal atrophy and Sanfilippo syndrome. Treatments for these disorders would therefore be expected to replace the lost cells (of the substantia nigra, cortex or hippocampus), clear the pathological hallmarks (e.g. synuclein or beta amyloid deposition) or repair cell function. By contributing or promoting neurite, particularly axonal, regeneration or outgrowth, the present invention provides a composition that contributes to repairing or improving nerve cell function and thus to the effective treatment of neurological disorders involving a loss of nerve tissue caused by a nerve injury or by a neurodegenerative disease. In this sense, we herein show that a substantially pure population of exosomes derived from or obtained from a culture of MenSCs can promote the growth of the longest neurite of a cortical neuron (fig 3 B-C, fig 4B), and even surpass an inhibitory effect of microvesicles on this same parameter and on the ramification pattern of these neurons (fig 3 B-C). Consequently, the invention has the clear potential to promote neurite outgrowth and therefore to be used to treat different diseases related with loss of neural tissue, in particular for the treatment of neurodegenerative disorders or of nerve injuries.

To our knowledge, this is the first time that a substantially pure population of exosomes derived from MenSCs are shown to have a specific therapeutic effect in neurodegenerative disorders. It is further noted and just a mere example, the exosomes can be obtained from the MenSCs by following serial centrifugation steps of the MenSCs culture method or, more specifically, by a method which comprises the following steps:

1. Centrifugation of MenSCs cultured in DMEM media and collection of the supernatant.

2. Serial centrifugations of the supernatant (300g for 10 min, 2000g for 10 min and 10,000g for 30 min at 4 °C).

3. Ultracentrifugation of the supernatant at 100,000g for 70 min at 4 °C.

4. Resuspension (in the final excipient of the composition) of the pellet containing the extracellular vesicles enriched in exosomes and centrifugation again at 100,000g for 1 h at 4°C.

5. Resuspension of the pellet in the final excipient of the composition.

It is a further aspect of the present invention (a second aspect) to provide a safe and more effective preparation or composition of exosomes that is suitable for the treatment of diseases and conditions that involve neurodegeneration. In this sense, a second aspect of the invention refers to a preparation or composition comprising a substantially pure population of exosomes obtained or derived from MenSCs for use in the treatment of diseases and conditions that involve neurodegeneration, such as a disease selected from the list consisting of Alzheimer^'s disease, Parkinson disease, glioblastoma multiforme, Epilepsy, Huntington's Disease, amyotrophic lateral sclerosis and Multiple Sclerosis.

Other objects of the present invention will become apparent to the person of skill upon studying the present description of the invention. In a preferred embodiment of the second aspect of the invention, the preparation or composition is for use in the treatment of neurodegenerative disorders.

It is a further aspect of the present invention (a third aspect) to provide a safe and more effective preparation or composition of exosomes that is suitable for the treatment of diseases and conditions that involve nerve injury. In this sense, a third aspect of the invention refers to a preparation or composition comprising a substantially pure population of exosomes obtained or derived from MenSCs for use in the treatment of diseases and conditions that involve nerve injury, such as a disease selected from the list consisting of ischemic stroke or a head and spinal cord trauma.

Other objects of the present invention will become apparent to the person of skill upon studying the present description of the invention. In a preferred embodiment of the second aspect of the invention, the preparation or composition is for use in the treatment of neural injury.

A fourth aspect of the invention refers to a pharmaceutical preparation comprising a substantially pure population of MenSCs derived exosomes. The pharmaceutical preparation according to this aspect of the present invention is preferably enriched for exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or chromatographically enrichment using the monolithic technology (e.g. CDVI®, BIA separations, Austria) as stationary phases instead of columns packed with porous particles. Monoliths are continuous stationary phases that are cast as a homogeneous column in a single piece and prepared in various dimensions with agglomeration- type or fibrous microstructures. (see Iberer, G., Hahn, R., Jungbauer, A. LC-GC, 1999, 17, 998). Using these methods, surprisingly active fractions containing exosomes could be obtained. Then, in order to identify the most suitable fraction according to the invention, fractions being enriched with exosomes are tested for their in vitro neurite regenerative effect, and can further be analyzed, in microbiological, in virulence and in pyrogen tests to, for example, excluded possible contaminations. In addition, these fractions can be studied with regard to protein content, and particle size.

It could be found that fractions being enriched with exosomes were particularly useful in any of the diseases according to the second or third aspect of the present invention, if they exhibited strong in vitro effects in neuro, in particular neurite or axonal, regeneration (understood as the renewal of any nerve tissue through the internal processes of a body or system in activity tests), where, following the addition of said exosome-enriched fraction, an increased length of the longest neurite, or on the ramification pattern of neurons is observed. The present invention is thus based on the novel concept for an improved prevention and treatment of diseases by using a substantially pure population of MenSCs derived exosomes, in particular in patients suffering from a disease according to the second and third aspect of the invention. The pharmaceutical preparation according to the present invention is preferably enriched with exosomes having a size of between about 30 to 200 nm, 30 to 150 nm, preferably 40 to 120 nm, more preferably between 50 and 100 nm in size. "About" shall mean a +/- 10% deviation. Further preferred, the substantially pure population of exosomes are positive for cellular exosome markers.

The pharmaceutical composition may preferably contain at least 500 ng, more preferably at least 1 ug, of exosomes. The exosome amount can be measured by protein amount, for example, by using a Bradphore assay (BioRad) or a BCA protein assay kit (Pierce). Yet, the optimal dose will be selected according to the administration route, treatment regime and/or administration schedule, having regard to the existing toxicity and effectiveness data. In a preferred embodiment the substantially pure population of exosomes is in a dosage capable of providing a neuro regenerative effect, in particular neurite or axonal regeneration, in the absence of toxic effects. There are insufficient data from human and animal studies to establish a Safe Upper Level for this substantially pure population of exosomes, although the available data from this invention indicates that it is of low toxicity in vitro. Accordingly, in the present invention, the dosage of the pharmaceutical composition is not particularly restricted and may vary with the weight and health of the patient and might be estimated by a person skilled in the art, taking into account the experimental data from this invention.

In addition, said one or more pharmaceutical compositions are formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. This includes, for example, injections, by parenteral routes such as intralesional, intravascular, intravenous, intraarterial, subcutaneous, intra-cerebral, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically contemplated, by such means as depot injections or erodible implants. A preferred route of administration is intralesional administration, which is herein understood as the administration within the injured tissue. Alternatively, the administration can be intravascular, which is herein understood as the administration within a vessel or vessels and typically includes intravenous or intraarterial administration. In another aspect of the present invention, the pharmaceutical preparation according to the present invention, is suitable for i.v. administration, such as for example, intravenous administration or infusion into a patient in need thereof, or for intralesional administration. Another aspect of the present invention then relates to a method for producing a pharmaceutical preparation according to the present invention, comprising the following steps: a) providing a cell culture medium supernatant from MenSCs comprising exosomes, b) enriching substantially pure populations of exosomes, c) preferably determining an in vitro neuro-regenerative, in particular neurite or axonal regeneration properties, and selecting those substantially pure population of exosomes that exhibit a neuro-regenerative effect, and e) admixing said substantially pure population of exosomes of step c) with at least one suitable pharmaceutical excipient and/or carrier.

The method for producing a pharmaceutical preparation according to the present invention comprises the step of specifically enriching for substantially pure populations of exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or a monolithic method (see above), since using these methods, surprisingly active fractions containing substantially pure populations of exosomes could be obtained. Preferred is a method for producing a pharmaceutical preparation according to the present invention, wherein fractions that were enriched for substantially pure populations of exosomes are further analyzed in microbiological tests, virulence tests, protein content, pyrogen tests, and particle size, in order to identify the most suitable fraction according to the invention. It could be found that fractions that were enriched for exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro neuro-regenerative, in particular neurite or axonal regeneration, effects in activity tests.

A further aspect of the present invention refers to an in vitro method to promote neuritic regeneration or outgrowth or to promote axonal growth. In particular, such further aspect of the invention refers to an in vitro method to promote neuritic regeneration or outgrowth or axonal regeneration, the method comprising contacting a composition which in turn comprises a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of nerve cells, preferably with a partially or totally differentiated neuron culture; more preferably with a neuron culture wherein neurons are cortical neurons or with a culture of spinal or dorsal root ganglia.

A still further aspect of the invention refers to a substantially pure composition comprising nerve cells, preferably neurons, obtained or obtainable by the method of the precedent paragraph. A yet further aspect refers to a method for treating a neurodegenerative disorder or a nerve injury, the method comprising administering to the subject the composition of the previous paragraph, such that the neurodegenerative disorder or nerve injury in the subject is treated.

The following examples serve to illustrate the invention but they do not limit the same. Examples

Materials and methods

All experimental procedures were approved by the Institutional Review Board of Universidad de los Andes. Primary neuronal cultures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals ( IH Publications No. 80-23), revised 1996. In turn, the obtention of human stem cells was conducted in accordance with the Declaration of Helsinki.

Purification of microvesicles and exosomes from MSCs.

The MSC cultures from 3 different human donors were performed from human menstrual fluid, UC, chorion (Chor) and BM, as described in our previous studies (Alcayaga-Miranda et al., 2015; Gonzalez et al., 2015). MSCs were obtained and characterized in our laboratory according to the International Society for Cellular Therapy guidelines; i.e. plastic adherence, specific surface antigen expression of the mesenchymal markers CD 105, CD73 and CD90, negative expression of hematopoietic cell surface markers CD34, CD133 and CD45 and multipotent differentiation potential toward osteogenic, chondrogenic and adipogenic lineages, as described in our previous studies (Alcayaga-Miranda et al., 2015; Gonzalez et al., 2015). When indicated, MenSCs (5,000 cells) were seeded on 0.4-lm microporous membrane (Transwell, BD Bioscience), separated from cortical neurons. For extracellular vesicles purification, MSCs (3 X 10⁷ cells) at passages 2 to 6 were supplemented with serum-free DMEM (Gibco), 2 mM L- glutamine (Life Technologies, Santiago, RM, Chile) and 1% penicillin/ streptomycin (Life Technologies, Santiago, RM, Chile) for 72 h. The supernatant (conditioned media) was collected and subjected to serial centrifugations (300g for 10 min, 2,000g for 10 min and 10,000g for 30 min at 4 ° C). The microvesicle-enriched fraction was obtained from the 10,000g pellet, washed in PBS and centrifuged again at 10,000g, as previously described (Theryet al.,2006). In parallel, the supernatant from the 10,000g centrifugation was ultracentrifuged at 100,000g for 70 min at 4 °C to obtain the pellet, i.e. the exosome-enriched fraction, as previously described (Thery et al., 2006; Lopez- Verrilli et al., 2013). Protein concentration was quantified byBCAProtein Assay Kit (Pierce). To study the effect of the secretome on neurite outgrowth, the conditioned media were centrifuged at 300g for 10 min, 2,000g for 10 min and concentrated with Amicon Ultra 3 kDa tubes (Millipore) at 4 °C.

Characterization of exosomes and microvesicles

Electron microscopy. Microvesicles and exosomes were fixed with 2% PFA, deposited on Formvar-carboncoated grids and visualized by electron microscopy (EM) as previously described (Thery et al, 2006; Lopez- Verrilli et al., 2013). Flow cytometry. For flow cytometry analysis, the samples were prepared as described elsewhere (Alcayaga-Miranda et al., 2015). Briefly, the samples were acquired on a FACS Canto II Tm (BD Biosciences, San Jose, CA, USA) after performing the daily calibration recommended by the manufacturer with BDFACSDiva Cytometer Setup & tracking beads (BD Biosciences, San Jose, CA, USA). Once the daily calibration was done, either the micro vesicles or cells stained with the following conjugated mouse anti-human antibodies CD90 (clone 5E10), CD73 (clone AD2) CD 105 (clone 266), CD49a (clone TS2/7) and CD49f (rat anti Human) from BD Pharmigen; HLA-ABC (clone W6/32) from Biolegend; and CD63 (clone MX-49.125.5) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), were acquired in low flow rate to avoid the formation of aggregates (n=4, 10,000 events per sample). In all experiments, matching isotope controls were used as staining controls to compare with the positive signal of the conjugated antibodies. Results were analyzed with Flowjo software (Tree Star Inc, Ashland, OR, USA).

Western blot. Twenty micro gram of exosomes and MSC lysates was loaded into 10% SDS- PAGE as previously described (Lopez-Verrilli et al., 2013). The following antibodies were used: anti-CD63 (rabbit polyclonal 1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti- heat shock protein 70 (Hsp70, mouse monoclonal 1 : 1000, Stressgen), anti-heat shock protein 90 (Hsp90, rabbit polyclonal 1 : 1000, Stressgen), anti- TSG101 (mouse monoclonal 1 :500, Abeam), anti-Rab5 (rabbit polyclonal 1 :500, Abeam, Santiago, RM, Chile). Secondary antibodies used were goat anti-mouse HRP and goat anti-rabbit HRP (BioRad). Western blots were revealed by enhanced chemiluminescence (Amersham).

Neuronal cell cultures

Cortical neuron cultures. Were obtained from embryonic Sprague Dawley rats (El 8) as previously described (Sandoval et al., 201 1). Neurons (5000) were maintained inNeurobasalmedium(Life Technologies, Santiago, RM, Chile) supplemented with B27 (Life Technologies), 2 mM glutamine (Life Technologies) and penicillin/ streptomycin (Life Technologies). After 1 day in vitro, experiments were initiated with the addition of 3 μg of exosomes to a final volume of 500 μΐ of culture medium to assess neuronal morphology 72 h later in four separate experiments.

In all cases, n=150 neurons per experimental group were analysed using Sholl analysis(Image J software). Each concentric radius was at 5 lm from each other. The following parameters were obtained: length of the longest neurite, critical value (i.e. the radius or distance from the soma at which the maximum number of neurite crossings occurred) and the total number of crossings of concentric circles, a parameter that reflects branching.

Dorsal root ganglia (DRG) cultures. Were dissected from the spinal cord of embryonic C57 mice (El 6) as previously described (Lopez- Verrilli et al., 2013). Briefly, DRGs were plated on coverslips and maintained in Neurobasal medium (Invitrogen, Santiago, RM, Chile) containing 2% B27 (Invitrogen, Santiago, RM, Chile), 2 mM L-glutamine (Life Technologies), 50 ng/mL human nerve growth factor (Invitrogen, Santiago, RM, Chile), 1% penicillin- streptomycin (Life Technologies, Santiago, RM, Chile) and 10 1M cytosine arabinoside (Sigma- Aldrich, Santiago, RM, Chile) to eliminate non-neuronal cells.

The next day, DRGs were supplemented with 3 lg of exosomes to a final volume of 500 11 of culture medium on a daily basis during 4 days. Experiments were repeated 3 times with five replicates per condition and the length of the 10 longest neurites was measured in mm after 1, 3 and 4 days of treatment using the Image J software. This methodology is validated in previous papers in the field (Lopez- Verrilli et al., 2013).

Immunofluorescence

Neurons were fixed with 4% paraformaldehyde/4% sucrose and processed as previously described (Lopez- Verrilli et al., 2013). Neurons were stained with anti-MAP2 (mouse monoclonal 1 : 1000, Millipore), anti-acetylated-tubulin (mouse monoclonal 1 : 1000, Sigma- Aldrich, Santiago, RM, Chile), anti-a-tubulin (rabbit polyclonal 1 : 1000, Abeam) and nuclei with DAPI. The following secondary antibodies were used: Alexa555- conjugated goat anti-mouse IgG and Alexa488- conjugated goat anti-rabbit diluted at 1 : 1000 in PBS (Molecular Probes, Santiago, RM, Chile). Statistical analysis

Data are presented as mean ± SEM. Statistical significance was assessed by a one-way analysis of variance (ANOVA) followed by Tukey post-test and linear regression (GraphPad Software, Inc., San Diego, CA, USA). P values <0.05 were considered significant.

Example 1. MenScs contact with neurons inhibits neurite outgrowth, while their secretome increases it

First, we investigated whether the MenSCs or their secretomes modulate neuritic growth in primary cultures. Neurons were cultured alone or with MenSCs at a ratio of 1 : 1 in direct contact condition, or separated by a 0.4-μπι microporous membrane (Transwell condition) (Fig. 1A).

In Fig. IB, the following parameters are quantified: the length of the longest neurite (71 ±6 lm in contact vs 1 19±9 lm in control and 147±8 lm in the Transwell condition), indicating that cell contact not only reduced the longest neurite but also that the secretome (represented by the Transwell condition) stimulated its growth with respect to control (n=150 neurons per condition, p<0.05). The maximal number of processes decreased in neurons in contact with MenSCs (6.6 ±0.42 processes/neuron in contact vs 8.4±0.39 processes/neuron in the secretome and 8.4±0.30 processes/neuron in the control condition, n=150 neurons per condition, p<0.05), with ramifications closer to the cell soma (15±0.7 lm in contact vs 19 ±1.1 lm in the Transwell condition). We thus focused our study on identifying the specific components involved in the effect induced by the secretome by analysing independently its constituents, which were operationally defined as the pellet after a centrifugation at 10,000g (i.e. pellet enriched in microvesicles) and the pellet after centrifugation at 100,000g (i.e. pellet enriched in exosomes).

Example 2 Characterization of microvesicle- and exosome-like fractions secreted by MenSCs

MenSC microvesicles showed a heterogeneous size ranging from 200 to 1040 nm of diameter with a mean of 552±20 nm, as assessed by EM (Fig. 2A, C). We further characterized MenSC microvesicles by flow cytometry. To assess the validity of the differential centrifugation steps to isolate microvesicles, the following fractions were compared in the dot blots of Fig. 2D: the total secretome containing cell debris, cells and microvesicles but excluding exosomes (Fraction 1 , i.e. the pellet of 10,000g without subfractioning), microvesicles (Fraction 2) and exosomes (Fraction 3). The comparative analysis of both 10,000g pellets confirmed the relevance of discarding the contaminating products (cell debris and cells) by serial centrifugations to obtain a cleaner analysis of microvesicles. We then evaluated the presence of MenSC-specific surface markers in microvesicles by flow cytometry. Microvesicles derived from MenSC were positive for transmembrane and adhesion proteins, such as CD73, CD90, CD105, al integrins, as expected (Bruno et al., 2009), together with CD63 and HLA-I (Fig. 2E).

In turn, the 100,000g pellet presented spheric shapes, with an average size of 90±2 nm compatible with the morphology and size expected for exosomes derived from different cell types, including MSCs (Lai et al., 2010b) (Figs. 2B, C). Although a novel high-resolution flow cytometry-based method has been developed for quantitative analysis of nano-sized vesicles (Nolte-'t Hoen et al, 2013; van der Vlist et al., 2012), conventional flow cytometers cannot distinguish between vesicles with a diameter less than 300 nm. Thus, we characterized exosomes by Western blot (Fig. 2F). Exosomes showed typical markers such as CD63, TSG101, Hsp70 and Hsp90, which were enriched with respect to the cell lysate, while the early endosome marker ab5 was not present in the exosome fraction, as expected (Lopez- Verrilli et al., 2013). Taken together, these results show that MenSCderived exosome and microvesicle fractions carrying the characteristic protein repertoire as well as of characteristic size and shape were obtained. Example 3 Effect of the total and fractionated MenSC secretome on neuronal outgrowth

We then compared the effect of the MenSC total secretome, or its exosomal and microvesicular fractions on neuronal outgrowth. The fractionation process described in Fig. 3 A shows the different centrifugation protocols used to obtain the following fractions from the conditioned medium: extracellular vesicles together with soluble proteins (secretome), microvesicles alone, exosomes alone or microvesicles with exosomes. At one day following cortical neurons' isolation and culture, the medium of each culture condition was supplemented with 3 lg of protein of one of the obtained fractions and incubated for three days. Conversely to neurons in the Transwell condition, where an increased extension of the longest neurite was observed, the morphology of neurons supplemented with the secretome showed no difference when compared to the control condition (Fig. 3B, C). This might be due to the fact that in the case of the Transwell condition, neurons are continuously supplied with the secretome while in the second case, a fixed quantity of protein was added only once to cultures at one day in vitro. Microvesicles induced a striking decrease on the length of the longest neurite (64±5 lm in microvesicles vs 143±9 lm in control condition, n=150 neurons per condition, p<0.05), of the maximal branch number (4.8 ±0.30 processes/neuron in microvesicles vs 7.8±0.23 processes/neuron in the control condition, n=150 neurons per condition, p<0.05) and affected the ramification of neurons as revealed by the radius at which the maximal number of neurite crossings occured (12 ±0.7 lm in microvesicles vs 17±0.9 lm in control condition, p<0.05). The differential effect of microvesicles on neuron morphology was also statistically significant when compared to each of the different experimental conditions. When neurons were supplemented with the combination of microvesicles and exosomes, neurons restored their maximal neurite branch number and ramification pattern with respect to control condition, indicating that exosomes can block the inhibitory effect of microvesicles. Moreover, the combination induced an increase of the longest neurite compared to the control condition (198±19 lm in microvesicles plus exosomes vs 143±9 lm in control condition, p<0.05), indicating that exosomes can surpass the inhibitory effect of microvesicles on the growth of the longest neurite. Exosomes alone showed the same effects as their combination with microvesicles, being exosomes per se beneficial for neuronal outgrowth.

Example 4. Screening of MSC exosomes on neurite outgrowth

Taking into consideration the beneficial effect observed for MenSC-derived exosomes on neurite outgrowth of central nervous system neurons, we isolated exosomes from different sources of MSCs, i.e., UC, Chor and BM. The aim was to establish a comparative study classifying the potential effect of exosomes derived from the most clinically relevant MSC sources. EM analysis of exosomes showed the expected round shape and mean diameters of 87±3 nm for BM-, 101 ±5 for Chor- and 62±2 nm for UC-MSC-derived exosomes, respectively, together with the presence of the exosome markers CD63 and TSG101 (Fig. 4A). Exosomes from these MSC sources were supplemented to the cortical neurons culture at a concentration of 3 lg exosomes/5000 neurons. Unlike the effect of exosomes derived from MenSCs, exosomes from BM, UC and Chor did not alter the length of the longest neurite (Fig. 4B, C). Chor-SC exosomes decreased the total branch number with respect to control condition (7.1 ±0.19 processes/neuron in Chor-SC exosomes vs 9.7 ±0.26 processes/neuron in the control condition, p<0.05), while BM-SC-derived exosomes induced an increase of the distance to the maximal number of ramifications from the cell soma compared to control neurons (Fig. 4B, C). A similar protocol was followed to assess the comparative effect of exosomes on sensory neurons from DRG (Fig. 4C). DRGs were supplemented with vehicle (PBS) or 3 lg of exosomes on a daily basis for 4 days. On days 1, 3 and 4, the extension of the longest neurite was measured followed by a quantification of the growth rate. We observed that MenSC and BM-SC exosomes increased the rate of neuritic growth compared to control (304±5 and 323±8 lm/day with MenSC and BM-SC exosomes, respectively) in comparison with the control (226±6 lm/day), (n=3 independent experiments, n=5 ganglia per experiment, n=15 neurites per neuron, p<0.05) (Fig. 4D, E). Chor-SC and UC-SC exosome-treated neurons did not present any difference in comparison with untreated control condition (245±7 and 238 ±81m/day, respectively). Taking together, these results indicate that MenSC- and BM-SC-derived exosomes promote neurite growth both in cortical neurons and in sensory neurons, thus adding clear evidence that the cell type of origin is an essential factor in defining the biological activity of exosomes.

CONCLUSIONS TO EXAMPLES

Here we show for the first time that MSCs purified from menstrual fluid, release microvesicles and exosomes, and that exosomes present in the conditioned medium exert a beneficial effect on neuronal outgrowth, an action that could not be fully reproduced by exosomes derived from other MSC sources or by other MenSC culture components. Although regenerative approaches in neurological diseases using MSC injection of living cells have revealed biosafety, the use of alternative approaches involving MSC derivatives, such as extracellular vesicles, is of great promise as therapeutic conveyors for neurological diseases.

Our results provide the basis for stating that exosomes are associated with beneficial effects observed in cell therapy, but circumvent its multiple risks such as adverse immune responses, tumor formation and vascular occlusion, just to mention a few. Supporting this, extracellular vesicles did not evoke acute immune responses or immune cell infiltration after systemic administration in mice (Doeppner et al., 2015). In addition, peripherally administered exosomes can reach the central nervous system (Xin et al., 2013; Ridder et al., 2014; Zhang et al., 2015b), and as such will represent an invaluable tool to treat neurological disorders in the future. The originating cell type is essential to define the biological activity of exosomes. Thus, our findings support the potential therapeutic application of exosomes from MenSCs to promote axonal regeneration after nerve injury in the central as well as peripheral nervous system References

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Claims

1. A method for treating a neurodegenerative disorder or a nerve injury, the method comprising administering to the subject a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), such that the neurodegenerative disorder or nerve injury in the subject is treated.

2. The method of claim 1, wherein the exosomes are isolated from Mesenchymal stem cells derived from menstrual fluid.

3. The method of claim 1, wherein the method is for treating a nerve injury.

4. The method of claim 1, wherein the method is for treating a neurodegenerative disorder.

5. The method of claim 4, wherein the neurodegenerative disorder or nerve injury is selected from the group consisting of Alzhemer^'s disease, Parkinson disease, glioblastoma multiforme, Epilepsy, Huntington's Disease, amyotrophic lateral sclerosis and Multiple Sclerosis.

6. The method of claim 3, wherein the nerve injury is derived from an ischemic stroke or a Head and Spinal Cord Trauma.

7. The method of claim 2, wherein the method is for treating a nerve injury.

8. The method of claim 2, wherein the method is for treating a neurodegenerative disorder.

9. The method of claim 8, wherein the neurodegenerative disorder or nerve injury is selected from the group consisting of Alzhemer^'s disease, Parkinson disease, glioblastoma multiforme, Epilepsy, Huntington's Disease, amyotrophic lateral sclerosis and Multiple Sclerosis.

10. The method of claim 7, wherein the nerve injury is derived from an ischemic stroke or a Head and Spinal Cord Trauma.

11. The method of any of the precedent claims, wherein the composition is administered intra-lesionally, intravenous, intra-arterial or intra-cerebrally.

12. The method of claim 1, wherein the composition comprises at least about 1 Dg of exosomes as measured by the total protein amount contained in the exosomes.

13. An in vitro method to promote neuritic regeneration or outgrowth, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a partially or totally differentiated neuron culture.

14. An in vitro method to promote neuritic regeneration or outgrowth, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a neuron culture.

15. An in vitro method to promote neuritic regeneration or outgrowth, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of nerve cells.

16. An in vitro method to promote neuritic regeneration or outgrowth, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of cortical neurons.

17. An in vitro method to promote neuritic regeneration or outgrowth, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of spinal or dorsal root ganglia.

18. An in vitro method to promote axonal regeneration, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a partially or totally differentiated neuron culture.

19. An in vitro method to promote axonal regeneration, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a neuron culture.

20. An in vitro method to promote axonal regeneration, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of nerve cells.

21. An in vitro method to promote axonal regeneration, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of cortical neurons.

22. An in vitro method to promote axonal regeneration, the method comprising contacting a composition comprising a substantially pure population of exosomes isolated from Mesenchymal stem cells derived from menstrual fluid (MenSCs (menstrual stem cells)) or bone marrow (BMSCs (bone marrow stem cells), with a culture of spinal or dorsal root ganglia.

23. A substantially pure composition comprising nerve cells, preferably neurons, obtained or obtainable by the method of any of claims 13 to 22.

24. A method for treating a neurodegenerative disorder or a nerve injury, the method comprising administering to the subject the composition of claim 23, such that the neurodegenerative disorder or nerve injury in the subject is treated.