The present invention, in some embodiments thereof, relates to methods of ex vivo differentiating mesenchymal stem cells towards astrocytic cells using microRNAs.
Mesenchymal stem cells (MSCs) are a heterogeneous population of stromal cells that can be isolated from multiple species, residing in most connective tissues including bone marrow, adipose, placenta, umbilical cord and perivascular tissues. MSCs can also be isolated from the placenta and cord's Wharton's jelly.
The concentration of MSCs in all tissues, including bone marrow and adipose tissue is very low but their number can be expanded in vitro. Typically, expansion of MSCs using up to 15 passages does not result in mutations indicating genetic stability.
MSC can differentiate into cells of the mesenchymal lineage, such as bone, cartilage and fat but, under certain conditions, have been reported to acquire the phenotype of cells of the endodermal and neuroectodermal lineage, suggesting some potential for “transdifferentiation”.
Within the bone marrow compartment, these cells are tightly intermingled with and support hematopoiesis and the survival of hematopoietic stem cells in acquiescent state (7). In addition, after expansion in culture, MSCs retain their ability to modulate innate and adaptive immunity (8). Furthermore, MSCs migrate actively to sites of inflammation and protect damaged tissues, including the CNS, properties that supported their use as new immunosuppressive or rather immunoregulatory or anti-inflammatory agents for the treatment of inflammatory and immune-mediated diseases including autoimmune disorders (9). These features of MSCs merited their use to control life-threatening graft-versus-host-disease (GVHD) following allogeneic bone marrow transplantation, thus controlling one of the most serious complications of allogeneic bone marrow transplantation, helping to lower transplant-related toxicity and mortality associated with multi-system organ injury (10).
Several studies have shown that MSCs following exposure to different factors in vitro, change their phenotype and demonstrate neuronal and glial markers [Kopen, G. C., et al., Proc Natl Acad USA. 96(19):10711-6, 1999; Sanchez-Ramos, et al. Exp Neurol. 164(2): 247-56. 2000; Woodbury, D., J Neurosci Res. 61(4): 364-70, 2000; Woodbury, D., et al., J Neurosci Res. 69(6):908-17, 2002; Black, I. B., Woodbury, D. Blood Cells Mol Dis. 27(3):632-6, 2001; Kohyama, J., et al. Differentiation. 68(4-5):235-44, 2001; Levy, Y. S. J Mol Neurosci. 21(2):121-32, 2003].
Accordingly, MSCs (both ex-vivo differentiated and non-differentiated) have been proposed as candidates for cell replacement therapy for the treatment of various neurological disorders including multiple sclerosis, Parkinson's disease, ALS, Alzheimer's disease, spinal cord injury and stroke.
As an alternative to neuronal cell replacement strategy, in order to increase the survival of existing functional and morphologically normal cells, cell therapy may be aimed at restoring or reestablishing the normal anatomy (e.g. connectivity) and physiology (e.g. appropriate synaptic contacts and functioning neurotransmitters and neuroregulators) of a diseased or damaged tissue.
Astrocytes are the most abundant type of glial cells in the central nervous system and play major roles in the development and normal physiological functions of the brain. Mature astrocytes are divided into two types: fibrous and protoplasmic astrocytes.
Fibrous astrocytes populate the white matter and typically have a ‘star-like’ appearance with dense glial filaments that can be stained with the intermediate filament marker glial fibrillary acidic protein (GFAP). Protoplasmic astrocytes are found in the grey matter, have more irregular, ‘bushy’, processes and typically have few glial filaments. These cells come into contact with and ensheath thin processes, some of which also contact blood vessels.
Astrocytes also regulate water balance, redox potential and ion and neurotransmitter concentrations, secrete neurotrophic factors, remove toxins and debris from the cerebrospinal fluid (CSF) and maintain the blood-brain bather. They also participate in cell-cell signaling by regulating calcium flux, releasing d-serine, producing neuropeptides and modulating synaptic transmission.
Since astrocytes provide structural and physiological support in the central nervous system, differentiation of MSCs towards an astrocytic lineage has been proposed for the treatment of neurological disorders.
Various cells type produce GDNF including glia cells (oligodendrocytes and astrocytes), neuroblastoma and glioblastoma cell lines. It has been shown that rat BMSCs cultured in DMEM supplemented with 20% fetal bovine serum, at passage 6 express GDNF and NGF [Garcia R, et al., Biochem Biophys Res Commun. 316(3):753-4, 2004].
International Patent Publications WO2006/134602 and WO2009/144718 teach differentiation of mesenchymal stem cells into cells which produce neurotrophic factors.
International Patent Publication WO2010/111522 teaches mesenchymal stem cells which secrete and deliver microRNAs for the treatment of diseases.
International Patent Publication WO2010/144698 teaches expression of miRNAs in mesenchymal stem cells to induce neuronal differentiation thereof.
International Application No. IL2011/000660 teaches expression of miRNAs in mesenchymal stem cells to induce oligodendrocytic differentiation thereof.
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