Use of oligodendrocytes from oral neuroectodermal stem cells in the repair of the nervous system

Abstract

This invention concerns a new method for differentiating oral neuroectodermal stem cells (CSO-NE), in particular human gingival neuroectodermal stem cells (CSGh), into oligodendrocytes (OL), and their use in the repair of the nervous system, in particular of head injuries.

Claims

1. A method for inducing differentiation of oral neuroectodermal stem cells into oligodendrocytes, said method comprising: a) a step of differentiating said neuroectodermal oral stem cells into neuroepithelial stem cells in a neurogenic culture medium comprising DMEMf12, N 1 supplement, FGFb and EGF. b) a step of differentiating the neuroepithelial stern cells obtained in step a) into neural stem cells in a neurogenic culture medium comprising DMEMf12, N1 supplement, Noggin protein, FGFb, retinoic acid and SHH factor. c) a step of differentiating the neural stem cells obtained in step b) into oligodendrocyte pre-precursor cells in a neurogenic culture medium comprising DMEMf12, N1 supplement, B27 supplement and FGFb, d) a step of differentiating the oligodendrocyte pre-precursor cells obtained in step c) into oligodendrocyte precursor cells in a neurogenic culture medium comprising DMEMf12, PDGF-AA and SHH factor. e) a step of differentiating oligodendrocyte precursor cells obtained in step d) into oligodendrocytes, and maturing said oligodendrocytes, in a neurogenic culture medium comprising DMEMf12, ICE-1 , biotin, CAMP, PDGF-AA, L-glutamine or Cilutamax™ and NT3.

2. A method according to claim 1, wherein the neuroectodemic oral stem cells are derived from gingival tissue.

3. A cell population comprising oligodendrocytes obtained by the method according to claim 1, said oligodendrocytes expressing the Msx1 gene.

4. Cell population as defined in claim 3, for use in cell therapy.

5. Cell population for use according to claim 4, for the treatment of head injuries.

6. Culture medium for the differentiation of oral neuroectodermal stem cells into neuroepithelial stem cells comprising DMEMf12, N 1 supplement, FGFb and EGF.

7. Culture medium for the differentiation of neuroepithelial stem cells into neural stem cells comprising DMFMf12, N1 supplement, Noggin protein, FGFb, retinoic acid and factor.

8. Culture medium for the differentiation of neural stem cells into oligodendrocyte pre-precursor cells comprising DMFMf12, N1 supplement, B27 supplement and FGFb.

9. Culture medium for the differentiation of oligodendrocyte pre-precursor cells into oligodendrocyte precursor cells comprising DMEMf12, PDGF-AA and SHH factor.

10. Culture medium for the differentiation of oligodendrocyte precursor cells into oligodendrocytes, and for the maturation of said oligodendrocytes, comprising DMEMf12, IGF-1 biotin, CAMP, PDGF-AA, L-glutamine or Glutamax™ and NT3.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0036] FIG. 1 shows the different steps of oligodendrocyte generation from CSGh.

[0037] FIG. 2 shows optical microscopy photos representing the neurospheres obtained from CSGh (stade 1 of the method of the present invention) and their characterization using different staining methods well-known to the skilled person. The neuroepithelial character was confirmed by the positive Nestin staining (specific for neurons). Cellular integrity was verified by nuclear staining with DAPI. The photo “merged” indicated the organization into neurospheres with a lot of Nestin staining at the periphery and a slightly less differentiated reserve of cells in the center. The size of the neurospheres can be measured. (40× magnification)

[0038] FIG. 3 shows optical microscopy photos representing the neural stem cells obtained after differentiation of the neuroepithelial cells obtained in stade 1 (stade 2 of the method of the present invention) and their characterization using different staining methods well-known to the skilled person. The organization into rosette (Day 15, no staining) was specific to neural stem cells cultures. Cellular integrity was verified by nuclear staining with DAPI (+Nestin staining). Neural stem cells were further characterized by a positive Pax6 staining with DAPI.

[0039] FIG. 4 shows optical microscopy photos (no staining) representing (A) the ventral neural tube precursor cells obtained after differentiation of the neural stem cells obtained in stade 2 (stade 3 of the method of the present invention). The precursor cells of the ventral neural tube were characterized by positive OLIG2 staining (B) oligodendrocyte precursor cells (pre-OPC and OPC) differentiated from ventral neural tube precursor cells obtained in stade 3 (stade 4 of the method of the present invention). OL precursors (pre-OPC and OPC) were characterized by a positive NKX2.2 staining.

[0040] FIG. 5 shows optical microscopy photos (no staining) representing the mature oligodendrocytes obtained after differentiation of the oligodendrocyte precursors obtained at stade 4 (stade 5 of the method of the present invention). Mature oligodendrocytes have a characteristic cell morphology (small, branched central cell body.

[0041] FIG. 6 represents the principles and objectives of the invention: method of differentiation of CSG into oligodendrocytes, test of oligodendrocyte functionality and their use in cellular therapy for the repair of head injuries.

EXAMPLES

Example 1

Differentiation Protocol of CSGh into Oligodendrocytes

[0042] The different experiences were based on the differentiation of: PPSCs (pluripotent stem cells) (Hu et al., 2009 [20]), hESCs: (Human embryonic stem cells) (Nistor et al., 2005 [18]), and MSCs (mesenchimatous stem cells) (Leite et al., 2014 [22]).

TABLE-US-00001 Stade 1 Differentiation into neuroepithelial cells (CNE): neurospheres (FIG. 2) Cell Culture Type of Treatment of Culture Cell Culture Protocol line medium Supplements culture culture dish dish dissociation time CSO-NE CSO-NE MN N1, FGFb(50 ng/ml), Suspended Poly-Heme 65 mm Accutase 5 days EGF(10 ng/ml) Observations: Suspended culture in a neurogenic medium composed of: DMEMf12, N1 supplement, 0.5%, non-essential amino acids, 0.5%, antibiotic 0.5%, FGFb 50 ng/ml, EGF 10 ng/ml. Remarks: In this part of suspended cell culture: 4 cell populations may appear: floating spheres, cell populations that adhere to the bottom of the dish despite treatment, adherent spheres and a last population of cells that have lipid vacuoles related to the presence of insulin in the N1 supplement (0.5 mg/ml) (generally these are cells very sensitive to serum). For the passage to the next stade, only floating spheres containing cells capable of going beyond this first emancipation to the neural line are recovered. It should be noted that these spheres exhibit different sizes and densities and that dense spheres give rise to small spheres (5-10 cells) that proliferate over time. The culture time has been adjusted to 5 days to avoid any cell necrosis in the central part of the spheres and which seems sufficient to move to the next stade. These spheres are positive in Nestin, PIIITub (partially).

TABLE-US-00002 Stade 2 Differentiation into neural stem cells (NSC): neuroepithelial stem cells (FIG. 3) Cell Culture Type of Treatment of Culture Cell Culture Protocol line medium Supplements culture culture dish dish dissociation time CSO-NE CSO-NE MN N1, Adherent untreated 65 mm Yes, 10 days Noggin(100 ng/ml), Accutase FGFb(50 ng/ml), RA(20 nM), SHH(100 ng/ml) Observations: Adherent cultures in a neurogenic environment composed of: DMEMf12, supplement N1. The cells that are well dissociated at first, begin to organize radially by forming what are called rosettes (simulating the organization of neural tube cells). The cells at this stade are in proliferation and still keep the fusiform shape. After 5 days, the rosettes become mature rosettes and they begin to connect with other rosettes until the confluence of the cells. Remarks: Consideration was given to adding RA to neuroepithelial cells (mature rosette stade in the differentiation protocol) that carry the identity of cells derived from the dorsal anterior part of the neural tube and to differentiate them into OPCs that are in themselves derived from the ventral part of the tube; caudalizing factors such as the SHH factor or the RA that induces the expression of the Hox PG1 genes, the Nlz-1 and -2 genes, and vHnfl; these genes are essential for the development of this part and the development of OPC in vivo.

TABLE-US-00003 Stade 3 Differentiation of oligodendrocytes into pre-precursor cells (Pre-OPC) (FIG. 4A) Cell Culture Type of Treatment of Culture Cell Culture Protocol line medium Supplements culture culture dish dish dissociation time CSO-NE CSO-NE MN N1, B27, FGFb Adherent Laminin 65 mm In continuity (3 days) with stade 4 Observations: The cells were sensitive to spherical culture: a very significant loss of cells between stade 2 and 4. The cells were kept adherent between these two stades with the changes of medium in order to direct them towards OPC, knowing that from this stade onwards several cells present a bipolar or multipolar shape with small branches reminiscent of the shape of oligodendrocyte progenitors. Remarks: Cells tend to form spheres after the 10th day of culture, which explains why some authors use the suspended technique for this stade of differentiation.

TABLE-US-00004 Stade 4 Differentiation into oligodendrocyte precursor cells (OPC) (FIG. 4B) Cell Culture Type of Treatment of Culture Cell Culture Protocol line medium Supplements culture culture dish dish dissociation time CSO-NE CSO-NE MN 20 ng/ml, PDGF-AA, Adherent Polyornithine, 12-well Accutase 20 days 200 ng/ml SHH or laminin plate suspended Observations: OPCs are in the form of small cells with bipolarity and several terminal branches, some of these cells have lipid vacuoles. These OPCs are better cultivated in suspension which promotes the differentiation of these cells and their maturation. FGFb and EGF were removed because these factors block cell differentiation. Remarks: OPCs send extensions to connect with other OPCs and tend to cluster in 3-dimensional clusters and some reproduce the organization of the cells of the ventral neural tube.

TABLE-US-00005 Stade 5 Differentiation of OPCs into oligodendrocytes and their maturation (OL) (FIG. 5) Cell Culture Type of Treatment of Culture Cell Culture Protocol line medium Supplements culture culture dish dish dissociation time CSO-NE CSO-NE MN IGF-1, T3, biotin, Adherent PDL, 12-well Accutase >15 days cAMP, PDGFAA, I- polyornithine plate glutamine 2 mM and laminin Glutamax ™, NT3 Observations: Cells with a small central cell body and several branches Remarks: The cells at this stade are cultured in islets: 10000 cells per 100 μl of medium. [0043] PPSCs: pluripotent stem cells [0044] hESCs: Human embryonic stem cells [0045] MSCs: Mesenchymal stem cells [0046] CSO-NE: Oral neuroectodermal stem cells [0047] DMEMf12: DMEM F12 Media|Dulbecco Modified Eagle Medium, Gibco® [0048] MN: Neurogenic medium [0049] N1: Supplement N1 (used to produce a complete growth medium for N1 neuronal cells or a closely related medium) [0050] EGF: epidermal growth factor [0051] FGFb (or FGF2): basic fibroblast growth factor [0052] B27: Supplement allowing the proliferation of cells, e.g. neuronal cells, without differentiation. [0053] IGF or IGF-1: insulin-like growth factor 1 [0054] PDL: poly-D-lysine [0055] T3: T3 factor (thyroid hormone T3) inducer for oligodendrocyte line precursors [0056] RA: retinoic acid [0057] SHH: protein factor Sonic HedgeHog (role in regulating the fate of neural stem cells) [0058] Noggin: (NOG) protein involved in the development of many tissues, including nervous tissues [0059] PDGF: Platelet-derived growth factor (regulates cell growth and division) [0060] PDGF-AA: AA isoform of platelet-derived growth factor (regulates cell growth and division)

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