WO2008031451A1

WO2008031451A1 - A post-natal periodontal-derived neural stem cell

Info

Publication number: WO2008031451A1
Application number: PCT/EP2006/066221
Authority: WO
Inventors: Barbara Kaltschmidt; Darius Widera; Wolf-Dieter Grimm; Christian Kaltschmidt
Original assignee: Institut Für Molekulare Diagnostik Und Innovative Therapie - Molthera - Gmbh
Priority date: 2006-09-11
Filing date: 2006-09-11
Publication date: 2008-03-20

Abstract

The present invention refers to a post-natal neural stem cell, characterized by: the stem cell is derived from the oral cavity, provided that the stem cell is not derived from dental follicle or dental pulpa; the stem cell is Nestin positive and Sox2 positive when cultured in suspension culture; and the stem cell is Scleraxis negative. Further it is provided a method for the preparation and/or proliferation of a neural stem cell, comprising the following steps: a) provision of a tissue sample from periodontium or palatum containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is Scleraxis negative.

Description

A post-natal periodontal-derived neural stem cell

[0001] The present invention refers to a post-natal periodontal-derived neural stem cell, a method for preparation and its use.

[0002] Neural stem cells are a potential source of cells for cell therapy of neurodegenerative diseases, applications in cell biology and/or drug screening and regenerative medicine in periodontology, dentistry, and/or surgery. Beside the potential benefit, ethical and practical considerations limit the application of neural stem cells derived from human embryonic stem cells or adult brain tissue.

[0003] Thus, alternative sources of human neural stem cells are of high interest for both basic research and potential clinical use as well. These sources have to satisfy the claims of readily accession, rapid expansion in chemically defined media, and reliably induction to a neural fate and/or mesodermal or endodermal fate.

[0004] Background of the present invention

[0005] During mammalian central nervous system (CNS) development, multipotent precursor cells (stem cells) undergo division, cell fate specification, and finally maturation in response to extrinsic stimuli. These neural stem cells (NSCs) are characterized by the capability to undergo several cell divisions and to differentiate into multiple cell types e.g. neuronal or glial cells. Within the adult, there are two major sources of adult neural stem cells - the subgranular zone of the hippocampus and the subventricular zone (SVZ) (Doetsch et al., 1999; Johansson et al., 1999a). Adult NSCs can be cultured as self adherent cell clusters called neurospheres (Johansson et al., 1999a). These 3D neurospheres can be cultured for several passages without loosing their proliferation, migration and differentiation capabilities. [0006] Up to date several isolation and culture protocols for adult neural stem cells from SVZ and the subgranular zone have been established (Gage, 2000; McKay, 1997; Rao, 1999; Reynolds and Weiss, 1992). Furthermore, more restricted multipotent neuronal progenitor cells with limited capacity to give rise to both neuronal and glial progeny in vitro have been isolated from numerous adult brain regions in rodents and humans (Johansson et al., 1999b; Pagano et al., 2000; Roy et al., 2000). Since NSCs are more lineage-restricted than embryonic stem cells (ES cells), they represent less of a risk for tumor (teratoma) formation following transplantation Bithell and Williams (Clinical Science 108, 13-22 2005). Indeed, several animal transplantation studies carried out, showed that there is little evidence of their spontaneous tumorigenicity.

[0007] Recently, several studies showed a wide differentiation potential of somatic stem cells (Clarke et al., 2000; Eglitis and Mezey, 1997; Jiang et al., 2003; Jiang et al., 2002; Joannides et al., 2004; Joannides et al., 2003; Martin-Rendon and Watt, 2003).

[0008] The periodontal ligament represents a cell renewal system in steady state. In a perivascular location, progenitor cells exhibiting features of somatic stem cells have been identified (Gronthos et al., 2000; Miura et al., 2003; Seo et al., 2004; Shi et al., 2005; Shi et al., 2001). In order to take advantage of potential therapeutic opportunities of somatic stem cells as a source for adult human neural stem cells, novel methods for efficient isolation, cultivation in chemically defined environment (serum-free), rapid expansion and induction into neural fate are required.

[0009] Adult human neural stem cells are difficult to obtain due to their limited accessibility. Therefore, there is need for alternative sources for neural stem cells. These sources might include somatic stem cells from non-neuronal organs and tissues. [0010] Therefore, the object of the present invention was to provide a stem cell from a different source which preferably is capable of producing progeny that are capable of differentiating into neuronal cells or glial cells.

[0011] Summary of invention

[0012] The technical problem was solved by a post-natal neural stem cell, characterized by:

- the stem cell is derived from the oral cavity, provided that the stem cell is not derived from dental follicle or dental pulpa;

- the stem cell is Nestin positive and Sox2 positive when cultivated in suspension culture;

- the stem cell is Scleraxis negative.

[0013] In a preferred embodiment the present invention provides for the first time a post-natal periodontal tissue derived neural stem cell and which is not derived from dental follicle or dental pulpa, wherein the stem cell shows in suspension culture expression of the markers Nestin and Sox2, wherein the stem cell is Scleraxis negative, and wherein the cell can be obtained by minimal- invasive surgical procedure.

[0014] The expression of the markers Nestin and Sox2 characterize the stem cell as being neural or neuroectodermal. Accordingly, the stem cell of the present invention stains positive for Nestin and Sox2 and does not stain positive for Scleraxis. According to the present invention the stem cell provided or its progeny is able to differentiate into cells and/or tissues of the nervous system, including neurons, glial cells, Schwann cells, astrocytes and oligodendrocytes, by in vivo or in vitro induction.

[0015] In a preferred embodiment the stem cell is derived from periodontium or palatum, preferably from human periodontium or human palatum. The advantage is the easy access of the respective tissues containing the stem cells of - A -

the present invention by minimal-invasive surgical methods. In particular, the present invention provides for the first time a periodontal tissue derived neural stem cell (hereinafter called "pdNSC") and which is not derived from dental follicle or dental pulpa, wherein the stem cell shows in suspension culture expression of the markers Nestin and Sox2, and wherein the stem cell is Scleraxis negative. The expression of the markers Nestin and Sox2 means that the respective cells either can be stained positive for Nestin and Sox2 and/or the respective mRNA for Nestin and Sox2 can be detected, within a short time period sufficient for transcription and translation of the respective genes (Nestin and Sox2) after the cells have been brought into contact with above mentioned culture conditions and this expression was detectable at least after 14 days.

[0016] The stem cells of the present invention may be preferably isolated from periodontium or palatum. In contrast to the some prior art methods the present invention exclusively makes use of post-natal or adult, i.e. non-fetal, stem cells. By using periodontium or palatum as a source of the stem cell the collection of said cells will be of no harm for the donor.

[0017] Furthermore, it has been shown for the first time that cells of the nervous system may be generated by using stem cells isolated from periodontium or palatum and for inducing the differentiation of these cells to neural cells.

[0018] A population of cells having the phenotype of neural stem cells

(according to the present invention) have "multipotent" developmental potential and they can give rise to cells and/or tissues of the nervous system as was found by the inventors of the present invention. The term "multipotent" stem cells refers to cells that are capable of self-generation during propagation, and which have the capacity in vitro or in vivo to differentiate into lineage committed cells that further proliferate and terminally differentiate into cells of the nervous system. Such multipotent neural stem cell progenitors may differentiate into cells of the nervous system, including neurons, glial cells, Schwann cells, astrocytes and oligodendrocytes, through in vitro or in vivo induction. [0019] The stem cells can be isolated with low effort and are used for the generation of human cells and/or tissues of the nervous system. The generated cells may be used for therapeutic treatment for example in autologous, allogenous or exogenous transplantation. The transplantation of the isolated cells or the transplantation of their progeny cells which were generated by in vitro cultivation allows the substitution of damaged or dead cells in tissues which have limited self- renewal capacities.

[0020] Furthermore, the stem cells according to the present invention are able to survive and to integrate when transplanted into rat organotypic hippocampal slice cultures.

[0021] In a further preferred embodiment the stem cell shows expression, preferably expression, of the markers Nestin and Sox2, if the stem cell is cultivated in suspension culture subsequently after dissociation of the tissue sample in which the stem cell is contained, under culture conditions using serum-free media containing at least one proliferation-inducing growth factor.

[0022] Further, preferred is that the stem cell has the capability to proliferate into a spheroid aggregation of cells in a suspension culture using serum-free medium, wherein each of said spheroid aggregation of cells contains stem cells, which are capable of producing progeny that are capable of differentiating into neuronal cells or glial cells or osteoblasts or other mesenchymal cells. It is particularly preferred that the serum-free medium is containing basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) and a supplement containing putrescine, retinyl acetate, transferrin and insulin.

[0023] It is further preferred that the stem cell is capable of producing progeny that are capable of differentiating into neuronal cells or glial cells or osteoblasts or cells of the periodontium or teeth. [0024] In another preferred embodiment the stem cell is capable of producing progeny that are capable of differentiating into neuronal cells under culture conditions using serum-free media containing at least one proliferation- inducing growth factor. For this it is particularly preferred that the medium contains basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF) and retinoic acid. The cells cultivated this way are then expressing β-lll-tubulin, neurofilament M, H and L; Map-2, and GAD67. In another preferred embodiment the stem cell is capable of producing progeny that are capable of differentiating into periodontal cells, or periodontal ligament or osteoblasts or cementoblasts under known culture conditions suitable for differentiating these cells.

[0025] In an alternative embodiment the stem cell is capable of producing progeny that are capable of differentiating into glial cells under culture conditions using media containing serum in the absence of growth factors. The cells cultivated this way are then expressing GFAP.

[0026] The stem cell according to the present invention preferably expresses each of the following markers: Nestin, Sox2, CNPase, Emx2 and CD117. It is further preferred that the stem cell does not express the following markers: L1 , LeX, A2B5, CD34, CD45, CD90, CD133, PSA-NCAM, β-lll-tubulin, GFAP, neurofilament 3 (NF-H/NF-200KD), Neuro D1 , GMNN, Notchi , Oct4.

[0027] In a preferred embodiment the stem cell of the present invention is characterized in that the stem cell has a significant shorter doubling time than stem cells derived from dental pulpa. Preferably, the stem cell according to the present invention has a doubling time which is by at least a factor 1.2 shorter, preferably by at least a factor 1.5 shorter, most preferred by at least a factor 2.0 shorter than the doubling time of stem cells derived from dental pulpa.

[0028] Further preferred, the stem cell has the capability to migrate when stimulated with one or more of the following substances: macrophage chemoattractant protein MCP-1 , stem cell factor (SCF) and stromal-derived factor 1 alpha (SDF-1 alpha).

[0029] In another preferred embodiment the stem cell is defined as proliferative cell and as self renewing cell capable of producing progeny that are capable of differentiating into cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm.

[0030] In still a further preferred embodiment the stem cell is genetically modified.

[0031] The present invention also provides a spheroid aggregation of cells in suspension culture derived from the stem cell according to the present invention. In particular, the present invention also provides a spheroid aggregation of cells in suspension culture derived from a post-natal periodontal-derived neural stem cell, wherein the stem cell is derived from the oral cavity, provided that the stem cell is not derived from dental follicle or dental pulpa; wherein the stem cell shows in suspension culture expression of the markers Nestin and Sox2; and wherein the stem cell is Scleraxis negative.

[0032] The present invention further provides a medical product comprising the stem cell or the above mentioned spheroid aggregation of cells according to the present invention.

[0033] The present invention further provides a medicament comprising the stem cell or the above mentioned spheroid aggregation of cells according to the present invention.

[0034] In addition the present invention also refers to the use of the stem cell according to the present invention or of the spheroid aggregation of cells according to the present invention for the manufacture of a medicament for the treatment of the diseases selected from the group consisting of: Periodontal Diseases and periimplantitis/perimucositis, Parkinson disease, Alzheimer^'s disease, muliple sclerosis.

[0035] Further, it is provided the use of the stem cell according to the present invention or of the spheroid aggregation of cells according to the present invention for the manufacture of a medicament for the regeneration of the dental innervation, or the regeneration of periodontium and of dental implant sites and of alveolar ridge augmentations.

[0036] The present invention also provides the use of the stem cell for the differentiation of cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm. The inventors have shown that the stem cell according to the invention can be differentiated into cells of ektoderm, endoderm or mesoderm and that the stem cell according to the invention can be used for the isolation and enrichment of human neural crest- derived cells.

[0037] The present invention further provides a method for the preparation and/or proliferation of a neural stem cell, comprising the following steps: a) provision of a tissue sample from periodontium or palatum containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is Scleraxis negative.

[0038] The method is surgical minimal-invasive and does not involve the provision of an isolated tooth or penetration into a tooth; this means that the method according to the present invention does not include a tooth extraction. Therefore, this new method has the advantage to isolate stem cells without having to exfoliate third molars, or to access the dental pulp.

[0039] Preferably, in step c) a serum-free medium is used containing basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) and a supplement containing putrescine, retinyl acetate, transferrin and insulin.

[0040] In a preferred method after step c) the cells are cultivated under culture conditions using a serum-free medium containing at least one proliferation- inducing growth factor to induce differentiation into neuronal cells. Preferably, the serum-free medium is containing basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF) and retinoic acid.

[0041] In an alternative method after step c) the cells are cultivated under culture conditions using a medium containing serum and in the absence of proliferation-inducing growth factors, to induce differentiation into glial cells.

[0042] In a further preferred alternative method after step c) the cells are cultivated under culture conditions, wherein the expression of NF-kappaB in said cells is manipulated, to induce differentiation into mesodermal, ectodermal or endodermal cells.

[0043] A preferred embodiment provides after step c) that the cells are modified by introduction of nucleic acids and or a pharmaceutical compound, for providing a drug-delivery system. Preferably, the nucleic acid introduced into said cell is expressing a substance selected from the group peptide drug, polypeptide drug, an anti-sense RNA, an RNA providing RNAi-effect.

[0044] According to the present invention the spheroid aggregation of cells obtained in step c) is derived from human somatic stem cells present in the tissue sample from human periodontium or human palatum. [0045] Preferably, the method is performed for the isolation and/or enrichment of human neural crest-derived cells. Further preferrred the method is performed for the preparation and differentiation of cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm.

[0046] The present invention further refers to a cell culture obtainable by the method according to the present invention. Further, it is provided a neuronal cell or glial cell or osteoblast obtainable by the method according to the present invention.

[0047] Particularly, the present invention provides a method of transplanting a neural stem cell into a host, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual by minimal-invasive periodontal surgery containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of neuronal cells, glial cells, cells of the periodontium, periodontal ligament, osteoblasts, cementoblasts, osteoblasts; e) transfer of the cells obtained in c) or d) into the host; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is

Scleraxis negative.

[0048] In a further embodiment a method is provided for the treatment of periodontal diseases, periimplantitis or perimucositis, or for the regeneration of the dental innervation, or for the regeneration of periodontium, periodonal ligament, osteoblasts, cementoblasts, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of neuronal cells, glial cells, cells of the periodontium, periodontal ligament, cementoblasts, osteoblasts; e) transfer of the cells obtained in c) or d) into the site of intervention of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0049] In another embodiment of the present invention a method is provided for implant dentistry, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the alveolar ridge developmental site of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0050] In still another embodiment of the present invention a method is provided for augmentation in maxillofacial surgery, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of surgical maxillo-facial interventions of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0051] In a further embodiment of the present invention a method is provided for alveolar ridge augmentation, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of augmentation/ interventions of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0052] In still another embodiment a method is provided for the treatment of

Parkinson^'s disease or Alzheimer^'s disease, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neuarl stem cells; d) optionally, differentiation of the cells obtained in c) into neuronal cells and/or glial cells; e) transfer of the cells obtained in c) or d) into the central nervous system of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is Scleraxis negative. [0053] In a further alternative embodiment a method is provided of the treatment in maxillo-facial surgery, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of maxillo-facial surgery of the respective individual; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0054] In a further embodiment a method is provided for the treatment of diseases involving defects in cartilage or bone, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of osteoblasts, osteoclasts and/or chondrocytes; e) transfer of the cells obtained in c) or d) into the host; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

[0055] The present invention describes for the first time a method for efficient minimal invasive isolation, serum-free cultivation and reliable neural fate induction of periodontal derived neural stem cells (pdNSCs). Furthermore, the stem cells according to the present invention (pdNSCs) are able to survive and to integrate when transplanted into rat organotypic hippocampal slice cultures. [0056] Using minimal-invasive periodontal surgery (Gassmann and Grimm,

2006) the inventors isolated human somatic stem cells from the human periodontium. These periodontal stem cells could be propagated as neurospheres in serum-free media, which underscores their cranial neural crest cell origin.

[0057] Tissue isolated from biopsy samples of adult human periodontium was cultured and expanded in the presence of the mitogens epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2/bFGF). Cultured periodontal tissue derived neural stem cells (pdNSCs) are highly proliferative and are able to migrate in response to chemokines described to induce migration of neural stem cells, lmmunocytochemical techniques, and molecular biology based marker analysis have been used to assess neural differentiation after the treatment of expanded cells with a novel induction media. Initial characterization of cultured periodontal cells confirmed the presence of nestin, a neural precursor marker. Sequential culture in EGF and FGF-2 in serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive neural stem cells. Adherence to substrate and growth factor deprivation resulted in cells of neuronal morphology and stable expression of markers of neuronal differentiation.

[0058] The studies furthermore showed that retinoic acid treatment greatly enhances the neuronal induction of the cells to more than 90 % of cells with neuronal differentiation markers. Thus the method according to the present invention might provide nearly limitless numbers of neural precursors from a readily accessible autologous adult human source which could be used as a platform for further experimental studies and has potential therapeutic implications.

[0059] Detailed description of the present invention / Best mode for carrying out the invention

[0060] According to the present invention it is provided for the first time a periodontal tissue derived neural stem cell and which is not derived from dental follicle or dental pulpa, wherein the stem cell shows in suspension culture expression of the markers Nestin and Sox2, and wherein the stem cell is Scleraxis negative. Furthermore, the stem cells according to the present invention are able to survive and to integrate when transplanted into rat organotypic hippocampal slice cultures. This new stem cell has the advantage to be derived from accessible tissue without having to exfoliate third molars, or to access the dental pulp.

[0061] Further, it is described for the first time a method for the preparation and/or proliferation of a neural stem cell, comprising the following steps: a) provision of a tissue sample from periodontium or palatum containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is Scleraxis negative. Preferably, in step c) a serum-free medium is used containing basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) and a supplement containing putrescine, retinyl acetate, transferrin and insulin.

[0062] In particular, the present invention provides for the first time a minimal-invasive periodontal surgical method for isolation of adult periodontal derived human neural stem cells. This new method has the advantage to access autologous stem cell containing tissue without the necessity to exfoliate third molars if still obtainable, or to access the dental pulp. Furthermore a method for serum-free culture, rapid expansion and subsequent efficient induction of neural phenotype of periodontium-derived neural stem cells (pdNSCs) is described.

Figure 9 summarizes a scheme for the isolation and differentiation procedures applied.

[0063] The isolated and cultured, highly proliferative cells were positive for neural sternness markers nestin and Sox2 and negative for differentiation markers β-lll-tubulin for neurons and GFAP for glial cells. In addition, pdNSCs migrated if exposed to chemokines described as migration inducing in NSCs isolated from SVZ. RA treatment efficiently induced neuronal fate of pdNSCs as showed by high levels (>90%) of neuron specific markers as β-lll-tubulin, Map2 and neurofilaments M, H (Fig. 6) and L (data not shown). If cultured in presence of 10% FBS, pdNSCs differentiated into glial lineage as demonstrated by glial morphology and robust GFAP expression after four days of differentiation.

[0064] Finally, the present invention demonstrates pdNSCs to be able to survive and to integrate if transplanted into rat organotypic hippocampal slice cultures. Demonstrably, no ossification and other teratoma-like structures were observed in this cross-species transplantation assay. Despite the heterologous nature of the transplanted neural stem cells they survived and integrated. Further studies should investigate functional physiology.

[0065] Cellular therapies using adult human neural stem cells are promising approaches for the treatment of several chronic or acute neurological diseases such as Parkinson's, Alzheimer^'s or Huntington's diseases. One main problem relates to the potential use of human neural stem cells is the very limited accessibility of the source. Thus there is high interest on alternative sources for human NSCs.

[0066] Recently, some reports provided evidence for efficient isolation of adult human neural stem cells from skin (Joannides et al., 2004) or post-mortem human retina (Mayer et al., 2005).

[0067] For potential therapeutic use, it is eminent that cells are cultured in chemically defined, serum-free media. Demonstrably, cells isolated from skin biopsies were cultured in chemically non-defined media, namely in media containing 10% FBS. Similarly, studies providing evidence for plasticity of mesenchymal stem cells (MSCs) and differentiation into neural fate utilize serum- containing media (Deng et al., 2006; Wislet-Gendebien et al., 2005a; Wislet- Gendebien et al., 2005b; Zemchikhina and Golubeva, 2005).

[0068] Gronthos and co-workers reported stem cell properties of human dental pulp cells isolated from human third molars (wisdom teeth) and cultivated in serum-containing media (Gronthos et al., 2000; Seo et al., 2004; Shi et al., 2001 ). Ex-vivo expanded dental pulp stem cells (DPSC), stem cells from human exfoliated deciduous teeth (SHEDs) and periodontal ligament stem cells (PDLSCs) expressed a heterogeneous assortment of markers associated with mesenchymal stem cells (MSCs), dentin, bone, smooth muscle, neural tissue and endothelium. Interestingly, the dental pulp stem cells were also described as nestin positive. Nestin, one of the intermediate filaments constituting the cytoskeleton is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine (Fujita et al., 2006). Similarly, expression of nestin was detected during tooth development, e.g. in odontoblasts (Terling et al., 1995). Furthermore, in carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions (About et al., 2000). Miura et al. demonstrated that stem cells from dental pulp of human exfoliated deciduous teeth (SHEDs) are able to differentiate into cells with neuronal and glial phenotype (Miura et al., 2003). PDLSCs were also found to express the tendon specific marker, Scleraxis (Shi et al., 2005).

[0069] Although cells from dental pulp and dental follicle are often defined as ectomesenchymal cells, it is noteworthy that a number of implantation and tissue recombination studies demonstrated that periodontal tissues, including cementum, were tooth related but neural crest derived (Hildebrand et al., 1995; Lumsden, 1988). In addition, Pierret et al proposed eloquently in a recent review, that all adult stem cells might be progeny of the neural crest (Pierret et al., 2006). Thus the existence of neural precursor in dental adjacencies might be explained by the ontogeny of the human teeth. In this context it is noteworthy, that adult teeth are richly innervated, thus the turnover necessitates a local neural plasticity (for review see (Hildebrand et al., 1995)). In addition, Obara et al. reported strong expression of neural cell adhesion molecule (NCAM) in the dental follicle of developing mouse (Obara, 2002; Obara et al., 2002) - a further evidence for neural character of resident cells within the periodontal tissue. Furthermore, it has been described that several neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or glial cell-line derived neurotrophic factor are highly expressed within the developing human teeth (Nosrat et al., 2002). All this reports indicate the possible existence of neural stem/ or progenitor cells in the dental adjacencies or the common ontogeny of neural stem cells and cells generating dental tissues. The physiological role of these cells in adult humans may be addressed by a potential replacement of degenerated neuronal tissue and/or dental tissues as a result of diseases such as periodontal defects as described recently by (Bartold et al., 2006).

[0070] Although the capacity of periodontium derived neural cells to function in vivo remains unresolved, the ability to generate large numbers of neural precursors in a serum-free media from a readily accessible, autologous adult human source has important implications for potential therapeutic use and drug discovery purposes.

[0071] The present invention is further described in more detail in the following figures and examples.

[0072] Figures

[0073] FIGURE LEGENDS

[0074] Figure 1 shows that cultured periodontium derived neural stem cells form neurospheres. A. Cultured human pdNSCs form self adherent clusters similarly to SVZ derived neural stem cells, when cultured for 14 days in serum-free media in presence of FGF-2 and EGF. Bar, 200μm. B. Blow-up image of pdNSCs. Bar represents a size of 20μm. [0075] Figure 2 shows that pdNSCs express high level of intermediate filament nestin and the transcription factor Sox2. Cytospin preparations of pdNSCs were fixed and stained for nestin, Sox2, β-lll-tubulin and GFAP. Cells were negative for differentiation markers β -lll-tubulin for neurons and GFAP for glial cells.

[0076] Figure 3 shows RT-PCR analysis of cultured pdNSCs. Note the high expression of nestin and Sox2 in both - dental pulp stem cells (DPSC) and pdNSCs and the absence of hematopoietic markers CD45, CD34 and CD133 (marker for hematopoietic stem cells). H2O was used as PCR negative control. cDNA as normalized via the β-actin housekeeping gene.

[0077] Figure 4 shows that pdNSCs are highly proliferative. Proliferation of pdNSCs was analyzed by counting the total cell number. In order to compare the self-renewal capacity of pdNSCs and DPSC both cell types were investigated. In comparison to cultures prepared from the dental pulp of human third molares, pdNSCs from periodontal tissue show a significantly higher increase in cell numbers after 72 and 96 h of culture (p< 0.001 ). Population doubling time was determined as -24 h.

[0078] Figure 5 shows that pdNSCs migrate in response to chemoattractants inducing NSC migration. A. Schematic diagram of the Boyden chamber assay. B. The migratory behavior of pdNSCs after exposure to chemokines was assayed. All three tested chemokines, namely MCP-1 , SCF and SDF-1α significantly induced migration, compared to BSA controls (Fig. 5) in both - pdNSCs and DPSC as well.

[0079] Figure 6. Panel A shows that pdNSCs differentiate spontaneously into neuronal lineage if plated on poly-D-lysine/laminin in absence of cytokines. After 4 days of adherent culture all cells showed neuron-like morphology (see upper panel). Up to 30 % of the cells expressed the early neuronal marker β-lll- tubulin as shown by immunocytochemical staining (middle panel). Please note that the expression of the stem cell marker nestin is still observed in about 70% of the cells. B. Neural fate of pdNSCs can be efficiently induced by culture in media containing 5μM retinoic acid (RA). Cells were cultured in FGF-2/EGF/RA containing media for 4 days followed by removal of the cytokines and plating on PDL/laminin. The figure shows the high expression of β-lll-tubulin, Map2, NF-M, NF-H (>90% of the total cell number) and homogenous neuronal morphology.

[0080] Figure 7 shows that pdNSCs can be differentiated into glial lineage, when cultured in absence of cytokines and presence of 10%FBS. The figures shows the glial morphology and the robust expression of GFAP in more than 90% of the cells.

[0081] Figure 8. Panel A shows genetic manipulation of pdNSCs. pdNSCs can be efficiently transfected using electroporation-based methods resulting in efficacies of about 78%. Panel B shows cross-species transplantation of pdNSCs. GFP-transfected pdNSCs were stimulated into neural fate with RA and transplanted into rat organotypic hippocampus cultures. 13 days after transplantation several viable cells were detected in principal hippocampal neuron layer, suggesting migration to the proper anatomic region (upper panel). Blow-up images show two examples of transplanted cells, integrated into the hippocampal tissue.

[0082] Figure 9 shows a schematic diagram of the isolation and differentiation protocols. Minimal-invasive periodontal surgery was used to harvest donor-specific stem cells during routine periodontal surgery. After enzymatic tissue dissociation cells were cultured in presence on FGF-2 and EGF. After app. 10 days of culture free floating aggregates of pdNSCs were observed. These neurospheres can be very efficiently differentiated into neuronal fate, when treated with RA. pdNSCs cultured with 10%FBS differentiate into glial lineage.

[0083] Figure 10 shows the dental differentiation of pdNScs. The figure shows the differentiation of the stem cells derived from periodontium. Isolated stem cells from periodontium were cultured as self adherent 3D clusters and are able to form structures (described by Lopez-Cazaux et al.) as multicellular nodules formed by dental pulp cells. The dentin cluster is marked by an arrow.

[0084] Figure 11 shows the differentiation of pdNScs into osteoblasts. The figure shows the differentiation of the stem cells derived from periodontium.

Isolated stem cells from periodontium and mesenchymal stem cells (MSCs) were cultured for 20 days in NH OsteoDiff Medium (Miltenyi Biotec GmbH/Germany), which is a medium suitable for differentiation of cells into osteoblasts. As result it is shown that the stem cell according to the present invention stain positive for alkaline phosphatase which is specific for osteoblasts.

[0085] Examples

[0086] Example 1 : Minimal-invasive isolation of periodontal tissue

[0087] Periodontal tissue was isolated during standard surgical therapy under approved local guidelines from 7 patients with periodontal defects (23-54 years old). All patients gave informed consent. For the tissue isolation, a surgical technique that utilizes small incisions and a limited minimal-invasive access approach was used. Namely, intrasulcular incisions were made on the teeth adjacent to the defect followed by periodontal tissue dissection using microsurgical instrumentation (Gassmann, and Grimm, 2006).

[0088] Isolated adult periodontal tissue was collected in ice cold HBSS- glucose solution (HBSS from Gibco, Eggenstein, Germany) containing 300 mg/ml D-glucose (Sigma, Deisenhofen, Germany) followed by digestion with 1 ,33 mg/ml trypsin (Sigma, Deisenhofen, Germany), 0,7 mg/ml hyaluronidase (Sigma, Deisenhofen, Germany), 200u/ml DNAse (Sigma, Deisenhofen, Germany) and 0,2 mg/ml kynurenic acid (Sigma, Deisenhofen, Germany) to dissociate tissue at 37°C. Tissue was passed through a 70μm cell strainer (BD Falcon; Heidelberg, Germany) and transferred to ice cold EBSS containing 15mM HEPES and 0.04g/ml BSA to stop trypsin activity. After additional centrifugation step at 212 x g cells were re-suspended in DMEM/F12, Gibco, Eggstein, Germany) containing basic fibroblast growth factor (FGF-2; 20ng/ml, Chemicon, Hofheim, Germany), epidermal growth factor (EGF; 20ng/ml; R&D Systems, Wiesbaden, Germany) and B27 supplement (60μl/ml, Gibco, Eggenstein, Germany) followed by culture (see below).

[0089] Example 2: Isolation of dental pulp stem cells (DPSC)

[0090] Human dental pulp stem cells (DPSC) were isolated as previously described by Gronthos et al. (Gronthos et al., 2000).

[0091] Example 3: Culture of pdNSCs and DPSC

[0092] pdNSCs from periodontal tissue, isolated as described above or

DPSC were cultured in serum-free media (DMEM/F12, Gibco, Eggstein, Germany) containing basic fibroblast growth factor (FGF-2; 20ng/ml, Chemicon, Hofheim, Germany), epidermal growth factor (EGF; 20ng/ml; R&D Systems, Wiesbaden, Germany) and B27 supplement (60μl/ml, Gibco, Eggenstein, Germany). After appearance, primary periodontium derived neurospheres were dissociated at day 8-10 using Accutase (PAA, Pasching, Austria) to derive secondary neurospheres. Accutase combines protease and collagenolytic activities without a need of additional washes or enzyme inhibitors after dissociation. The sub-culturing protocol consisted of neurosphere passaging every 3-4 days with whole culture media change (with freshly added growth factors).

[0093] Periodontal tissue samples were isolated from 7 human donors.

Seven cultures could be propagated as 3D spheroids. For cultivation a culture paradigm using serum-free conditions in presence of FGF-2 and EGF has been used. The techniques applied resulted in a population of spheroid cultures which is shown in Fig. 1. The spheroids can be cultured as self adherent cell clusters and can be kept in culture for several passages without loosing their proliferation, migration and differentiation capabilities.

[0094] Example 4: Characterization of the isolated stem cells by lmmunocytochemistry

[0095] Periodontium derived neurospheres were harvested on microscope slides by cytospin centrifugation (212g, 5 min., Shandon, Thermo, Dreieich, Germany). Fixation was done by 3.7 % PFA for 60 min at 4°C and washed 3x in 1x PBS for 5 min. Blocking was done in 5% appropriate serum (goat or rabbit) for 30 min. followed by incubation with anti-Nestin (1 :100, Chemicon, Temecula, USA); anti-GFAP (1 :100, BD Pharmingen, Heidelberg, Germany), anti-β-lll-tubulin (1 :50, Promega, Mannheim, Germany), anti-LeX (1 :100, Developmental Hybridoma Bank, Iowa City, USA), anti Sox2 (1 :100, Sigma, Deisenhofen, Germany), anti-Musashi (1 :100, Chemicon, Temecula, USA), anti-L1 (1 :100, Developmental Hybridoma Bank, Iowa City, USA), anti-PSA-NCAM (1 :50, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-Notch1 (1 :100, Developmental Hybridoma Bank, Iowa City, USA), anti-NF-L (1 :100, Chemicon, Temecula, USA), anti-NF-H (1 :100, Chemicon, Temecula, USA), anti NF-M (1 :100, Chemicon, Temecula, USA), anti MAP2 (1 :100, Chemicon, Temecula, USA), anti GAD67(1 :100, Chemicon, Temecula, USA), anti-CD34 (1 :11 , Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD117 (1 :11 , Miltenyi Biotec, Bergisch Gladbach, Germany), anti CD133 (1 :20, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-A2B5 (1 :100, Chemicon, Temecula, USA). Detection was done with Cy3 conjugated antibody (1 :300, Jackson lmmuno Research Laboratories, distributed by Dianova, Hamburg, Germany). Nuclear staining was done with SYTOX (1 :10000, Molecular Probes, Gδttingen, Germany). Antibody staining was visualized using confocal laser scanning microscopy (LSM Pascal, Zeiss, Jena, Germany).

[0096] For characterization the propagated population of pdNSCs, the expression of several biomarkers was assayed. Protein expression of biomarkers was studied using antibodies against neural stem cell specific markers, read: sternness markers such as nestin and Sox2 (see Fig. 2). As result it was shown that the majority of the pdNSCs was anti-nestin immuno-reactive, characteristic for neural stem cells. In addition the cells expressed neural stem cell specific transcription factor Sox2 (Graham et al., 2003). No expression was detected for the population markers L1 , LeX and for the oligodendrocytic lineage marker A2B5 (data not shown). In addition, no expression of markers specific for hematopoietic stem cells, CD34 and CD117 was detected (data not shown). Moreover moderate expression of PSA-NCAM, a marker for migrating neuronal precursor cells (data not shown) has been detected. Demonstrably, no expression of differentiation marker β-lll-tubulin for neurons and very low expression of GFAP (glial cell marker) were detected.

[0097] Example 5: Characterization of the isolated stem cells by reverse transcription polymerase chain reaction

[0098] Total RNA of CD133⁺ HSC, pdNSCs and DPSC was extracted using the NucleoSpin RNA Il Kit (Macherey & Nagel, Dueren, Germany) according to manufacturer's instructions. cDNA was synthesized in a 30μl reaction mixture containing 300 U Superscriptll, 1x first strand buffer (0.3 μM, Invitrogen, Karlsruhe, Germany), dNTP (200 μM each, PEQLAB, Erlangen, Germany), Oligo(dT)18 (5 μg) and (N6) random hexamere (0.2 μg, both Metabion, Martinsried, Germany). Human normal adult brain cDNA, human fetal brain cDNA and human adult liver cDNA (each with 1 μl per PCR reaction) were purchased from Biochain Institute, lnc (Hayward, CA, USA). PCR was performed in a 25 μl reaction mixture containing 1.25 units Taq polymerase, 1x reaction buffer, 2 mM MgCl2 (Fermentas International Inc., Burlington, Ontario, Canada), 200 μM of each dNTP (PEQLAB, Erlangen, Germany) and 200 nM primers (Metabion).

[0099] The cycling conditions comprised an initial denaturation of 5 min at

94°C and 30 cycles of 1 min at 94°C, 1 min at the appropriate temperature and 1 min at 72°C followed by a final elongation of 10 min at 72°C. For primer sequences and annealing temperatures see Table 1. [00100] The results of the RNA expression of lineage markers using RT-PCR is summarized in Fig. 3). CD45, a marker for blood cells was detected in human adult CD 133⁺ hematopoietic stem cells and in RNA derived from human liver, but not in pdNSCs from different origins. No expression was detected in pdNSCs of the hematopoietic stem cell markers CD34 and CD90. Expression of the marker for primitive lymphoid and myeloid hematopoietic bone marrow progenitor cells and mesenchymal stem cells CD117 (stem cell factor receptor/c-kit) was only weakly detected in pdNSCs. In contrast the neural stem cell markers Nestin and Sox-2 were strongly expressed in pdNSCs. Surprisingly the pre-oligodendrocytic marker 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was expressed in all samples from human organs (adult brain, fetal brain and adult liver) and in CD133 stem cells and pdNSCs. Unlike CD133⁺ hematopoietic stem cells, pdNSCs express Emx2, an early marker for developing neocortex. Demonstrably, no expression of differentiation markers GFAP for glial cells and neurofilament 3 (NF- H/NF-200kD) for neurons was detected. Neuro D1 a marker for adult neurons was not expressed in pdNSCs. Interestingly, in contrast to brain, liver or even CD133 HSCs, pdNSCs do not express geminin (GMNN), - a well described negative regulator of replication, indicating highly proliferative character of the cells. In addition the cells were negative for Notchi and CD133. The marker for embryonic stem cell pluripotency Oct4 was not expressed in pdNSCs.

[00101] Example 6: Determination of proliferation of pdNSCs and cell number determination

[00102] Periodontium derived neural stem cells or dental pulp stem cells (DPSC) were plated at 1.00 E+5 cells/mL as triplicate for each condition. Spheres were collected at 6, 24, 48 and 72 hours after plating, dissociated and total cell numbers were counted. Results were expressed as the mean +; SEM. Statistical significance was determined using two-way ANOVA followed by post hoc t-test with Bonferroni correction. Differences between two conditions at P<_0.05 were considered as statistically significant. [00103] The results shown that pdNSCs are highly proliferative. Proliferation of pdNSCs was analyzed by total cell number determination to avoid potential artifacts of BrdU incorporation such as labeling of DNA repair sites. In order to compare the self-renewal capacity of pdNSCs and DPSC (dental pulp derived stem cells) both cell types were assayed. In comparison to cultures prepared from dental pulp of human third molars pdNSCs from periodontal tissue show a significantly higher increase in cell numbers after 72 and 96 h of culture (p<0.001) which is summarized in Fig. 4. Population doubling time was determined as -24 h for pdNSCs.

[00104] Example 7: Characterization of migration behaviour of the stem cells by Boyden migration assay

[00105] In vitro migration assay was performed using a Boyden chamber system. Cell culture inserts with a pore size of 8 μm (Falcon) were coated with 0.1 % gelatin for 48 hours at 37°C and air dried. MCP-1 (R&D Systems 279-MC), SCF or SDF-1α was diluted in media to a final concentration of 20ng/ml. 500μl of the dilution was placed in the lower chamber (24well cell culture plate) of the modified Boyden apparatus. As control 20ng/ml BSA was used. 1.00 E+4 cells in 200μl_ of serum-free medium containing B27 supplement were seeded in the upper chamber. The chamber was incubated for 4 hours at 37°C in a humidified incubator with 5% CO2. After the incubation period, the filter was removed, and the upper side of the filter, containing non-migrating cells, were cleaned with a rubber cell lifter and washed with PBS 3 times. The filter containing migrated cells on the underside was fixed with 3.7% PFA and stained with DAPI solution (1 μg/ml). Cell migration was quantified by counting migrated cells on 4 independent fields of view on each filter. All experiments were performed at least in triplicates. Differences in migratory activity between control and treated pdNSCs or DPSC were assessed by two-way ANOVA followed by post hoc t-test with Bonferroni correction. P< 0.05 was considered significant. [00106] The results show that pdNSCs migrate in response to chemoattractants inducing NSC migration. Migration of neural stem cells is guided by gradients of several chemokines such as macrophage chemoattractant protein MCP-1 (Widera et al., 2004), stem cell factor (SCF) (Sun et al., 2004) and stromal- derived factor 1 α (SDF-α) (Imitola et al., 2004). In order to test this second crucial property of neural stem cells - the ability to migrate (for review see (Muller et al., 2006) and (Martino and Pluchino, 2006)), the migratory behavior of pdNSCs after exposure to these chemokines has been tested. The assay outlay is depicted in Fig. 5A. All three tested chemokines, namely MCP-1 , SCF and SDF-1α significantly induced migration, compared to BSA controls (Fig. 5B) in both - pdNSCs and DPSC as well.

[00107] Example 8: Spontaneous neuronal differentiation of pdNSCs

[00108] Secondary periodontium derived neurospheres were harvested and dissociated as described above followed by plating on Poly-D-Lysine/Laminin coated culture slides (BD Biocoat, Heidelberg, Germany) in serum-free media containing B27 supplement. 4 days after the plating cells were fixed, stained and analyzed as described above.

[00109] Example 9: Induction of neuronal differentiation of pdNSCs by retinoic acid

[00110] For increased neuronal differentiation pdNSCs were harvested and dissociated followed by cultivation in EGF/FGF-2 containing media with 5μM retinoic acid (RA). Dissociated Neurospheres re-aggregate rapidly in presence of FGF-2 and EGF (Widera et al., 2006). Re-aggregated spheres were dissociated after four days of RA treatment and plated in serum-free media containing B27 on Poly-D-Lysine/Laminin coated culture slides (BD Biocoat, Heidelberg, Germany).

[00111] The results show that the pdNSCs of the present invention can be efficiently differentiated into cells with neuronal phenotype. For spontaneous neuronal induction pdNSCs were dissociated and single cells were plated on poly- D Lysine/Laminin coated culture dishes. Already one day after plating neuron-like morphology (bipolar cell bodies with elaborated processes) was evident. After four days of adherent culture up to 30 % of the cells are expressing the early neuronal marker β-lll-tubulin. Interestingly the expression of the stem cell marker nestin is still observed in about 70% of the cells (Fig. 6A).

[00112] In order to enhance the frequency of neuronal cells a differentiation protocol for somatic neural stem cells was developed. Cultures were kept as aggregates in presence of growth factors (FGF-2 and EGF) and 5 μM retinoic acid (RA) for four days. After dissociation cells were plated and analyzed. 24 h after plating the frequency of neuronal marker positive cells was greatly enhanced in comparison to the cells without RA treatment (as showen in Fig. 6). In addition the cells expressed neurofilament M, neurofilament H, Map-2 (Fig. 6 B), GAD67 (data not shown) and neurofilament L (not shown).

[00113] Example 10: Glial differentiation of pdNSCs

[00114] Secondary periodontium derived neurospheres were harvested and dissociated, followed by subsequent culture in DMEM/F:12 (Gibco) and 10% FBS (PAA, Pasching, Austria). After 4 days of culture cells were fixed, stained and analyzed as described above. The results show that the pdNSCs can be induced to differentiate into glial cells. In order to determinate the glial differentiation potential of pdNSCs cells were cultured for 4 days in presence of 10% FCS. After 4 days of culture a typical glial morphology was observed, lmmunohistochemical analysis verified glial lineage of differentiated pdNSCs as shown by high expression of GFAP (Fig. 11 ).

[00115] Example 11 : Differentiation of pdNSCs into osteoblasts

[00116] Isolated stem cells from periodontium and mesenchymal stem cells (MSCs) were cultured for 20 days in NH OsteoDiff Medium (Miltenyi Biotec GmbH/Germany), which is a medium suitable for differentiation of cells into osteoblasts. After 20 days of culture cells were fixed, stained and analyzed as described above. As result it is shown that the stem cell according to the present invention stain positive for alkaline phosphatase which is specific for osteoblasts. The results show that the pdNSCs can be induced to differentiate into osteoblasts.

[00117] Example 12: Organotypic hippocampal slice cultures

[00118] Organotypic hippocampal slice cultures were prepared and maintained according to Stoppini et al. (Stoppini et al., 1991 ). Briefly, hippocampi from rats (postnatal day 5) were cut perpendicularly to the longitudinal axis into 400μm thick slices by using a Mcllwain tissue chopper (Mickle Laboratory Engineering, Gomshall, Surrey, UK) and kept in ice-cold MEM HBSS, pH 7.3, containing 25mM HEPES and 2mM L-glutamine. Slices were put on top of Millipore membranes (diameter 30mm, pore size 0.4μm), transferred to six-well plates containing 750 μl culture medium (50% MEM HBSS, 25% heat inactivated horse serum, 2mM L-glutamine, 5mM NaHCOs) per well (refreshed twice a week), and incubated in humidified 93% air/5% CO2 atmosphere at 37°.

[00119] Example 13: Transplantation of pdNSCs into organotypic hippocampal slice cultures

[00120] Periodontium derived neurospheres were dissociated as described above and transfected using Rat NSC Nucleofector Kit (Amaxa, KoIn, Germany) with pmaxGFP (Amaxa, KoIn, Germany) according to manufacturer's instruction. After 24h of culture at 37°C in presence of 5μM RA, cells were harvested via cytospin centrifugation and dissociated using Accutase. 1.00 E+3 cells were dropped on the slice and cultured for 13 days. After 13 days of culture at 37°C and 5% CO2, slices containing transfected, transplanted pdNSCs were fixed for 1 h in 4% PFA followed by 2 wash steps in IxPBS. Transplanted, GFP positive cells were visualized using confocal laser scanning microscopy (LSM Pascal, Zeiss, Jena, Germany). [00121] The results show that pdNSCs survive and integrate when transplanted into organotypic hippocampal slice cultures. GFP transfected pdNSCs (Fig. 8A) were transplanted into organotypic hippocampal slice cultures. After 13 days of culture GFP positive cells remain detectable within the slice (see Fig 8B). Blow-up images showed viable and integrated pdNSCs indicating that pdNSCs might integrate into brain tissue after transplantation.

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Wislet-Gendebien, S., F. Wautier, P. Leprince, and B. Rogister. 2005b. Astrocytic and neuronal fate of mesenchymal stem cells expressing nestin. Brain Res

Bull. 68:95-102. Zemchikhina, V.N., and O.N. Golubeva. 2005. The capacity of mesenchymal stem cells for neural differentiation in vitro. Tsitologiia. 47:644-8.

Claims

1. A post-natal neural stem cell, characterized by:

- the stem cell is derived from the oral cavity, provided that the stem cell is not derived from dental follicle or dental pulpa; - the stem cell is Nestin positive and Sox2 positive when cultured in suspension culture;

- the stem cell is Scleraxis negative.

2. The stem cell of claim 1 , wherein the stem cell is derived from periodontium or palatum.

3. The stem cell of claim 1 or 2, wherein the stem cell shows expression of the markers Nestin and Sox2, if the stem cell is cultivated in suspension culture subsequently after dissociation of the tissue sample from which the stem cell is derived, under culture conditions using serum-free media containing at least one proliferation-inducing growth factor.

4. The stem cell of any one of claims 1 to 3, wherein the stem cell has the capability to proliferate into a spheroid aggregation of cells in a suspension culture using serum-free medium, wherein each of said spheroid aggregation of cells comprises stem cells, which are capable of producing progeny that are capable of differentiating into neuronal cells or glial cells or osteoblasts or other mesenchymal cells.

5. The stem cell of any one of claims 1 to 4, wherein the stem cell is capable of producing progeny that are capable of differentiating into neuronal cells or glial cells or osteoblasts or cells of the periodontium or teeth.

6. The stem cell of any one of claims 1 to 5, wherein the stem cell is capable of producing progeny that are capable of differentiating into neuronal cells under culture conditions using serum-free media containing at least one proliferation- inducing growth factor.

7. The stem cell of any one of claims 1 to 5, wherein the stem cell is capable of producing progeny that are capable of differentiating into glial cells under culture conditions using media containing serum in the absence of growth factors.

8. The stem cell of any one of claims 1 to 7, wherein the stem cell expresses each of the following markers: Nestin, Sox2, CNPase, Emx2 and CD117.

9. The stem cell of any one of claims 1 to 8, wherein the stem cell does not express the following markers: L1 , LeX, A2B5, CD34, CD45, CD90, CD133, PSA- NCAM, β-lll-tubulin, GFAP, neurofilament 3 (NF-H/NF-200KD), Neuro D1 , GMNN, Notch 1 , Oct4.

10. The stem cell of any one of claims 1 to 9, wherein the stem cell has a significant shorter doubling time than stem cells derived from dental pulpa.

11. The stem cell of any one of claims 1 to 10, wherein the stem cell has a doubling time which is by at least a factor 1.2 faster than that of stem cells derived from dental pulpa.

12. The stem cell of any one of claims 1 to 11 , wherein the stem cell has the capability to migrate when stimulated with one or more of the following substances: macrophage chemoattractant protein MCP-1 , stem cell factor (SCF) and stromal-derived factor 1 alpha (SDF-1 alpha).

13. The stem cell of any one of claims 1 to 5, wherein the stem cell is defined as proliferative cell and as self renewing cell capable of producing progeny that are capable of differentiating into cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm.

14. The stem cell of any one of claims 1 to 13, wherein the stem cell is genetically modified.

15. A spheroid aggregation of cells in suspension culture derived from a stem cells of any one of claims 1 to 14.

16. A medical product comprising the stem cell of any one of claims 1 to 14 or comprising the spheroid aggregation of cells according to claim 15.

17. A medicament comprising the stem cell of any one of claims 1 to 14 or comprising the spheroid aggregation of cells according to claim 15.

18. The use of the stem cell of any one of claims 1 to 14 or of the spheroid aggregation of cells according to claim 15 for the manufacture of a medicament for the treatment of the diseases selected from the group consisting of: periodontal diseases, periimplantitis, permucositis, or neurodegenerative diseases such as Parkinson^'s disease, Alzheimer^'s disease, MuMpIe Sclerosis.

19. The use of the stem cell of any one of claims 1 to 14 or of the spheroid aggregation of cells according to claim 15 for the manufacture of a medicament for the regeneration of the dental innervation, or the regeneration of periodontium and of dental implant sites or of alveolar ridge augmentations.

20. The use of the stem cell of any one of claims 1 to 14, for the differentiation of cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm.

21. A method for the preparation and/or proliferation of a neural stem cell, comprising the following steps: a) provision of a tissue sample from periodontium or palatum containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum- 5 free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture (step c), and wherein the stem cell is Scleraxis negative.

io 22. The method according to claim 21 , wherein after step c) the cells are cultivated under culture conditions using a serum-free medium containing at least one proliferation-inducing growth factor, to induce differentiation into neuronal cells.

23. The method according to claim 21 , wherein after step c) the cells are is cultivated under culture conditions using a medium containing serum and in the absence of proliferation-inducing growth factors (or in the absence of FGF-2 and EGF), to induce differentiation into glial cells.

24. The method according to claim 21 , wherein after step c) the cells are cultivated under culture conditions using a medium suitable for differentiation of

20 the cells into osteoblasts, to induce differentiation into osteoblasts.

25. The method according to claim 21 , wherein after step c) the cells are cultivated under culture conditions, wherein the expression of NF-kappaB in said cells is manipulated to induce differentiation into mesodermal, ektodermal or endodermal cells.

25 26. The method according to claim 21 , wherein after step c) the cells are modified by introduction of nucleic acids and or a pharmaceutical compound, for providing a drug-delivery system.

27. The method according to claim 26, wherein the nucleic acid introduced into said cell is expressing a substance selected from the group peptide drug, polypeptide drug, an anti-sense RNA, an RNA providing RNAi-effect.

28. The method according to any one of claims 21 to 27, wherein the spheroid aggregation of cells obtained in step c) is derived from human somatic stem cells present in the tissue sample from human periodontium or human palatum.

29. The method according to claim 21 , for the isolation and/or enrichment of human neural crest-derived cells.

30. The method according to claim 21 , for the preparation and differentiation of cells of one or more germ layers selected from the group consisting of ektoderm, endoderm and mesoderm.

31. A cell culture obtainable by the method according to any one of claims 21 to 30.

32. A neuronal cell obtainable by the method according to any one of claims 21 , 22, 26 to 28.

33. A glial cell obtainable by the method according to any one of claims 21 , 26 to 28.

34. An osteoblast obtainable by the method according to any one of claims 21 , 24, 26 to 28.

35. A method of transplanting a neural stem cell into a host, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum- free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of neuronal cells, glial cells, cells of the periodontium, periodontal ligament, osteoblasts, cementoblasts, osteoblasts; e) transfer of the cells obtained in c) or d) into the host;

wherein the neural stem cell is Nestin positive and Sox2 positive when cultured in suspension culture, and wherein the stem cell is Scleraxis negative.

36. A method for the treatment of periodontal diseases, periimplantitis or perimucositis, or for the regeneration of the dental innervation, or for the regeneration of periodontium, periodonal ligament, osteoblasts, cementoblasts, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of neuronal cells, glial cells, cells of the periodontium, periodontal ligament, cementoblasts, osteoblasts; e) transfer of the cells obtained in c) or d) into the site of intervention of the respective individual;

37. A method for implant dentistry, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the alveolar ridge developmental site of the respective individual;

38. A method for augmentation in maxillofacial surgery, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of surgical maxillo-facial interventions of the respective individual;

39. A method for alveolar ridge augmentation, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of augmentation/ interventions of the respective individual;

40. A method for the treatment of Parkinson^'s disease or Alzheimer^'s disease, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; d) optionally, differentiation of the cells obtained in c) into neuronal cells and/or glial cells; e) transfer of the cells obtained in c) or d) into the central nervous system of the respective individual;

41. A method of the treatment in maxillo-facial surgery, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one nerual stem cell; b) dissociating said tissue to obtain a cell suspension comprising said stem cell; c) cultivation in serum-free medium containing at least one proliferation-inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of fibroblasts, osteoblasts, osteoclasts, neuronal cells and cementoblasts; e) transfer of the cells obtained in c) or d) into the sites of maxillo-facial surgery of the respective individual;

42. A method for the treatment of diseases involving defects in cartilage or bone, comprising the following steps: a) provision of a tissue sample from periodontium or palatum of an individual containing at least one neural stem cell; b) dissociating said tissue to obtain a cell suspension comprising said neural stem cell; c) cultivation in serum-free medium containing at least one proliferation- inducing growth factor, to obtain a suspension culture comprising spheroid aggregations of cells, each of said spheroid aggregations of cells comprising neural stem cells; d) optionally, differentiation of the cells obtained in c) into cells selected from the group consisting of osteoblasts, osteoclasts and/or chondrocytes; e) transfer of the cells obtained in c) or d) into the host;