Abstract:
The present invention relates to a simplified process, which is shorter in time, for propagation of proliferating cells, such as e.g. progenitor or stem cells, by means of a biphasic culturing system having a differentiation supporting component and a proliferation supporting component, and to the use of the stem cell cultures obtained in this way for cell therapy purposes. The present invention invention describes a method, which is highly efficient to prime stem or progenitor cells to differentiation using non-attachment matrices and differentiation supporting component. The cells produced therefrom may be used to treat a variety of neurodegenerative disorders.

Description:
FIELD OF THE INVENTION 
     The present invention relates to in vitro methods for expansion of nervous system-derived progenitor and stem cells (NSC&#39;s) into neuronal cells. Specifically, methods of ex-vivo expansion of nervous system-derived progenitor and stem cells, expanded populations of renewable progenitor and stem cells and their uses in therapeutic applications such as treatments of a variety of diseases, including neurotrauma and/or neurodegenerative diseases. 
     BACKGROUND OF THE INVENTION 
     The mammalian, including human, central nervous system (CNS, brain and spinal cord) has an extremely low ability for spontaneous anatomical and functional recovery after injury. This inability of the CNS to regenerate is due to a lack of a natural way to replace lost neurons and to establish intense functional connections between different neuronal populations after trauma. It has been demonstrated that central nervous system contains multipotent progenitor cells. These multipotent cells proliferate and differentiate into neurons, astrocytes and oligodendrocytes in vitro and in vivo. Progenitor cells or neuronal derivatives of these cells can be used for transplantation to stimulate anatomical and functional regeneration. Cell replacement and neuronal circuitry reconstruction strategies in human neurological conditions require a well-established source of neuronal cells. Techniques have been developed to isolate, propagate and differentiate neuronal stem cells from the fetal and adult human central nervous system. Unfortunately, propagation of these stem cells is time consuming (many months) and during propagation many cells loose their multipotency to differentiate in variety of neuronal types. 
     One of the crucial problems in cell therapy using autologous transplantable cells is propagation of neural stem cells in conditions that will result in a large number of multipotent cells whereas cells maintain the capacity to differentiate into variety of neural cell types. Numerous data clearly show that there is a balance between multipotent neural stem cell population and populations of neural progenitor cells (NPC) that are committed to certain differentiation pathway both in vivo and in vitro. 
     With regard to development, it is well established that different signaling routes (Wnt, Shh and BMP/TGFbeta, Notch and TK signaling cascades) are critical for proper gene expression at appropriate times since antagonistics biological processes, such as proliferation-differentiation and survival-apoptosis, are all integrated in the formation of a three-dimensional nervous (brain) tissue, whose function changes with time. In embryonic stages, the growth and proper development of nervous structures is dependent on the interaction between the glial cells and neurons and require the concerted actions of various bioactive peptides and hormone-like substances, and cell-cell and cell-substrate interactions. The necessity for these complex structural and hormonal interactions provide a challenge for the development of in vitro cell culture models that more accurately mimic the developing nervous system. 
     Similarly, the mammalian central nervous system (CNS; brain and spinal cord) has a limited ability for spontaneous recovery following an injury. This inability of the CNS to regenerate is caused by the lack of a natural pathway to replace lost neurons and re-establish the functional connections between different neuronal populations after trauma. 
     However, it has been demonstrated that the CNS contains multipotent progenitor cells (nervous system-derived progenitor and stem cells; NSC&#39;s) that can proliferate and differentiate into neurons, astrocytes and oligodendrocytes both in vitro and in vivo. Progenitor cells (or neuronal derivatives of these cells) can be transplanted to stimulate anatomical and functional regeneration. Techniques have been developed to isolate, propagate and differentiate neuronal stem cells from the CNS, but propagation of these stem cells is time consuming (typically many months) and many cells lose their multipotency to differentiate as a result of the process. 
     Current culture models using continuous exposure of cultured NSC&#39;s to high levels of bFGF (basic Fibroblast Growth Factor), LIF (Leukemia Inhibitory Factor) and EGF (Epidermal Growth Factor) have not yielded substantial numbers of human NSC&#39;s (hNSC&#39;s) in vitro, thus preventing the use of propagated hNSCs for autologous transplantation purposes. Therefore, there exists a need for methods of expansion of hNSC&#39;s into neuronal cells. 
     SUMMARY OF THE INVENTION 
     Embodiments of the present invention provide methods and systems for biphasic culturing of hNSCs and further differentiation. The differentiation can result in neuronal cells or glial cells, and can provide related benefits. In some embodiments a pluripotent stem cell is obtained. In some embodiments a multipotent stem cell is obtained. In some embodiments a cell having characteristics of a neuronal cell is obtained. In some embodiments a cell having characteristics of a neural crest stem cell is obtained. In other embodiments neuronal cells or glial cells are obtained. The cells obtained by methods of the present invention can be used in various medical treatments where repair or formation of neurons is desired. In some embodiments, cells according to the present invention are used to treat or reduce neuron degradation or the rate of neuron degradation. In some embodiments cells of the present invention are useful for the treatment or prevention of, for example, neurodegenerative disorders, neurotrauma, Alzheimer&#39;s disease, Parkinson&#39;s disease, and the like. In other embodiments compounds suspected of affecting differentiation or development of neural stem cells or neuronal cells are studied using the methods or cells of the present invention. Differentiated cells of the present invention can be obtained by providing a population of neural stem cells/neurospheres and exposing the neural stem cells/neurospheres to conditions that affect or direct differentiation potential. Such conditions can include, for example, altering the surrounding growth factor composition of the media or environment and providing or eliminating an appropriate supporting growth matrix or substrate. 
     Embodiments of the invention provide methods of optimized in vitro growth (propagation) of hNSC&#39;s, enabling rapid growth without loss of multipotency. 
     Embodiments of the invention provide techniques to propagate human neural stem cells (“hNSCs”) as multipotent cells in significant numbers and then, for example, hNSCs differentiate the cells into particular types of nervous system cells. Certain embodiments include a method of fast propagation of hNSCs utilizing dynamic incubation of cells in a biphasic culture system where cells are initially grown for a set time interval, such as, for example, 1 day, 2 days, 3 days, 4 days, 6 days, 7 days, or more, or the like. 
     In certain embodiments, during the initial growth period the cells can be cultured with, for example, combinations of different bioactive molecules such as growth factors epidermal growth factor (“EGF”), transforming growth factor-alpha (“TGFα”), neuregulin-1, sonic hedgehog (“SHH”), Wnt3a, Wnt 5, ciliary neutrophic factor (“CNTF”), Notch ICD, or the like. 
     In some embodiments, the initial growth period can be followed by a second growth period of a set time interval, such as, for example, 1 day, 2 days, 3 days, 4 days, 6 days, 7 days, or more, or the like. During this second growth period, cells can be cultured with, for example, serum (FBS, HBS or synthetic serum substitutes) or a mix of bFGF, EGF and leukemia inhibitory factor (“LIF”), or any combination thereof, or the like. Likewise, in some embodiments the second growth period can be followed by a third growth period, or more. In certain embodiments, the type of cell culture media used during the third growth period can be the same as the type used during the initial growth period. 
     In one aspect of the present invention, a method for differentiation of a neural stem cell to a neuronal cell is provided, including providing a neural stem cell population and incubating the cell population under conditions suitable for differentiation of neural stem cells to neuronal cells. In one embodiment the differentiation conditions include initial priming for 7 days in the absence of attachment matrix and medium containing DMEM/F12 and B27 supplement and 1 uM all-trans-retinoic acid and/or 1 mM dBcAMP. After priming, the further differentiation conditions include the presence of a laminin attachment matrix and medium containing DMEM/F12 and B27 supplement. The progress of differentiation can be monitored by detecting one or more markers distinguishing neuronal cells from glial cells. In one embodiment the detectable marker is beta-III-tubulin. In another embodiment the detectable marker is MAP2. In still another embodiment the marker is GAD65. In still another embodiment the marker is pTH. In still another embodiment the marker is GFAP. In other embodiments, the marker includes one or more detectable markers. In other embodiments, the present invention includes a neuronally differentiated cell. 
     Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. 
         FIG. 1  shows the experimental protocol regarding media modification as performed in Example 1. 
         FIG. 2  depicts the growth rate of hNSCs grown as neurospheres according to the method of Example 1. 
         FIG. 3  shows the experimental protocol regarding media modification as performed in Example 2. 
         FIG. 4  shows the growth rate of hNSCs grown as neurospheres according to the method of Example 2. 
         FIG. 5  shows the experimental protocol regarding media modification as performed in Example 3. 
         FIG. 6  depicts growth rate of hNSCs grown as neurospheres according to the method of Example 3. 
         FIG. 7  shows the differentiation of hNSCs cultured according to the method of Example 3 using various growth factors. 
         FIG. 8  depicts differentiation of hNSC&#39;s to neurons and astrocytes, cultured according to the methods of Examples 1 and 2. 
         FIG. 9  shows a graph illustrating the changes (in percentage) in numbers of β-TubIII immunoreactive cells upon differentiation of hNSC&#39;s according to Example 4. 
         FIG. 10  shows expression of genes functioning in the biosynthesis of dopamine neurotransmitter systems. Upon differentiation according to the method of Example 5, cells were subjected to RT-PCR analysis of expression and semiquantitative levels of 3 genes governing the dopamine biosynthesis pathway, namely NURR1, EN1, and PTX3. The graph shows RT-PCR analysis data of 4 analyzed genes. Samples were normalized to the amount of GAPDH. 
         FIG. 11  shows an expression analysis of genes functioning in the biosynthesis of GABAergic neurotransmitter systems. Upon differentiation according to the method of Example 5, cells were subjected to RT-PCR analysis of expression of 1 gene governing in the GABAergic biosynthesis pathway, namely PITX2. The graph shows RT-PCR analysis of 2 analyzed genes. Samples were normalized to the amount of GAPDH. 
         FIG. 12  shows an expression analysis of genes functioning in the biosynthesis of cholinergic and GABAergic neurotransmitter systems. Upon differentiation according to the method of Examples 5, cells were subjected to qPCR analysis of expression and quantitative levels of 4 genes functioning in the cholinergic biosynthesis pathway, and of 9 genes functioning in the GABAergic biosynthesis pathway. The graph shows qPCR cycle threshold values of 13 analyzed genes. Samples were normalized to the amount of GAPDH. 
         FIG. 13  shows an expression analysis of genes functioning in the biosynthesis of glycinergic, glutamatergic, serotonergic, and dopaminergic neurotransmitter systems. Upon differentiation according to the method of Examples 5, cells were subjected to qPCR analysis of expression and quantitative levels of 19 genes functioning in the biosynthesis pathway of different neurotransmitter systems. The graph shows qPCR cycle threshold values of 19 analyzed genes. Samples were normalized to the amount of GAPDH. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides methods and systems for rapid amplification of human neural stem cells and subsequent differentiation into desired neuronal cell types. Examples of cell types obtained using methods of the present invention include neural stem cells (NSC), neuronal cells, pluripotent stem cells, multipotent stem cells, and cells having characteristics similar to those listed. The invention illustrates methods for rapid propagation by providing a human neural stem cell and incubating the cell under biphasic conditions that selectively support either proliferation or differentiation. Cell types are confirmed by examining the presence or absence of cell markers corresponding to the particular cell type or stage and optionally by viewing cell morphology or shape. Further, human neural stem cells that have been propagated in biphasic conditions are shown to selectively differentiate upon the addition of differentiation medium or conditions. The present invention is not limited to human neural cells obtained from human tissue but may be performed on cultured neural stem cells such as cell lines, any mammalian neural stem cells or any neural stem cells obtained from a vertebrate. 
     As will be appreciated by one skilled in the present art, the present invention provides pluripotent stem cells useful for a variety of medical applications and has a wide variety of therapeutic applications in medicine including human and veterinary medicine. Non-limiting examples of applications for the cells and methods of the present invention include cell based therapeutics of neurodegenerative disorders or neurotrauma, Alzheimer&#39;s disease and Parkinson&#39;s disease. Additional applications include treatment for patients suffering from stroke, brain injury or spinal cord injury. The methods of the present invention may be used to provide a replacement for defective or absent cells or may be used to develop a delivery method for therapeutic products. 
     In other aspects, the present invention is used for drug screening such as high throughput drug screening of potential prophylactic or therapeutic candidates. The methods and compositions of the present invention may be used to determine the effect of a compound on a neuronal cell by administering a compound to a neuronal cell generated using the methods of the present invention and detecting at least one phenotypic change. In still other embodiments, the methods of the present invention are used to monitor the progression of a precursor cell to a neuron. In other embodiments the methods of the present invention may be used to study the effect of a compound on a neural stem cell. 
     Terms used in the present invention may be found throughout texts and research articles within the fields of cell biology, developmental biology, stem cell biology, molecular biology, biochemistry and the like. Where there is a conflict in terminology, the specification and more preferably the following definitions shall control. 
     The term “cell population” as used herein refers to a group of cells having the same identifiable characteristics. The identifiable characteristics may include morphology, presence of a surface antigen, or relative abundance of particular mRNA and the like. A cell population may include a single cell, a group of cells grown from a single cell, or a common cell and the like. 
     The terms “detectable marker” or “cell marker” as used herein refers to characteristics that alone or in combination identify a cell or group of cells as belonging to a particular cell type or cell population. Markers may be detected by binding to a binding partner, such as an antibody having a label conjugated thereto, then observing directly or indirectly the label, by RT-PCR techniques with specific primers and the like. 
     The terms “develop”, “differentiate” and “mature” as used herein all refer to the progression of a cell from the stage of having the potential to progress via the lineage to become a more specialized cell. The terms may be used interchangeably. 
     The terms “incubation under suitable conditions” as used herein refers to an incubation step where the cell is maintained in an environment to result in the desired effect. The environment may include compounds such as growth factors, substrates for cell attachment or cell growth, and the like. 
     The term “lineage” as used herein refers to all of the stages of development of a cell type, from the earliest precursor cell to a completely mature cell (i.e. specialized cell). 
     The term “medium capable of” as used herein refers to cell culture medium that includes the presence of compounds in an amount sufficient to provide the desired cell phenotype. The compounds and their amount can vary depending on cell type, desired cell phenotype, incubation time and can include growth factors and the like. Where particular concentrations are provided in the specification, the amounts are not intended to be specific limitations to the present invention but instead exemplary or preferred embodiments, because variations that produce the same effect are also intended to be encompassed by the present invention. Where particular concentrations are provided the present invention includes variations within 10%, 20%, 25% and 30% so long as the variations allow the desired result. Results may be tested using methods described herein. 
     The term “multipotent” as used herein refers to cell that can give rise to several other cell types, but those types are limited in number. 
     The term “neural stem cell” or “NSC” as used herein refers a cell derived from the nervous system which is characterized by having the ability of self-renewal and asymmetric division, which includes the ability to divide to produce two daughter cells with one being a self-renewed cell and the other having a development potential less than the renewed cell. The foregoing however is not to be construed to mean that each cell division of an NSC gives rise to asymmetrical division. It is possible that a division of an NSC can result only in self-renewal, or in the production of more developmentally restricted progeny only, or in the production of a self-renewed stem cell and a cell having restricted developmental potential. NSC&#39;s can be identified by detecting corresponding surface markers or by detecting relative abundance of mRNA compared to differentiated progeny. NSC&#39;s can be identified in comparison to neuronal cells using RT-PCR or immunoanalysis. More specifically, NSC&#39;s have a lower abundance of bIIItubulin, MAP2 and GFAP. NSC&#39;s can have a greater abundance of SOX1 mRNA. 
     The term “precursor cell” as used herein refers to any cell in a cell differentiation pathway that is capable of differentiating into a more mature or more specialized cell. 
     The term “pluripotent” as used herein refers to a cell that can develop into a variety of lineages. A pluripotent cell can create all cell types except for extra embryonic tissue, unlike a totipotent cell. 
     The present invention provides methods of obtaining neural stem cells, neuronal cells, or cells having characteristics of glial cells. 
     Embodiments of the invention provide techniques to propagate human neural stem cells (“hNSCs”) as multipotent cells in significant numbers and then, for example, hNSCs differentiate the cells into particular types of nervous system cells. Certain embodiments include a method of fast propagation of hNSCs utilizing dynamic incubation of cells in a biphasic culture system where cells are initially grown for a set time interval, such as, for example, 1 day, 2 days, 3 days, 4 days, 6 days, 7 days, or more, or the like. 
     In certain embodiments, during the initial growth period the cells can be cultured with, for example, combinations of different bioactive molecules such as growth factors epidermal growth factor (“EGF”), transforming growth factor-alpha (“TGFα”), neuregulin-1, sonic hedgehog (“SHH”), Wnt3a, Wnt 5, ciliary neutrophic factor (“CNTF”), Notch ICD, or the like. 
     In some embodiments, the initial growth period can be followed by a second growth period of a set time interval, such as, for example, 1 day, 2 days, 3 days, 4 days, 6 days, 7 days, or more, or the like. During this second growth period, cells can be cultured with, for example, serum (FBS, HBS or synthetic serum supplements) or a mix of bFGF, EGF and leukemia inhibitory factor (“LIF”), or any combination thereof, or the like. Likewise, in some embodiments the second growth period can be followed by a third growth period, or more. In certain embodiments, the type of cell culture media used during the third growth period can be the same as the type used during the initial growth period. 
     Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. 
     EXAMPLES 
     The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and a like or similar result obtained without departing from the spirit and scope of the invention. 
     Example 1 
     Propagation of hNSC&#39;s 
     hNSCs were alternately cultured in the presence (EFL+FBS) or absence (EFL−FBS) of fetal bovine serum (see  FIG. 1 ). The media change was carried out on days 7 and 14 after plating. Growth of the cells included initial incubation in EFL−FBS, followed by incubation in EFL+FBS. Experimental analysis of hNSCs was carried out on days 5, 10, 15, and 20, if not otherwise indicated. The following abbreviations were used to designate specific growth factors: E=EGF; F=bFGF; L=LIF. 
     Medium 1 (−FBS): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA). EGF (20 ng/ml, Peprotech), bFGF (20 ng/ml, Peprotech), LIF (10 ng/ml, Chemicon), heparin (5 ug/ml, Sigma). 
     Medium 2 (+FBS): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA), 1% FBS (Gibco), EGF (20 ng/ml, Peprotech), bFGF (20 ng/ml, Peprotech), LIF (10 ng/ml, Chemicon), heparin (5 ug/ml, Sigma). 
     After 10 days, cells were counted using a hemocytometer and 150,000 cells from all samples were seeded in new wells. 
       FIG. 2  shows the growth rate of hNSCs grown as neurospheres according to the method of Example 1. On days 5, 10, 15, and 20, if not otherwise indicated, neurospheres were dissociated using trypsin and single cells were counted using a hemocytometer. As controls, hNSCs were continuously grown either in the presence of serum and growth factors (FBS ON) or absence of serum, but in the presence of growth factors (control). 
     Example 2 
     Propagation of hNSC&#39;s 
     hNSC&#39;s were alternately cultured in media containing a certain growth factor and serum (GF+FBS) or in the media containing EFL but lacking the presence of fetal bovine serum (EFL−FBS) as shown in  FIG. 3 . The media was changed on days 7 and 14 after plating the cells. This example utilized an initial incubation in EFL−FBS, followed by incubation in media including FBS. Experimental analysis of hNSCs was carried out on days 5, 10, 15, and 20, if not otherwise indicated. 
     Human Neural stem cells were seeded at density of 100,000-150,000 cells/200 mm 2 . Cells were grown alternately in 2 different conditions, each period lasting for 5 to 7 days. 10 to 14 days after plating, cells were counted and seeded to new plates at the same density (100,000-150,000 cells/200 mm 2 ). 
     Medium 1 (−GFs): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA), EGF (20 ng/ml, Peprotech), bFGF (20 ng/ml, Peprotech), LIF (10 ng/ml, Chemicon), heparin (5 μg/ml, Sigma). 
     Medium 2 (+GFs): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA), 1% FBS (Gibco)+one of the combination of growth factors (CNTF, Jagged, SHH, TGFa, Wnt1, Wnt5A, or all) 
     7 different combinations of growth factors were used: 2.1. CNTF (20 ng/ml, Peprotech); 2.2. Jagged (20 ng/ml, Peprotech); 2.3. SHH (20 ng/ml, Peprotech); 2.4. TGFa (20 ng/ml, Peprotech); 2.5 Wnt1 (20 ng/ml, R&amp;D); 2.6 Wnt5A (20 ng/ml, R&amp;D); and 2.7 all above. 
     After 14 days, cells were counted using hemocytometer and again 150,000 cells from all samples were seeded in new wells. 
       FIG. 4  shows the growth rate of hNSCs grown as neurospheres according to the media schedule of  FIG. 3 . On days 5, 10, 15, and 20, if not otherwise indicated, neurospheres were dissociated using trypsin and single cells were counted using a hemocytometer. As a control, hNSCs were continuously grown in the absence of serum, but in the presence of growth factors EFL (control). The color code indicates the specific growth factor (GF) that was used in the GF+FBS mode. Abbreviations used: GF, growth factor; CNTF, ciliary neurotrophic factor; Jagged, SHH, sonic hedgehog; TGFα, transforming growth factor alpha; Wnt1, wingless-type MMTV integration site family, member 1; Wnt5A wingless-type MMTV integration site family, member 15a; or combined (CNTF, Jagged, SHH, TGFα, Wnt1, Wnt5A), E, EGF; F, bFGF; L, LIF. 
     Example 3 
     Propagation of hNSC&#39;s 
     hNSC&#39;s were alternately cultured in media containing EGF or TGFα and bFGF and serum (E*F+FBS) or in media containing growth factors but lacking FBS (E*F−FBS) as shown in  FIG. 5 . Incubation mode C refers to initial incubation in E*F−FBS, followed by incubation in E*F+FBS. Experimental analysis of hNSCs was carried out on days 5, 10, 15, and 20, if not otherwise indicated. As a control, hNSC&#39;s were continuously grown in the absence of serum, but in the presence of growth factors EFL (control). 
     hNSC&#39;s were seeded at a density of 100,000-150,000 cells/200 mm 2 . Cells were grown alternately in 2 different conditions, each period lasting for 5 to 7 days. 10 to 14 days after plating, cells were counted and seeded to new plates at the same density (100,000-150,000 cells/200 mm 2 ). 
     Medium 1 (−T, TE, E or C): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA). EGF (20 ng/ml, Peprotech), bFGF (20 ng/ml, Peprotech), LIF (10 ng/ml, Chemicon), heparin (5 ug/ml, Sigma). Abbreviations used: T, TGFα; E, EGF; C, EGF+bFGF+LIF. 
     Medium 2 (+T, TE, E or C): DMEM/F12 (1:1), 2% B27 (Gibco), 1% Pen-Strep (PAA), 1% FBS (Gibco)+one of the combination of growth factors (T, TE, E or C). Abbreviations used: T, TGFα; E, EGF; C, EGF+bFGF+LIF. 
     The 4 different combinations of growth factors were: 3.1. TGFα (20 ng/ml, Peprotech) (T); 3.2. TGF α (20 ng/ml, Peprotech), EGF (20 ng/ml, Peprotech) (TE); 3.3. EGF (20 ng/ml, Peprotech) (E); 3.4. EGF (20 ng/ml, Peprotech), bFGF (20 ng/ml, Peprotech), LIF (10 ng/ml, Chemicon), and heparin (5 ug/ml, Sigma) (C). Abbreviations used: T, TGFα; E, EGF; C, EGF+bFGF+LIF. 
     After 14 days, cells were counted using a hemocytometer, and 150,000 cells from all samples were seeded in new wells. 
       FIG. 6  shows the growth rate of hNSCs grown as neurospheres according to the media schedule of  FIG. 5 . On days 5, 10, 15, and 20, if not otherwise indicated, neurospheres were dissociated using trypsin and single cells were counted using a hemocytometer. As a control, hNSC&#39;s were continuously grown in the absence of serum, but in the presence of growth factors EFL (control). The color code indicates the specific growth factor that was used in the E*F+FBS mode or control (EFL). 
       FIG. 6  shows the differentiation efficiency of hNSC&#39;s when cultured according to the method shown in  FIG. 5 . Differentiation was initiated by plating cells onto laminin coated tissue culture plates in the growth media containing no growth factors. Cells were fixed with 4% paraformaldehyde in PBS, 14 days after plating, then immunostained. An antibody against bIII-tubulin was used to detect neurons, and anti-GFAP was used to detect astrocytes. Abbreviations: E*, EGF, TGFα, alone or combined; F, bFGF. P&lt;0.05 between different experiments. 
     Example 4 
     Differentiation of hNSC&#39;s 
     hNSC&#39;s derived via the methods of Example 1 and 2 were plated onto laminin coated (incubated with laminin solution [(Roche, 0.02 mg/ml) 60 minutes at 37 C)] slides. After coating, the slides were washed with 1×PBS once. Growth media containing no growth factors (DMEM/F12 (Gibco), 2% B27(Gibco), Pen-Strep) was used. The media was changed every other day. After 14 days, cells were fixed with 4% paraformaldehyde in PBS and immunostained. Antibodies against bIII-tubulin and MAP-2 were used to detect neurons, anti-GFAP to detect astrocytes. Then one of the following: β-TubIII (Covance MRB-435P, rabbit, 1:2000); GFAP (Chemicon MAB360, mouse, 1:400); MAP2 (Chemicon AB5622, rabbit, 1:800). Cells were then counterstained with DAPI. 
     The results are shown in  FIG. 8 . hNSCs differentiate to neurons and astrocytes being cultured according to conditions of the biphasic culture systems A and B. Antibodies against bIII-tubulin (green, top middle) and MAP-2 (green, top right) were used to detect neurons, anti-GFAP (green, top left) to detect astrocytes. Cells were counterstained with DAPI (blue). 
     Example 5 
     Differentiation of hNSC&#39;s 
     hNSC&#39;s (derived via the methods of Example 1 and 2) from ½ of a 75 cm 2  flask were used for each differentiation condition. 35 mm Petri dishes were used to avoid cells attaching to plastic during differentiation. Growth media used was: DMEM/F12 (Gibco), 2% B27 (Gibco), and Pen-Strep; plus one of 6 different conditions as in the Table 1 below. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Media Conditions 
               
             
          
           
               
                 No 
                 Factor(s) 
                 Concentration 
                 Code 
               
               
                   
               
               
                 1 
                 EGF + FGF (Peprotech) + 
                 20 ng/ml/20 ng/ml/ 
                 EFL 
               
               
                   
                 LIF (Chemicon) 
                 10 ng/ml 
               
               
                 2 
                 RA + cAMP (Applichem) 
                 500 nM/1 mM 
                 RC 
               
               
                 3 
                 RA + cAMP (Applichem) + BDNF 
                 500 nM/1 mM/50 ng/ml 
                 RCB 
               
               
                   
                 (Peprotech) 
               
               
                 4 
                 RA + cAMP (Applichem) + 
                 500 nM/1 mM/50 ng/ml 
                 RCG 
               
               
                   
                 GDNF (Peprotech) 
               
               
                 5 
                 RA + cAMP (Applichem) + 
                 500 nM/1 mM/50 ng/ml 
                 RCN 
               
               
                   
                 NGF (Peprotech) 
               
               
                 6 
                 RA + cAMP (Applichem) + 
                 500 nM/1 mM/50 ng/ml 
                 RCT 
               
               
                   
                 NT-3 (Peprotech) 
               
               
                   
               
             
          
         
       
     
     After 14 days, cells were fixed with 4% paraformaldehyde in PBS and immunostained. Antibodies against bIII-tubulin were used to detect neurons. The following were added: β-TubIII (Covance MRB-435P, rabbit, 1:2000); GFAP (Chemicon MAB360, mouse, 1:400); or MAP2 (Chemicon AB5622, rabbit, 1:800). 
     Cells were then counterstained with DAPI. The results are shown in  FIG. 9 . The figure illustrates changes (in percentage) in numbers of β-TubIII immunoreactive cells upon differentiation of hNSC&#39;s. Immunostaining revealed that “priming” of human neural stem cells in Petri dishes does not affect their potential to differentiate to neurons. Differentiation was initiated by harvesting cells in Petri dishes for 7 days after which cells were plated onto laminin coated tissue culture plates in the growth media containing no growth factors. As a control, differentiation was initiated by plating cells directly onto laminin coated tissue culture plates in the growth media containing no growth factors. Abbreviations used: RC=retinoic acid+dBcAMP; B=BDNF; G=GDNF; N=NGF, T=NT-3. 
     Example 6 
     Characterization of hNSC-Derived to TH+ and GABA+ Neurons 
     hNSC&#39;s were propagated according to the method of Example 1 and differentiated according to the method of Example 5. hNSC-derived bIIItubublin+cells. hNSC-derived bIIItubulin+ cells were immunostained with TH and GAD65 antibodies. Condition where cells were differentiated 14 days on laminin-coated tissue culture dishes without the addition of any growth factors nor sera (DM#0) were used as control. The results are shown in Table 2 below. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Quantitative analysis of TH +  and GAD65 +  neurons upon 
               
               
                 differentiation using DM#3 and DM#4 media. 
               
             
          
           
               
                   
                 Differentiation 
                 TH+/ 
                 GAD65+/ 
               
               
                   
                 Media 
                 bIIItubulin + cells (%) 
                 bIIItubulin + cells (%) 
               
               
                   
                   
               
               
                   
                 DM#1 
                 38 ± 6 
                 75 ± 4 
               
               
                   
                 DM#3 
                 43 ± 8 
                 ND 
               
               
                   
                 DM#4 
                 &lt;1 ± 1 
                 84 ± 9 
               
               
                   
                   
               
             
          
         
       
     
     The Table 3 illustrates that DM#3 supported TH+ differentiation and DM#4 enhanced GABA+ (GAD65+) neuronal differentiation. These differentiated cells were also positive for tubulin βIII and MAP2. In addition, hNSC-derived TH +  cells did not co-express GABA or markers for astrocytes and oligodendrocytes such as, GFAP and O4, respectively. Similar findings were true to GABA+ cells. Using DM#0, 1, 2, 5 and 6 treatments, the numbers of GABA+ and TH+ neurons did not reach beyond &lt;1% of total bIIItubulin+ cells. 
     During differentiating hNSCs to TH+ neurons, the change of several markers was analyzed by semiquantitative RT-PCR ( FIG. 10 ). The neural progenitor-marker Sox1 was strongly up-regulated already in proliferating hNSCs, whereas the expression of dopaminergic neuronal markers, such as NURR1, EN1, and PTX3 were prominent DM#3-differentiated neuronal cultures 7D post-treatment. 
     During differentiating hNSCs to GABA+ neurons, the change of several markers was analyzed by semiquantitative RT-PCR ( FIG. 10 ). In DM#4 differentiated cultures, a significant increase in GABAergic neuron-specific transcription factor PITX2 mRNA was detected 7D after induction of differentiation ( FIG. 11 ). Immunoanalysis GAD65 is one of the target genes of PITX2 activity. 
     The RT-PCR results support the IHC data indicating that the developed protocols are suitable for generation of dopaminergic (DA) and GABAergc neurons 
     Example 7 
     Phenotypic Analysis of Differentiated hNSC&#39;s Using RT-PCR Analysis 
     hNSC&#39;s were propagated according to the method of Example 1. Without triturating, spheres from ½ of 75 cm 2  flask were used for each differentiation condition. 35 mm Petri dishes were used to avoid cells attaching to plastic during differentiation. The media used included DMEM/F12 (Gibco), 2% B27 (Gibco), Pen-Strep; +RA, and the differentiation period was 7 days. 
     Cells were then lysed (directly on plastic) and the RNA extracted using RNA Micro Kit (Ambion). cDNA was synthesized with First Strand Kit (Invitrogen). 
     The PCR protocol used was as follows: Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). PCR program: U-incubation at 50° C. for 2 min; polymerase activation at 95° C. for 2 min; cycling at 95° C. for 5 sec, 60 C for 10 sec and 72 C for 10 sec; 45 cycles (CHAT, DBH, TH: 50 cycles). Primers used are listed in Table 4. All primers are downloadable from Primer Bank database: http://pga.mgh.harvard.edu/primerbank/index.html 
     
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Synthetic Primers 
               
             
          
           
               
                 SEQ 
                 Receptor 
                 Sense 
                 Antisense 
               
               
                   
               
             
          
           
               
                 Acetylcholine Receptors 
               
             
          
           
               
                  1 
                 CHRNA4 
                 GGCCTTCCTGCAAGTCACC 
                   
               
               
                  2 
                 CHRNA4 
                   
                 ATCGTCTCGGGGAACACAGT 
               
               
                   
               
               
                  3 
                 CHRNA5 
                 ACGTTTTGAAGGGACCAGTACG 
                   
               
               
                  4 
                 CHRNA5 
                   
                 CACTCACAATCTCCCATTCTCC 
               
               
                   
               
               
                  5 
                 CHRNB 
                 TGACATCGTGCTTTACAACAACG 
                   
               
               
                  6 
                 CHRNB 
                   
                 CGTGTGGTCATAGGTCCAGG 
               
               
                   
               
             
          
           
               
                 Benzodiazepine Receptors 
               
             
          
           
               
                  7 
                 GABRA2 
                 AGTGGCTGTTGCCAATTATGC 
                   
               
               
                  8 
                 GABRA2 
                   
                 GGACTGACCCCTAATACAGGTT 
               
               
                   
               
               
                  9 
                 GABRA3 
                 GTCACTGTTACATGACCAGCC 
                   
               
               
                 10 
                 GABRA3 
                   
                 CGTCCAGAAGACGATCCAAGAT 
               
               
                   
               
             
          
           
               
                 GABA-A Receptors 
               
             
          
           
               
                 11 
                 GABRA1 
                 AGAAAAACAACACTTACGCTCCA 
                   
               
               
                 12 
                 GABRA1 
                   
                 GGGCTTGACCTCTTTAGGTTC 
               
               
                   
               
               
                 13 
                 GABRB1 
                 GTACAAAATCGAGAGAGTCTGGG 
                   
               
               
                 14 
                 GABRB1 
                   
                 GCGAATGTCATATCCTTTGAGCA 
               
               
                   
               
               
                 15 
                 GABRB3 
                 CTTGACAATCGAGTGGCTGA 
                   
               
               
                 16 
                 GABRB3 
                   
                 CAATCCTTTCCACTCCGGTA 
               
               
                   
               
               
                 17 
                 GABRQ 
                 ACATCGTGATCGTGTAGTCCA 
                   
               
               
                 18 
                 GABRQ 
                   
                 TCCCGAAATTCCACTTCGAGT 
               
               
                   
               
               
                 19 
                 GABRR2 
                 TGCCTGCCAGAGTTTCACTG 
                   
               
               
                 20 
                 GABRR2 
                   
                 CACACATGCACGGGAACTTC 
               
               
                   
               
             
          
           
               
                 Galanin Receptors 
               
             
          
           
               
                 21 
                 GALR2 
                 GCCCTACCTGAGCTACTACC 
                   
               
               
                 22 
                 GALR2 
                   
                 GAGGATCATGCGTGTCACCTT 
               
               
                   
               
             
          
           
               
                 Glycine Receptors 
               
             
          
           
               
                 23 
                 GLRA1 
                 CAGTGAACGTGAGCTGCAAC 
                   
               
               
                 24 
                 GLRA1 
                   
                 TCCAGAGAGTCGTCAGGGTAT 
               
               
                   
               
               
                 25 
                 GLRA3 
                 TCGGGATTTTACTTCTGGGAAGC 
                   
               
               
                 26 
                 GLRA3 
                   
                 GATAGAGCCGAAACTGTTGATGA 
               
               
                   
               
             
          
           
               
                 Glutamate Receptors 
               
             
          
           
               
                 27 
                 GRIA1 
                 GGTCTGCCCTGAGAAATCCAG 
                   
               
               
                 28 
                 GRIA1 
                   
                 CTCGCCCTTGTCGTACCAC 
               
               
                   
               
               
                 29 
                 GRIN1 
                 AGGAACCCCTCGGACAAGTT 
                   
               
               
                 30 
                 GRIN1 
                   
                 CTCTCCAGTCGTCACCAGGT 
               
               
                   
               
             
          
           
               
                 Neuropeptide Receptors 
               
             
          
           
               
                 31 
                 GPR74 
                 TGGGTCCTAGCCATCACCATT 
                   
               
               
                 32 
                 GPR74 
                   
                 GCCCTGAAGAGTGAAATTCCA 
               
               
                   
               
             
          
           
               
                 Neuropeptide Y Receptors 
               
             
          
           
               
                 33 
                 NPY2R 
                 ATGGGTCCAATAGGTGCAGAG 
                   
               
               
                 34 
                 NPY2R 
                   
                 AGCAGTAGGCCAATATGAGAACA 
               
               
                   
               
               
                 35 
                 PPYR1 
                 GGTCCTGGGTAACCTCTGC 
                   
               
               
                 36 
                 PPYR1 
                   
                 GAGACCGTCACCGACATGC 
               
               
                   
               
             
          
           
               
                 Serotonin Receptors 
               
             
          
           
               
                 37 
                 HTR2A 
                 GCTCAACTACGAACTCCCTAATG 
                   
               
               
                 38 
                 HTR2A 
                   
                 AGAGGCACCCTTCACAGGAA 
               
               
                   
               
               
                 39 
                 HTR3A 
                 GCTTGCCAGAAAAGGTGAAATC 
                   
               
               
                 40 
                 HTR3A 
                   
                 GGCGGATGACCACATAGAACTT 
               
               
                   
               
             
          
           
               
                 Somatostatin Receptors 
               
             
          
           
               
                 41 
                 SSTR2 
                 GTCACCCGAATGGTGTCCATC 
                   
               
               
                 42 
                 SSTR2 
                   
                 ATTTGTCCTGCTTACTGTCACTC 
               
               
                   
               
             
          
           
               
                 Tachykinin Receptors 
               
             
          
           
               
                 43 
                 TACR1 
                 CACAACGAATGGTACTACGGC 
                   
               
               
                 44 
                 TACR1 
                   
                 CTCTGCTGGGCATGGTCTC 
               
               
                   
               
             
          
           
               
                 Neurotransmitter Biosynthesis 
               
             
          
           
               
                 45 
                 CHAT 
                 CGTAAGATGGCAGCAAAAACTC 
                   
               
               
                 46 
                 CHAT 
                   
                 GCCAGGCGGTTGTTGAGAT 
               
               
                   
               
               
                 47 
                 GAD1 
                 GCCAAACAGAAGGGATATGTTCC 
                   
               
               
                 48 
                 GAD1 
                   
                 GCCCATCATCTTGTGAGGGTT 
               
               
                   
               
               
                 49 
                 GAD2 
                 GGCTTTTGGTCTTTCGGGTC 
                   
               
               
                 50 
                 GAD2 
                   
                 TTCTCGGCGTCTCCGTAGAG 
               
               
                   
               
               
                 51 
                 TH 
                 GAGACGTTTGAAGCCAAAATCC 
                   
               
               
                 52 
                 TH 
                   
                 AGGTCAGGGTCGAACTTGGT 
               
               
                   
               
               
                 53 
                 DBH 
                 ACTGGCTACTGCACGGACAAG 
                   
               
               
                 54 
                 DBH 
                   
                 CTTTCTCCCAGTCAGGTGTGTGT 
               
               
                   
               
             
          
           
               
                 Neurotransmitter Secretion 
               
             
          
           
               
                 55 
                 SNPH 
                 CAGCAGCCGATGACACACT 
                   
               
               
                 56 
                 SNPH 
                   
                 GCACGAAGTCTGTCTGGATGG 
               
               
                   
               
               
                 57 
                 SYN3 
                 CAGCAGCAAAGGTCACCAG 
                   
               
               
                 58 
                 SYN3 
                   
                 GGCTGTTAGTCAGGGACTGAG 
               
               
                   
               
             
          
         
       
     
     Example 8 
     Proliferation of Neural Stem Cells Treated with Various Bioactive Molecules 
     Human neural stem cells are most routinely grown in medium supplemented with EGF and bFGF (and LIF). To analyze whether influencing signaling pathways with other growth factors can also support the proliferation of neural progenitor cells in undifferentiated state, growth conditions were alternated every 7 days. Cells were grown for 16 weeks and counted regularly every two weeks. During the first week (also third, fifth week, etc.) the cells were grown with different growth factors and 1% FBS in culture media. 8 different treatments were used: CNTF, Jagged-1, SHH, TGFa, Wnt1, Wnt5a, mix (CNTF, Jagged, SHH and Wnt-1) and combination of EGF, bFGF, LIF as control. During the second week (also fourth, sixth week, etc.) the cells were switched to routine growth medium (EGF, bFGF, LIF, no FBS). Cells were counted 8 times. During the first 8 weeks the cells proliferated at a quite similar rate, with only the control cells were growing slightly faster (data not shown). During the next 8 weeks, it was clearly seen that with control medium, cells were growing remarkably faster ( FIG. 4 ). At the last time point analyzed, 5 times more cells were grown with control medium than under any other conditions. Surprisingly, the mix of four factors (CNTF, Jagged, SHH and Wnt1) supported the cell growth most weakly. All the factors used alone gave quite similar results; Wnt1, TGFa and Jagged-1 being slightly more efficient than the others. From these results was concluded that none of the tested conditions was supporting the proliferation of neural progenitor cells as efficiently as the combination of EGF, bFGF and LIF. 
     Example 7 
     Differentiation of Neural Stem Cells to Neurons and Astrocytes 
     Next, how the stimulation of signaling pathways affects the commitment of human neural stem cells to neuronal and glial lineages was examined. For differentiation, samples of cells grown in all 8 conditions were removed from proliferating cell populations (after 8 and 12 weeks in culture) and plated onto laminin coated coverslips. All additional factors were excluded from culture medium and cells were grown for 14 days. Next, cells were fixed and stained for neural and glial markers as described previously. Examples of immunocytochemistry analysis can be seen on  FIG. 8 . In differentiated cultures, cells were found to be positive for neuronal (bTubIII, MAP-2) and astrocyte markers (GFAP). No cells were detected as positive for oligodendrocyte marker O4. After immunostaining, cells from 3 random fields were counted and their differentiation potential was estimated according to the number of bTubIII positive cells present in samples. Results can be seen on  FIG. 9 . It is interesting to note, that while cells grown with different growth factors had significantly lower proliferation rates as compared to control, most of the cells had still retained differentiation potential similar to control cells. Cells grown with Wnt5a had the least percentage of bTubIII positive cells (33%), all the other conditions gave 41 to 47% of bTubIII positive cells (in control 45%). Taken together these data show that treatments for 16 weeks did not change the potential of neural progenitors to differentiate to bTubIII positive neuronal cells.