MXPA00008071A - Method of promoting neuronal cell proliferation and diffrentiation - Google Patents

Abstract

The present invention provides methods, improved cell culture medium and kits for promoting neuronal cell proliferation and/or differentiation by growth in the presence of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.

Description

METHOD FOR PROMOTING PROLIFERATION AND NEURONAL CELL DIFFERENTIATION Field of the Invention The present invention relates to methods and equipment for accelerating the proliferation and / or differentiation of neuronal cells.

BACKGROUND OF THE INVENTION The pluripotent cells in the central nervous system (herein referred to as "CNS") have the potential to differentiate into neurons, astrocytes and oligodendrocytes and renew themselves. (McKay, Science 276: 66-71, 1997, incorporated herein by reference in its entirety). The CNS progenitor cells have a greater restricted potential than a pluripotential cell, while the CNS precursor cells comprise any CNS cell of the non-differentiated type (McKay, 1997). Fetal precursor cells of mammals that increase neurons and glia have been isolated (Frederi sen et al., Neuron 1: 439 (1988); Reynolds and Weiss, Science 255: 1707 (1992); Davis and Temple, Nature 372: 263 (1994)). Adult CNSs also contain multipotential precursor cells for neurons, astrocytes and oligodendrocytes (McKay, 1997). Cells grown from adult and fetal CNS cells that have proliferated in vitro can be differentiated to show characteristic morphological and electrophysiological characteristics of neurons (Griííi et al., J. Neurosci 16: 1091 (1996); Vicario-Abejón et al. ., Neuron 15: 105 (1995)). These data show the multipotential nature of the cells derived from the CNS. The multipotential cells of the fetal brain have been shown to be homogeneous and stable. (Jonh et al., Genes Dev. 10: 3129 (1996)). In vitro, these cells divide daily and efficiently generate neurons and glia in at least the first month of culture. These cells can be considered as pluripotent cells because they meet the criteria of multipotency and self-renewal. Adult brain cells proliferate and differentiate into neurons and glia in tissue culture with the same efficiency for neuronal differentiation as found in fetal pluripotent cells and the same response for extracellular ligands (McKay, 1997). Therefore, similar general mechanisms control the differentiation of pluripotent cells from the adult or fetal brain. The proliferation of precursor cells in the brain can be stimulated by the direct in vivo application of mitogenic growth factors and in animals treated by this route, proliferation cells in the subventricular zone differentiate into neurons and glia (Craig et al. ., J. Neurosci 16: 2649 (1996)). However, in vivo less than 3% of proliferation cells labeled with bromodeoxyuridine differ in neurons (McKay, 1997). The discrepancy between the efficient neuronal differentiation of adult pluripotent cells in vitro and their inefficient differentiation in vivo is an unresolved question in the field (Id). The lack of differentiation of neurons can not be a consequence of the lack of cells with appropriate potential but rather a function of the signaling environment in the adult brain (Id.).

The long-term delivery of proteins in the brain is an important goal in gene therapy. The transplantation of the cells directed to produce growth factors shows the potential of the grafted cells as vectors for the delivery of proteins (Beck et al., Nature 373: 339 (1995); Tomac et al., Nature 373: 335 (1995 ); Moore et al., Nature 382: 76 (1996)). It is possible to generate many different immortal cell lines from the development of the CNS. It is also possible to graft primary cells expanded in vitro. Experiments have suggested that primary adult cells derived from the hippocampus and cultured for long periods in vitro can still be differentiated in neurons when they are reimplanted in the migratory route used to replenish neurons in the adult olfactory bulb (Sushonen et al., Nature 383: 624 (1993)). Experimental grafts in animal models suggest that the integration of grafted neurons in the host circuitry may be possible, as other studies illustrate the use of in vitro manipulated donor cells that differentiate in vivo in oligodendrocytes (Tontsch et al., Proc. Nati, Acad. Sci. 91: 11616 (1994); Groves et al., Nature 362: 453 (1993)). Additionally, clinical evidence shows that neuron replacement therapies for neurodegenerative diseases, such as Parkinson's or Huntington's disease, are feasible (Kordower et al., New Engl. J. Med. 332: 1118 (1995); LindvAII et al. ., Ann. Neurol. 35: 172 (1994)). Therefore, for clinical applications, cell culture offers an important opportunity to use sophisticated genetics in cell-based therapies for neural disease (McKay, 1997). The CNS pluripotent cells have been expanded in vitro by the use of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) (McKay, 1997). In vitro, EGF has also been shown to be a differentiating factor for astrocytes. While these studies demonstrate the potential for pluripotent cells and neuron replacement therapy, only a careful analysis of adult neuronal pluripotent cells has been initiated. Furthermore, the characterization of the mechanisms that control multipotentiality, self-renewal and the restriction destiny of neuronal pluripotent cells is clearly important to develop new therapies for cell regeneration and replacement in the adult nervous system (Johe et al., 1996) . Methods that increase the proliferation and in vitro and ex vivo differentiation of neuronal progenitor and pluripotent cells will greatly increase the utility of neuron replacement therapy in various neurodegenerative conditions such as Parkinson's and Alzheimer's Disease and amyotrophic lateral sclerosis. Similarly, methods that increase proliferation and in vivo differentiation of neuronal progenitor and pluripotent cells improve the utility of neuron replacement therapy by rapidly increasing local concentrations of progenitor and neuronal pluripotent cells at the therapy site. .

SUMMARY OF THE INVENTION The present invention provides methods that increase the proliferation or differentiation of neuronal progenitor and pluripotent cells that are useful in rapidly providing a considerable population of such cells in neuron replacement therapy and to make a considerable population of cells transfected for use in neuron replacement therapy. In another aspect, the present invention provides methods that promote the proliferation or differentiation of neuronal cells by contacting the cells with angiotensinogen, angiotensin I (Al), Al analogs, Al fragments and analogues thereof, angiotensin II (All) , All analogues, All fragments or analogs thereof or type 2 All 2 AT2 receptor agonists, either alone or in combination with other growth factors and cytokines. In another aspect of the present invention, there is provided an improved cell culture medium for the proliferation or differentiation of neuronal cells, wherein the enhancement comprises adding to the cell culture medium an effective amount of angiotensinogen, Al, Al analogs. , Al fragments and analogs thereof, All, All analogues, All or analogous fragments thereof or All 2 All 2 AT2 receptor agonists. In a further aspect, the present invention provides equipment for the propagation or differentiation of neuronal cells, wherein the kits comprise an effective amount of angiotensinogen, Al, Al analogs and / or Al fragments and analogs thereof, All, analogs, etc. , All or analogue fragments thereof and / or agonists receptors of type 2 All AT2 and instructions for culturing the cells. Preferred embodiments of the kit further comprise the cell culture growth medium, a sterile container and an antibiotic supplement.

Brief Description of the Figures Figure 1. Effect of the All on the human neuronal result of the progenitor neurite, where: A = Units of result B = Days of Cultivation Figure 2. Effect of All, AII (1-7) and Ala4-AIII on the proliferation of normal human neural progenitors, where: C =% increase at the beginning D = Peptides Tested E = Control Detailed Description of Preferred Modalities As defined herein the term "neuronal cells" includes primary cells or established cell lines with the potential to differentiate into neurons, astrocytes and oligodendrocytes and for self-renewal and also for differentiated cells derived from the same, including completely differentiated CNS and cell types of the peripheral nervous system ("PNS"). Examples of neuronal progenitor and pluripotent cells include but are not limited to those described in Gritti et al., J. of Neuroscience 16: 1091-1100 (1996); Frederiksen et al., (1988); Reynolds and Weiss, (1992); Davis and Temple, (1994); McKay, (1997); Vicario-Abejón et al., (1995); Craig et al., (1996); Tontsch et al. (1994); Graves et al., (1993) and Johe et al., Genes and Develop. 10: 3129-3142 (1996), all references are incorporated in their entirety in this document. As defined in this document, "proliferation" comprises cell auto-renewal and cell proliferation with the differentiation that accompanies it. Unless indicated otherwise, the term "active agents" as used herein refers to the group of compounds comprising angiotensinogen, angiotensin I (Al), Al analogs, Al fragments and analogs thereof, angiotensin II. (All), All analogs, All or analogue fragments thereof and type 2 All 2 AT2 receptor agonists. In this application, unless stated otherwise, the techniques used can be found in any of several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press ), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutshcer, ed., (1990 ) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd. Edition (R. Freshney, 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp 109-128 ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX). The Patent of E.U.A. No. 5,015,629 to DiZerega (the full disclosure thereof is incorporated herein by reference) discloses a method for increasing the healing rate of tissue from a wound comprising the application to said tissue of angiotensin II (All) in an amount which is sufficient for this increase. The application of All to the wound tissue significantly increases the healing speed of the wound, leading to a faster re-epithelialization and repair of the tissue. The term "All" refers to an octapeptide present in humans and other species having the Asp-Arg-Val-Tyr-lle-His-Pro-Phe Sequence [SEQ ID NO: 1]. The biological formation of angiotensin is initiated by the action of renin in the angiotensinogen of the plasma substrate. The substance formed is a decapeptide called angiotensin I (Al) which is converted to All through the conversion of the enzyme angiotensinase that removes the residues of C-terminal His Leu from Al (Asp-Arg-Val-Tyr-lle-His- Pro-Phe-His-Leu [SEQ ID NO: 37]). All is a known precursor agent and is commercially available. There has also been described the use of the All analogs and fragments, AT2 agonists, as well as there and analogs there and fragments in the healing of the wound. (U.S. Patent No. 5,629,292, U.S. Patent No. 5,716,935, WO 96/39164, all references are incorporated herein in their entirety). Studies have shown that All increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair and also increases their release of extracellular growth factors and matrices (diZerega, US Pat. No. 5,015,629; Dzau et al. al., J. Mol. Cell, Cardiol., 21: S7 (Supp III), 1989, Berk et al., Hypertension 13: 305-14 (1989), Kawahara et al., BBRC 150: 52-9 (1988); Naftilan, et al., J. Clin Invest. 83: 1419-23 (1989); Taubman et al., J. Biol. Chem 264: 526-530 (1989); Nakahara, et al., BBRC 184: 811 -8 (1992), Stouffer and Owens, Circ. Res. 70: 820 (1992), Wolf, et al., Am. J. Pathol., 140: 95-107 (1992), Bell and Madri, Am. Pathol. 137: 7-12 (1990). In addition, the All proved to be angiogenic in rabbit corneal eye and chorioallantoic chicken membrane models (Fernandez, et al., J. Lab.Clin.Med. 105: 141 (1985); LeNoble, et al., Eur. J Pharmacol., 195: 305-6 (1991). Therefore, the All can accelerate wound repair through increased neovascularization, growth factor release, reepithelialization, and / or production of the extracellular matrix. The All has also been applied in cell growth and differentiation (M? Ffert et al., Mol.and Cellul.Endocrin.122: 59 (1996)). Two main classes of All, ATi and AT2 receptors have been identified. (Meffert, 1996) The effects of the promotion of the growth of the All have been attributed to the mediation of the AT1 receptor, even though some evidence suggests that the AT2 receptor can be involved in the mediation of the cellular differentiation effects of the All ( Bedecs et al., Biochem J. 325: 449 (1997).) The effects of the receptor All and the All receptor antagonists have been examined in two experimental models of vascular damage and repair that follow that both the All receptor subtypes (AT1 and AT2) play a role in wound healing (Janiak et al., Hypertension 20 : 737-45 (1992); Prescott, et al., Am. J. Pathol. 139: 1291-1296 (1991); Kauffman et al., Life Sci. 49: 223-228 (1991); Viswanathan, et al., Peptides 13: 783-786 (1992); Kimura, et al., BBRC 187: 1083-1090 (1992). Many studies have focused on AII (1-7) (residues All 1-7) or other All fragments to evaluate their activity. The All (1-7) produces some, but not the full range of effects produced by the All. Pfeilschiffer, ef al., Eur. J. Pharmacol. 225: 57-62 (1992); Jaiswal, et al., Hypertension 19 (Supp. Ll): ll-49-ll-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266: 506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265: 664-673 (1991); Jaiswal, et al., Hypertension 17: 1115-1120 (1991); Portsi, et al., Br. J. Pharmacol. 111: 652-654 (1994). Studies have shown that All inhibits the proliferation of an immortalized neuronal cell line (the pheochromocytoma derived PC12W, Meffert, 1996) and primary cultures dissociated from the retro-thalamic hypothalamus of 18-day rat embryos (Jirikowski et al., Develop. Res. 14: 179-183 (1984)). Other studies have shown that the proportion of ATi and AT2 receptors in rat brain changes during development, with fetal tissue expressing much more AT2 receptor subtype, while adult animals express much more ATi subtype (Meffert, 1996). However, little is known about the effects of All in most CNS or cell types of the peripheral nervous system ("PNS"). In addition, it is not known if the All receptor subtypes are expressed by neuronal progenitor and pluripotent cells, or what effect the All has on its proliferative capacity. A peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than for AT1) is p-aminophenylalanine6-AII ["p-NH2-Phe) 6-AII"], Asp-Arg-Val- Tyr-lle-Xaa-Pro-Phe [SEQ ID NO. 36] where Xaa is p-NH2-Phe (Speth and Kim, BBRC 169: 997-1006 (1990) .This peptide gave comparable binding characteristics to the AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol 256: 93-97 (1994); Bryson, et al., Eur. J. Pharmacol. 225: 119-127 (1992).

Active Al, Al analogs, Al fragments and analogs thereof, All analogs, All fragments and analogues thereof of particular interest according to the present invention are characterized in that they comprise a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of the general formula I R1 -R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, RA is appropriately selected from Asp, Glu, Asn, Acpc (acid 1-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, RB is appropriately selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D- Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc, Lys and Tyr; R4 is selected from the group consisting of Tyr, Tyr (P? 3) 2, Thr, Ser, homoSer, Ala and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R4 as a terminal group Tyr.

Compounds falling within the category of the AT2 agonists useful in the practice of the invention include the All analogs set forth above subject to the restriction that R6 is p-NH2-Phe. In addition to the peptide agents, various nopeptide agents (eg, peptodomimetics) that have the activity requirement of the AT2 agonist are further contemplated for use in accordance with the present invention. Particularly preferred cnations for RA and RB are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: All, There or AII (2-8), Arg-Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 2]; AII (3-8), also known as desl-AIII or AlV, Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 3]; AII (1-7), Asp-Arg-Val-Tyr-lle-His-Pro [SEQ ID NO: 4]; AII (2-7). Arg-Val-Tyr-lle-His-Pro [SEQ ID NO: 5]; AII (3-7), Val-Tyr-lle-His-Pro [SEQ ID NO: 6]; AII (5-8), lle-His-Pro-Phe [SEQ ID NO: 7]; AII (1-6), Asp-Arg-Val-Tyr-lle-His [SEQ ID NO: 8]; AII (1-5), Asp-Arg-Val-Tyr-lle [SEQ ID NO: 9]; AII (1-4), Asp-Arg-Val-Tyr [SEQ ID NO: 10] and AII (1-3), Asp-Arg-Val [SEQ ID NO: 11]. Other preferred embodiments include: Arg-norLeu-Tyr-lle-His-Pro-Phe [SEQ ID NO: 12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Another preferred embodiment comprised within the scope of the invention is a peptide having the Asp-Arg-Pro-Tyr-lle-His-Pro-Phe Sequence [SEQ ID NO: 31]. AII (6-8), His-Pro-Phe [SEQ ID NO: 14] and AII (4-8), Tyr-lle-His-Pro-Phe [SEQ ID NO: 15], were also tested and found that are not effective. In another preferred embodiment, the present invention provides a method for the promotion of neuronal cell proliferation or differentiation comprising contacting neuronal cells with an effective amount to promote the proliferation or differentiation of at least one active agent comprising a sequence consisting of of the general formula: R1-ARG-VAL-TYR-R2-HIS-PRO-R3 wherein R1 is selected from the group consisting of H or Asp; R2 is selected from the group consisting of lie, Val, Leu, norLeu and Ala; R3 is Phe or H. In a more preferred embodiment, the active agent is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26 and SEQ ID NO: 34. Another class of compounds of particular inferes according to the present invention are those of the general formula II R2-R3-R4-R5-R6-R7-R8 wherein R2 is selected from the group consisting of H, Arg, Lys, Ala , Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc and Tyr; R4 is selected from the group consisting of Tyr, Tyr (POs) 2, Thr, Ser, homoSer and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), He and Tyr. A particularly preferred sub-class of the compounds of the general formula II has the formula R2-R3-Tyr-R5-His-Pro-Phe [SEQ ID NO: 16] wherein R2, R3 and R5 are as previously defined. Angiotensin III of the formula Arg-Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 2] is particularly preferred. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18] . The AII fragment (4-8) was ineffective in the repeated tests; this is believed due to the tyrosine exposed in the N-terminus. In the above formulas, the standard three-letter abbreviations for amino acid residues are used. In the absence of an indication to the contrary, the L form of the amino acid is tried. Other waste is abbreviated as follows: TABLE 1 Abbreviation for Amino Acids It has been suggested that the All and its analogues adopt a gamma or beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).) In general, it is believed that the neutral side chains in position R3, R5 and R7 can be involved in maintaining the appropriate distance between the active groups in positions R4, R6 and R8 basically responsible for binding to receptors and / or intrinsic activity.The hydrophobic side chains in positions R3, R5 and R8 can also play an important role in the complete conformation of the peptide and / or contribute to the formation of a hypothetical hydrophobic cavity The appropriate side chains at the amino acid at the R2 position may contribute to the affinity of the compounds for target receptors and / or play an important role in shaping the For this reason, Arg and Lys are particularly preferred as R. For purposes of the present invention, it is believed that R3 may be involved in the formation of linear and non-linear hydrogen bonds with R5 (in the gamma model) or R6 (in the beta rotation model). R3 would also participate in the first turn in an antiparallel beta structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that the beta and gamma branches are equally effective in this position. In addition, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R3 may be appropriately selected from Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R3 is Lys. With respect to R4, conformational analyzes have suggested that the side chain in this position (as well as in R3 and R5) contributes to a hydrophobic cluster that is believed to be essential for receptor occupancy and stimulation. Therefore, R4 is preferably selected from Tyr, Thr, Tyr (P? 3) 2, homoSer and azaTyr. In this position, Tyr is particularly preferred to form a hydrogen bond with the receptor site capable of accepting a hydrogen of the phenolic hydroxyl (Regoli, et al. (1974), supra). In a further preferred embodiment, R4 is Ala. In the R5 position, an amino acid with an aliphatic or alicyclic β chain is particularly desirable. Therefore, while Gly is appropriate in the R5 position, it is preferred that the amino acid in this position be selected from lie, Ala, Leu, norLeu, Gly and Val. In Al, Al analogues, Al fragments and analogs thereof, All, Analogs All, fragments and analogs of fragments of particular interest according to the present invention, R6 is His, Arg or 6-NH2-Phe. The unique properties of the imidazole ring of histidine (eg, ionization at physiological pH, the ability to act as a proton donor or acceptor, aromatic character) are thought to contribute to its particular utility as R6. For example, conformational models suggest that His can participate in hydrogen bond formation (in the beta model) or in the second antiparallel structure spin by influencing the orientation of R7. Similarly, it is considered that R7 should be Pro to provide the most desirable orientation of R8. In the R8 position, a hydrophobic ring and a terminal anionic carboxyl are particularly useful in binding the analogs of interest to the receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention. Analogs of particular interest include the following: TABLE 2 Angiotensin II analogues The polypeptides of the present invention can be synthesized by methods such as those described in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chemical Co., Rockford, III. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York (1965) for the synthesis of the solution. The descriptions of the previous treaties are incorporated in this document as a reference. In general, these methods involve the sequential addition of protected amino acids to a chain of growth peptides (U.S. Patent No. 5,693,616), incorporated herein by reference in their entirety). Normally, the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is bound to an inert solid support or used in solution and the next amino acid in the Sequence, also appropriately protected, is added under treatable conditions for amide bond formation. After all the desired amino acids have been linked in the proper sequence, the protecting groups and any solid support are removed to produce the natural polypeptide. The polypeptide is desalted and purified, preferably chromatographically to produce the final product. In one aspect of the present invention, there is described a method of in vivo, in vitro and ex vivo increase of the proliferation of neuronal progenitor and pluripotent cells by exposure to angioinensinogen, Al, Al analogues, Al fragments and analogs thereof, analogues All, All or analogue fragments thereof or agonists of the All 2 All 2 receptor ("active agents"). Experimental conditions for isolation, purification, in vitro / ex vivo growth and in vivo mobilization of neuronal progenitor and pluripotent cells have been reported (Frederiksen et al., 1988; Reynolds and Weiss, 1992; Gritti et al., 1996 Vicario-Abejón et al., 1995; Johe et al., 1996; Craig et al., 1996; Suhonen et al., Nature 383: 624-627, 1996 and Tontsch et al., 1994).

Proliferation can be quantified using any of several techniques well known in the art, including, but limited to, incorporation of bromodeoxyuridine (Vicario-Abejón et al., 1995), incorporation of 3H-thymidine (Fredericksen et al., 1988) or labeling of antibodies of a protein present in higher concentration in proliferating cells than in non-proliferating cells. In a preferred embodiment, the proliferation of neuronal progenitor and pluripotent cells is determined by reactivity to an antibody directed against a known protein present in higher concentrations in proliferating cells than in non-proliferating cells, including but not limited to proliferating cell nuclear antigen ( PCNA or cyclin; Zymed Laboratories, South San Francisco, California). In one embodiment, neuronal cells are isolated from primary cell masses in accordance with standard methods (Jirikowski et al., 1984; Reynolds and Weiss, 1992; Johe et al., 1996; Gritti et al., 1996; Abejón et al., 1995, Kordower et al., 1995, Nauert and Freeman, Cell Transplant 3: 147-151, 1994, Freeman and Kordower, In: LindvAII et al., Eds, Intracerebral Transplantation in Movement Disorders, New York: Elsevier Science, 163-170, 1991), are suspended in a culture medium and incubated in the presence of, preferably, between about 0.1 ng / ml and about 10 mg / ml of the active agents of the invention. The cells are expanded for a period of between 8 and 21 days and cell proliferation is determined as described above. In a preferred embodiment, neuronal progenitor and pluripotent cells are isolated from primary cells that are isolated from the rat embryonic hippocampus or from the brain of an adult rat mammal (Johe ai al., 1996). The cell mass is dissociated by mechanical trituration or by incubation of sectioned tissue in a Hank Stabilized Saline Solution (HBSS). Cells are collected by centrifugation and resuspended in a serum-free medium containing Dulbecco's Modified Eagle Medium (DMEM) / F12, glucose, glutamine, sodium bicarbonate, 25 μg / ml insulin, 100 μg / ml human apotransferrin , 20 nm of progesterone, 100 μm of putrescena, 30 nm of sodium selenite (pH 7.2), plus 10 ng / ml of the recombinant basic fibroblast growth factor (bFGF, R &D, Inc.) (Johe et al. , nineteen ninety six). The cells were plated on plates of tissue culture plates precoated with cell binding factors, as is well known in the art. BFGF was added daily and the medium was changed every two days. The cells are passed to a 50% confluence by brief incubation of them in HBSS and scraping them with a cell scraper. Alternatively, the neuronal progenitor and pluripotent cells were isolated from the dissociated cell mass by antibody-mediated cell capture ("apanelation", Barres et al., Cell 70: 31-46, 1992). Antibodies that were used to isolate the neuronal precursor and pluripotent cells include, but are not limited to, nestin antibodies (Vicario-Abejón et al., 1995). The cells were subsequently treated as indicated above. The neuronal progenitor and pluripotent cells were exposed to the active agents, as described above can be used for neuron replacement therapy, to treat diseases including, but not limited to, Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Cells were cultured in vitro or ex vivo as described above. The cells were rinsed to remove all traces of the culture fluid, resuspended in an appropriate medium and subsequently granulated and rinsed several times. After final rinsing, the cells were resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and used for transplantation according to the methods described above. (Kordower et al., 1995; Freed et al., N. Engl. J. Med. 327: 1549-1555, 1992; Ann. Neurol. 31: 155-165, 1992; Peschanski et al., Brain 117: 487-499, 1994; Spencer at al., N. Engl. J. Med. 327: 1541-1548, 1992; Henderson et al., Arch. Neurol. 48: 822-827, 1992; Hitchcock et al., Exp. Neurol. 129: 3, 1994; LindvAII et al., Science 247: 574-577, 1990; Widner et al., N. Engl. J. Med. 327: 1556-1563, 1992; Bankiewicz et al., J. Neurosurg. 72: 231-244, 1990; Kordower et al., Ann. Neurol. 29: 405-412, 1991). In a preferred embodiment, the neuronal progenitor and pluripotent cells used for transplantation were transfected with an expression vector to express a therapeutic protein, including but not limited to glial cell line derived neurotrophic factor (GDNF; Beck et al., Nature 373: 339-341, 1995; Tomac et al., Nature 373: 335-339, 1995) after transplanfe. In a further aspect of the present invention, the effect of the active agents on differentiation of progenitor and neuronal pluripotent cells is determined by examining changes in gene expression, phenotype, morphology or any other method that distinguishes progenitor cells and / or pluripofenses of completely differentiated cells. Examples of such differentiation markers against which antibodies are available, include, but are not limited to, specific neuron-associated microtobular protein 2 (MAP2, Vicario-Abejón et al., 1995; antibody available from Boehringer Manheim, Germany), specific astroglial fibrillary acidic glial protein (GFAP, Vicario-Abejón et al., 1995, antibody available from Incstar); specific neuron tau protein (Johe et al., 1996; antibody available from Sigma, St. Louis, MO); neurofilaments L and m (Johe et al., 1996; antibody available from Noehringer Manheim) and specific oligondendrocyte 04 and galactocerebroside (GalC; Johe et al., 1996). The DNA sequences for all these specific differentiation markers are known and therefore PCR amplification and / or hybridization studies for evaluating gene expression of differentiation markers can be performed in accordance with standard methods in the art. (Johe ef al., 1996). In a preferred embodiment, neuronal progenitor and pluripotent cells were isolated and cultured as described above. Differentiation was initiated by contacting the cells with the active agents as described above, in a serum-free medium in the absence of bFGF. Differentiation was determined at various times by immunodetection of specific differentiation markers, using the antibodies described above (Johe et al., 1996). Alternatively, the differentiation was determined morphologically by measurement of the neurite result. In another aspect of the present invention the active agents are used to increase in vivo neuronal progenitor and pluripotential cell proliferation.

For use in increasing the proliferation of neuronal progenitor and pluripotent cells, active agents can be administered by any appropriate route, including orally, parenterally, by inhalation spray, rectally or topically in unit dose formulations containing conventional carriers, adjuvants and vehicles. pharmaceutically acceptable The term "parenteral" as used herein includes the subcutaneous, intravenous, intra-arterial, intra-ventricular, intramuscular, intra-tracheal, intrandinous, intra-spinal, intracranial, intrathoracic, infusion or intraperitoneally routes. The active agents can be manufactured in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions or emulsions) and can be subjected to conventional pharmaceutical operations such as sterilization and / or they can contain conventional adjuvants , such as preservatives, stabilizers, wetting agents, emulsifiers, regulators, etc. While the active agents can be administered as a single active agent, they can also be used in combination with one or more other compounds. When administered as a combination, the active agents and other compounds can be formulated as separate compositions that occur at the same time or at different times or the active agents and other compounds can be given as a single composition. For administration, the active agents ordinarily are combined with one or more adjuvants appropriate for the indicated route of administration. The compounds can be mixed with lactose, sucrose, starch powder, cellulose esters or alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide and sodium and calcium salts of sulfuric and phosphoric acids, acacia, gelatin, sodium alginate. , polyvinylpyrrolidine and / or polyvinyl alcohol and are tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention can be dissolved in saline, water, polyethylene glycol, propylene glycol, colloidal solutions of carboxymethyl cellulose, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum and / or several stabilizers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include a retarding material such as glyceryl monostearate or glyceryl distearate alone or with a wax or other materials well known in the art. Formulations suitable for topical administration include liquid or semi-liquid preparations for penetration through the skin (eg, liniments, lotions, ointments, creams or pastes) and appropriate drops for administration to the eye, ear or nose. The dose regimen for increased proliferation or in vivo differentiation of the neuronal progenitor or pluripotent cell with the acive agents of the invention is based on a variety of factors, including the type of injury or deficiency, age, weight , the sex, the medical condition of the individual, the severity of the condition, the route of administration and the particular compound used. Therefore, the dosage regimen can vary widely but can be routinely determined by a physician using standard methods. Dosage levels in the order of between 0.1 ng / kg and 10 mg / kg of angiotensinogen, Al, Al analogues, Al fragments and analogs thereof, All, All analogues, All fragments and analogues thereof and / or agonists of the same. Type 2 All AT2 receptor by body weight are useful for all methods of use described in this document. In a preferred embodiment of the present invention, the active agents are administered by unilateral infusion directly into the lateral ventricle of the mammalian brain using an osmotic pump (Alza Palo Alto, CA) linked to a 30 gauge cannula implanted at the co-ordinate of the injection, as described in Craig et al., J. of Neuroscience 16: 2649-2658 (1996). An appropriate injected dose of the active ingredient of the active agents is preferably about 0.1 ng / kg and about 10 mg / kg administered twice a day. The active ingredient can comprise from 0.001% to 10% w / w, for example, from 1% to 2% by weight of the formulation, although it can comprise as much as 10% w / w, but preferably not more than 5% enp / p and more preferably not more than 5% w / w and more preferably from 0.1% to 1% of the formulation. In another aspect of the present invention, an improved cell culture medium is provided for the proliferation and differentiation of neuronal cells, wherein the improvement comprises adding to the cell culture medium an effective amount of the active agents, as described above. . Any cell culture medium that supports the growth of neuronal progenitor and pluripotent cells can be used with the present invention. Said cell culture medium includes, but is not limited to, Eagle Basal Medium, Dulbecco's Modified Eagle Medium, Iscove Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Optically Reduced Serum Medium. MEM®, RPMI Medium and Macrophage Medium-SFM or combinations thereof. The improved cell culture medium can be provided in a concentrated or non-concentrated form (for example: 10X) and can be provided as a liquid, a powder or a lyophilisate. The cell culture can be chemically defined or it can contain a serum supplement. The culture medium is commercially available from many sources, such as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO). In a further aspect, the present invention provides equipment for the propagation of neuronal progenitor and pluripotent cells, wherein the equipment comprises an effective amount of the active agents described above. In a preferred embodiment, the kit comprises a cell growth culture medium. Any cell culture medium that can support the growth of neuronal progenitor and pluripotent cells can be used with the present invention. Examples of said cell culture media are described above. The improved cell culture medium can be provided in a concentrated or non-concentrated form (for example: 10X) and can be provided as a liquid, a powder or a lyophilisate. The culinary medium can be defined chemically or it can contain a serum supplement. In another preferred embodiment, the equipment further comprises a sterile container. The sterile container may comprise a sealed container, such as a cell culture flask, an expansion pack or a centrifuge tube or an unsealed container, such as a cell culture dish or chemical microconcentration dish (Nunc).; Naperville, IL). In a preferred embodiment, the kit further comprises an antibiotic supplement by inclusion in the reconstituted cell growth medium. Examples of suitable antibiotic supplements include, but are not limited to, actimonicin D, Fungizone®, kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO). The present invention can be better understood with reference to the accompanying examples which are for illustrative purposes only and should not limit the scope of the invention, as defined by the appended claims.

Example 1. Effect of All on the Prolation and Differentiation of Normal Human Neuronal Progenitor Cells Human progenitor cells were obtained from Clonetics (San Diego, CA) and cultured in a Neural Progenitor Cell Maintenance Medium (NPMM) (Neural Progenitor Basal Medium containing the beta factor of human recombinant fibroblast growth, human recombinant epidermal growth factor, neural survival factors, gentamicin and amphotericin B), The cells were thawed, diluted in NPMM and cultured for 24 hours in a 75 cm2 flask. The cells were cultured in dedifferentiated spheroids until studies were carried out to determine the differentiation. When the cells were cultured in a suspension culture in the presence of 1, 10 or 100 μg / ml All for 4-7 days before placing the cells in a culture substrate that allowed adhesion and differentiation (as described below) , an increase in the number of cells able to undergo differentiation (ie: prolation) was observed (Table I).

Table I. Effect of All on the Prolation of Human Neuronal Progenitor Cells Determination of Differentiation To determine the differentiation of neuronal cells, the cells were seeded in plates covered with 0.05% polyethyleneimine (PEI) substratum in a borate stabilizing solution. The plates of 96 plates per plate were covered with 0.05 ml of this solution overnight at room temperature. After incubation, the substrate was removed by aspiration, rinsed with sterile water and dried before seeding the cells. After cell culture for 4-7 days in the presence of All (to determine prolation), the cells were washed and placed on coated PEI plates to determine differentiation. Four days after plating, the number of cells experienced differentiation was counted and the neurite result was determined (see Table 1).

In a further study, the effect of All on the differentiation of neuronal progenitor cells was determined. After adhesion to the PEI substrate, the cells stopped prolation and experienced differentiation and neurite outgrowth. One thousand cells were placed in each plate in the presence and absence of various concentrations of All. Four and seven days after the start of the culture, the size of the neurites was determined (by measurement with an ocular micrometer) in the cells undergoing differentiation and the number of cells undergoing differentiation. Cell culture in the presence of All increased the proportion of the neurite result (see Figure 1) and the number of cells undergoing differentiation by approximately 50% (data not shown). These studies demonstrate that exposure to All promotes the prolation and differentiation of normal human neuronal progenitor cells.

Example 2. Effect of All, (AII (1-7) and Ala4-AIII on the prolation of normal human neural progenitors Normal human progenitor cells were obtained from Clonetics (San Diego, CA) and cultured in a Progenitor Cell Maintenance Medium Neuronal (NPMM) (Neural Progenitor basal medium containing beta factor of human recombinant fibroblast growth, human recombinant epidermal growth factor, neural survival factors, gentamicin and amphotericin B) .The cells were thawed, diluted in NPMM and cultured by 24 hours in a 75 cm2 flask: Until the studies to determine the differentiation, the cells were cultured in differentiated spheroids if the cells were cultured in a suspension culture in the presence of All 10 μg / ml (SEQ ID NO: 1) , AII (1-7) (SEQ ID NO) or Ala4-AIII (SEQ ID NO: 18) for 7 days before placing the cells in plates covered with collagen to allow adherence, was observed or an increase in the number of cells capable of experiencing prolation (Figure 2). The increase in the number of human neural progenitors in the plates was 300% in the presence of All, 175% in the presence of Ala4-AIII and 100% in the presence of AII (1-7), while the increase was only 33% in the control plates. These studies show that each of these peptides promoted the proliferation of normal human neuronal progenitor cells. The present invention, through a method to improve the proliferation of neuronal cells will greatly increase the clinical benefits of the progenitor and neuronal pluripotent transplantation. This is true for increased neuronal "self-renewal" pluripotent cells, which will provide a broad supply of pluripotent cells capable of differentiation into various types of neuronal cell and for proliferation with differentiation that will provide a broad supply of differentiated cells and neuronal progenitors in their appropriate site. Similarly, methods that increase in vivo the proliferation of neuronal differentiated, progenitor and pluripotent cells are beneficial in the treatment of many neurological diseases, including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis.

The method of the present invention also increases the potential utility of neuronal progenitor and pluripotent cells as vehicles for gene therapy in diseases of the central and peripheral nervous system by more efficiently providing a large number of cells for transfection and also by more efficient means to rapidly expand the transfected neuronal progenitor and pluripotent cells. The present invention is not limited by the particular preferred embodiments mentioned above. Various modifications may be made to the preferred embodiments without departing from the scope of the invention.

Claims (21)

1. A method for the promotion of neuronal cell proliferation or differentiation comprising contacting the neuronal cells with an effective amount to promote the proliferation or differentiation of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of the group R1-R8 in the sequence of the general formula I R1 -R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, RA is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, RB is selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc, Lys and Tyr; R4 is selected from the group consisting of Tyr, Tyr (P? 3) 2, Thr, Ser, homoSer, Ala and azaTyr; R5 is selected from the group consisting of 'lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding the sequences that include R4 as a terminal Tyr group.

2. The method according to claim 1, wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO. : 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEC ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

3. The method according to claim 1, wherein the active agent is SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 18.

4. The method according to claim 1, wherein the concentration of the active agent is between about 0.1 ng / kg and about 10.0 mg / kg.

5. An improved cell culture medium for the promotion of neuronal cell proliferation or differentiation, wherein the improvement comprises the addition to the cell culture medium of an amount effective to increase the proliferation or differentiation of neuronal cells of at least one active agent that comprises a sequence consisting of at least three contiguous amino acids of the groups R1-R8 in the sequence of the general formula I R1-R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, RA is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly , Asp (NH2) and Suc, RB is selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc, Lys and Tyr; R4 is selected from the group consisting of Tyr, Tyr (P? 3) 2, Thr, Ser, homoSer, Ala and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R4 as a terminal group Tyr.

6. The improved cell culture medium according to claim 5, wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEC ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO : 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

7. The improved cell culture according to claim 5, wherein the active agent is SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 18.

8. The improved cell culture medium according to claim 5, wherein the concentration of the active agent is between about 0.1 ng / ml and about 10.0 mg / ml.

9. An apparatus for the promotion of cell proliferation or differentiation comprising: (a) an amount effective to promote proliferation or cell differentiation of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of the R1 groups -R8 in the sequence of the general formula I R1 -R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, RA is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, RB is selected from Arg, Lys , Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc, Lys and Tyr; R4 is selected from the group consisting of Tyr, Tyr (P03) 2, Thr, Ser, homoSer, Ala and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R4 as a terminal group Tyr and (b) instructions for using the effective amount of the active agent to promote proliferation or neuronal cell differentiation.

10. The kit according to claim 9, wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO. : 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEC ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO : 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

11. The equipment according to claim 9, wherein the active agent is SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 18.

12. The equipment according to claim 9, wherein the concentration of the active agent is between about 0.1 ng / ml and about 10.0 mg / ml.

13. A method for the promotion of neuronal cell proliferation or differentiation comprising contacting the neuronal cells with an effective amount to promote the proliferation or differentiation of at least one active agent comprising a sequence consisting of the general formula: R1-ARG -VAL-TYR-R2-HIS-PRO-R3 wherein R1 is selected from the group consisting of H or Asp; R2 is selected from the group consisting of lie, Val, Leu, norLeu and Ala; R3 is Phe or H.

14. The method according to claim 13, wherein the. active agent is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26 and SEQ ID NO: 34.

15. The method according to claim 13, wherein the concentration of the active agent is between about 0.1 ng / kg and about 10.0 mg / kg.

16. An improved cell culture medium for the promotion of neuronal cell proliferation or differentiation, wherein the improvement comprises adding to the cell culture medium an amount effective to increase the proliferation or differentiation of neuronal cells of at least one active agent comprising a sequence consisting of the general formula I R1-ARG-VAL-TYR-R2-HIS-PRO-R3 wherein R1 is selected from the group consisting of H or Asp; R2 is selected from the group consisting of lie, Val, Leu, norLeu and To; R3 is Phe or H.

17. The improved cell culture medium according to claim 16, wherein the active agent is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 ,. SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26 and SEQ ID NO: 34.

18. The improved cell culture medium according to claim 16, wherein the concentration of the active agent is between about 0.1 ng / ml and about 10.0 mg / ml.

19. A device for neuronal cell differentiation or promotion comprising: (a) an amount effective to promote proliferation or neuronal cell differentiation of at least one active agent comprising a sequence of the general formula: R1-ARG-VAL-TYR -R2-HIS-PRO-R3 wherein R1 is selected from the group consisting of H or Asp; R2 is selected from the group consisting of lie, Val, Leu, norLeu and Ala; R3 is Phe or H and (b). instructions for using the effective amount of the active agent to promote proliferation or neuronal cell differentiation.

20. The kit according to claim 18, wherein the active agent is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 26 and SEQ ID NO: 34.

21. The equipment according to claim 18, wherein the concentration of the active agent is between about 0.1 ng / ml and about 10.0 mg / ml.