This invention relates to a method of selectively producing neural cells, including neurons or glial cells, in vitro or in vivo. Also provided are methods of treating or ameliorating neurodegenerative disease or medical conditions by producing neural cells. Thus, a combination of factors is used to achieve two steps: increasing the number of neural stem cells and instructing the neural stem cells to selectively become neurons or glial cells.

Description of the Invention

SUMMARY OF THE INVENTION

This invention relates to a two-step method of producing neural cells in vitro or in vivo. We discovered that neurogenesis and gliogenesis by multipotent neural stem cells (NSCs) involve proliferation and directed differentiation. As shown in FIG. 1 (see Original Patent), EGF (or its adult homolog TGF.alpha.) induces the self-renewal/expansion of the NSC population. The NSCs will undergo-spontaneous differentiation in a default pathway to become glial precursor cells (GPCs). This spontaneous differentiation can be attenuated by ciliary neurotrophic factor (CNTF). GPCs will differentiate into the glial cells, which differentiation is promoted by EGF. Alternatively, NSCs can be instructed by EPO and/or PACAP/cAMP to differentiate to neuronal precursor cells (NPCs), which make neurons only.

Therefore, a two-step process can be used to produce neurons or glial cells: (1) increasing the number of NSCs; and (2) promoting differentiation of the NSCs to either neurons or glial cells by subjecting the NSCs to appropriate conditions which selectively promotes production of neurons or glial cells.

Accordingly, one aspect of the present invention provides a method for producing neuronal precursor cells or glial precursor cells, comprising: (a) providing at least one neural stem cell; (b) contacting the neural stem cell with a factor selected from the group consisting of prolactin, growth hormone, estrogen, ciliary neurotrophic factor (CNTF), pituitary adenylate cyclase activating polypeptide (PACAP), fibroblast growth factor (FGF), transforming growth factor alpha (TGF.alpha.) and epidermal growth factor (EGF) in an amount sufficient to increase the number of neural stem cells; and (c) contacting the neural stem cells from step (b) to a factor selected from the group consisting of erythropoietin (EPO), PACAP, prolactin, serotonin, bone morphogenetic protein (BMP) and cAMP in an amount sufficient to enhance the production of neuronal precursor cells or glial precursor cells from the neural stem cells; with the proviso that when the factor in step (b) is EGF or FGF, the factor in step (c) is PACAP or prolactin.

Thus, step (b) is performed to increase the number of neural stem cells, which can be achieved by at least one of the following: (i) increasing proliferation of the neural stem cell, such as by providing EGF; (ii) inhibiting spontaneous differentiation of the neural stem cell, such as by providing CNTF; or (iii) promoting survival of the neural stem cell, such as by providing an estrogen.

These two steps, increasing NSCs numbers and enhancing neuron or glia production, may be performed sequentially or concurrently. It is preferable that step (b) is performed prior to step (c).

The factors can be provided by any method established in the art. For example, they can be administered intravascularly, intrathecally, intravenously, intramuscularly, subcutaneously, intraperitoneally, topically, orally, rectally, vaginally, nasally, by inhalation or into the brain. The administration is preferably performed systemically, particularly by subcutaneous administration. The factors can also be provided by administering to the mammal an effective amount of an agent that can increase the amount of endogenous factors in the mammal. For example, the level of prolactin in an animal can be increased by using prolactin releasing peptide.

When the factors are not directly delivered into the brain, a blood brain barrier permeabilizer can be optionally included to facilitate entry into the brain. Blood brain barrier permeabilizers are known in the art and include, by way of example, bradykinin and the bradykinin agonists described in U.S. Pat. Nos. 5,686,416; 5,506,206 and 5,268,164 (such as NH.sub.2-arginine-proline-hydroxyproxyproline-glycine-thienylalanine-seri- ne-proline-4-Me-tyrosine.psi.(CH.sub.2NH)-arginine-COOH). Alternatively, the factors can be conjugated to the transferrin receptor antibodies as described in U.S. Pat. Nos. 6,329,508; 6,015,555; 5,833,988 or 5,527,527. The factors can also be delivered as a fusion protein comprising the factor and a ligand that is reactive with a brain capillary endothelial cell receptor, such as the transferrin receptor (see, e.g., U.S. Pat. No. 5,977,307).

Although mammals of all ages can be subjected to this method, it is preferable that the mammal is not an embryo. More preferably, the mammal is an adult.

The mammal may suffer from or be suspected of having a neurodegenerative disease or condition. The disease or condition may be a brain injury, such as stroke or an injury caused by a brain surgery. The disease or condition may be aging, which is associated with a significant reduction in the number of neural stem cells. The disease or condition can also be a neurodegenerative disease, particularly Alzheimer's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, or Parkinson's disease.

Alternatively, the neural stem cell may be in a culture in vitro. The cell may be from an animal of any age. Preferably, the animal is not an embryo, and most preferably the animal is an adult.

Another aspect of the present invention provides a method of treating or ameliorating a neurodegenerative disease or medical condition, comprising (a) administering to a mammal a factor which is capable of increasing the number of neural stem cells; and (b) subjecting the mammal to a condition which enhances the production of a lineage restricted cell; whereby production of the lineage restricted cell is enhanced. For example, neurons can be produced to compensate for lost or malfunctioning neurons by administering EGF and EPO. Other factors which are capable of increasing the number of NSCs, such as CNTF, FGF, prolactin, growth hormone, IGF-1, PACAP or estrogen, can also be used instead of EGF or in addition to EGF. Likewise, other factors which can enhance neuron production, such as PACAP or factors which increases cAMP level, can be used in the place of EPO or in addition to EPO.

To produce glial cells to compensate for lost or malfunctioning glial cells, EGF can be administered, which stimulates NSC proliferation, and the resulting NSC will differentiate to glial cells by default. Optionally, inhibitors of the neuronal pathway, such as antibodies of EPO and cAMP signaling inhibitors, can be used to promote glial production. Preferably, a factor that promotes glial formation, such as BMP, is also used to further produce glial cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a method of selectively producing neural cells, including neurons or glial cells, in vitro or in vivo. Also provided are methods of treating or ameliorating neurodegenerative disease or medical conditions by producing neural cells. Thus, a combination of factors is used to achieve two steps: increasing the number of neural stem cells and instructing the neural stem cells to selectively become neurons or glial cells.

Methods

Neural stem cells (NSCs), such as the ones found in the adult forebrain, are the likely source of restricted neuronal and glial progenitors, which repopulate structures such as the olfactory bulb and corpus callosum, respectively. The mechanisms by which NSCs give rise to restricted progenitors have been unclear prior to this invention.

We found that EGF-responsive NSCs gradually become restricted to a glial lineage. This process is blocked by CNTF, acting through notchl, to maintain NSCs in a multipotent stage. We also found that erythropoietin (EPO) directs the production of restricted neuronal precursors through a mechanism that utilizes Mash1.

Thus, we infused either CNTF or EPO into the lateral ventricles of adult mice for six days, after which we either removed the entire adult ependyma/subependyma to examine the total number of EGF-responsive NSCs or examined the in vivo production of neuronal precursors. CNTF infusion resulted in a 20-25% increase in the number of NSCs, most likely by preventing NSC differentiation into glial precursors. EPO infusion resulted in a 50% reduction in the number of NSCs and a concomitant doubling in neuronal precursors. Infusion of anti-EPO antibodies resulted in a 20% increase in NSCs. Therefore, EGF-responsive NSCs turn over continuously in vivo, a sub-population of which spontaneously differentiates into restricted glial precursors, while another sub-population is directed to the neuronal restricted linage by EPO.

This mechanism is illustrated in FIG. 1 (see Original Patent). Thus, EGF (or its adult homolog TGF.alpha.) induces the self-renewal/expansion of the NSC population. The NSCs undergo spontaneous differentiation as a default pathway to become glial precursor cells (GPCs), which differentiate into glial cells. This spontaneous differentiation can be attenuated by CNTF. Alternatively, NSCs can be instructed by EPO and/or PACAP/cAMP to differentiate to neuronal precursor cells (NPCs), which make neurons only.

Based on this mechanism, we developed a two-step method to produce neural cells. The first step is to increase the number of neural stem cells, which can be achieved by, for example, proliferating neural stem cells (e.g., by EGF, FGF-1, FGF-2, TGF.alpha., estrogen, prolactin, PACAP, growth hormone, and/or IGF-1), inhibiting spontaneous differentiation of neural stem cells (e.g., by CNTF), and/or promoting survival of neural stem cells (e.g., by estrogen). The second step is to enhance neuronal or glial formation from neural stem cells. For example, erythropoietin, prolactin, serotonin, PACAP and/or cyclic AMP can be used to enhance neuron formation, while bone morphogenetic protein (BMP) can be used to enhance glial formation.

The present method can be used in vivo or in vitro. In vitro, the present invention will result in large quantities of neural cells, which can be used in research or therapeutical purposes. In particular, the neural cells can be used in transplantation treatment for neurodegenerative diseases or conditions. In vivo, the present method can increase the number of neural stem cells in situ, and enhance neuronal or glial formation from the enlarged pool of neural stem cells. The resulting neural cells can migrate to appropriate places in the nervous system to enhance neurological functions, or compensate for lost or dysfunctional neural cells. In addition, the in vivo and in vitro applications can be combined. Thus, neural cells, particularly neural stem cells produced by the present method in vitro, can be transplanted into an animal, and factors of the second step can be provided to the animal to enhance differentiation of neural cells in vivo. Optionally, factors of the first step may be provided to the animal as well to further increase the number of neural stem cells that can be subsequently turned to neurons or glial cells.

One particularly interesting neurodegenerative condition is aging. We have found that the number of neural stem cells in the subventricular zone is significantly reduced in aged mice. Accordingly, it will be of particular interest to ameliorate problems associated with aging by the present invention.

In addition, the neural stem cell in the subventricular zone is the source of olfactory neurons, and olfactory dysfunction is a hallmark of forebrain neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington's diseases. Disruption of neuronal migration to the olfactory bulb leads to deficits in olfactory discrimination, and doubling the new olfactory interneuons enhances new odor memory (Rochefort et al., 2002). Therefore, the present invention can be used to enhance olfactory discrimination or olfactory memory, as well as physiological functions that are associated with olfaction and olfactory discrimination, such as mating, offspring recognition and rearing.

Another particularly important application of the present invention is the treatment and/or amelioration of brain injuries, such as stroke (Example 2). A brain injury mimicking a stroke was introduced into the motor cortex of rats, and the injured rats showed abnormal behavioral conducts that correlated with the location of the injury. The rats then received prolactin or growth hormone for 7 days, both of which can increase neural stem cell proliferation. Subsequently, the rats received a vehicle control or erythropoietin for 7 days to enhance neuron formation. The rats were then observed for a period of time for behavioral testing, and sacrificed for anatomical analysis.

The results indicate that both prolactin and growth hormone treatments led to an improvement of motor functions in the injured rats. The addition of erythropoietin further enhanced the effect, particularly when combined with prolactin. The anatomical analysis also shows that the number of migrating neurons and/or neural stem cells was increased by every treatment comprising prolactin or growth hormone. In fact, the combination of prolactin and erythropoietin even resulted in complete or partial filling of the cavities created by the brain injury in a majority of the rats. Therefore, these factors, particular combinations of which, can be used to produce neural cells and restore neurological functions in animals with brain injuries.

An intriguing observation is that prolactin and growth hormone led to the restoration of different behavioral functions. Thus, the rats recovered from asymmetrical forelimb usage in balancing after receiving growth hormone, while prolactin acted to correct abnormal positioning of the forelimb during swimming. Therefore, different factors may lead to different cellular migration patterns or the production of different cells, which participate in different neural functions. Accordingly, it is preferable that multiple factors are combined in the treatment of diseases or conditions that have complicated symptoms. Preferred combinations include: (a) prolactin and at least one factor that enhances neuronal or glial differentiation, such as EPO, PACAP, cyclic AMP and/or BMP; (b) EGF and at least one factor that enhances neuronal or glial differentiation, such as prolactin, EPO, PACAP, cyclic AMP and/or BMP, particularly prolactin and/or PACAP; (c) at least one factor that increases neural stem cell number in conjunction with prolactin; (d) at least one factor that increases neural stem cell number in conjunction with PACAP; (e) at least one factor that increases neural stem cell number in conjunction with EPO; and (f) combinations of the above.

Particularly preferred combinations include EGF and EPO, EGF and prolactin, EGF and PACAP, EGF and growth hormone (and/or IGF-1), EGF and prolactin and growth hormone (and/or IGF-1), EGF and prolactin and PACAP, prolactin and growth hormone (and/or IGF-1), prolactin and growth hormone (and/or IGF-1) and EPO, prolactin and PACAP and growth hormone (and/or IGF-1). Most preferred combinations include EGF and PACAP, EGF and prolactin, and prolactin and PACAP. Preferably, FGF is not used.

Compositions

The present invention provides compositions comprising at least one factor that is capable of increasing neural stem cell numbers and at least one factor that is capable of enhancing differentiation of neural stem cells. It should be noted that some factors are capable of both functions, such prolactin. PACAP, in addition to enhancing neuronal differentiation, also enhances proliferation of neural stem cells in the presence of another mitogen.

The factors that are useful in the present invention include their analogs and variants that share a substantial similarity and at least one biological activity with the native factors. For example, although the major form of prolactin found in the pituitary gland has a molecular weight of 23 kDa, variants of prolactin have been characterized in many mammals, including humans. Prolactin variants can result from alternative splicing of the primary transcript, proteolytic cleavage and other post-translational modifications. A prolactin variant of 137 amino acids has been described in the anterior pituitary, which is likely to be a product of alternative splicing. A variety of proteolytic products of prolactin have been characterized, particularly the 14-, 16- and 22-kDa prolactin variants, all of which appear to be prolactin fragments truncated at the C-terminus. Other post-translational modification reported for prolactin include dimerization, polymerization, phosphorylation, glycosylation, sulfation and deamidation.

The prolactin useful in the present invention includes any prolactin analog, variant or prolactin-related protein which is capable of increasing neural stem cell number. A prolactin analog or variant is a polypeptide which contains at least about 30% of the amino acid sequence of the native human prolactin, and which possesses a biological activity of prolactin. Preferably, the biological activity of prolactin is the ability to bind prolactin receptors. Although several isoforms of the prolactin receptor have been isolated, for example the long, intermediate and short forms in rat, the isoforms share the same extracellular domain which binds prolactin. Therefore, any receptor isoform can be used to assay for prolactin binding activity. Specifically included as prolactins are the naturally occurring prolactin variants, prolactin-related protein, placental lactogens, S179D-human prolactin (Bernichtein et al., 2001), prolactins from various mammalian species, including but not limited to, human, other primates, rat, mouse, sheep, pig, and cattle, and the prolactin mutants described in U.S. Pat. Nos. 6,429,186 and 5,955,346.

Similarly, in addition to native EGF, an EGF analog or variant can also be used, which should share a substantial amino acid sequence similarity with the native EGF, as well as at least one biological activity with the native EGF, such as binding to the EGF receptor. Particularly included as an EGF is the native EGF of any species, TGF.alpha., or recombinant modified EGF. Specific examples include, but are not limited to, the recombinant modified EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51 (particularly EGF51gln51; U.S. Patent Application Publication No. 20020098178A1), the EGF mutein (EGF-X.sub.16) in which the His residue at position 16 is replaced with a neutral or acidic amino acid (U.S. Pat. No. 6,191,106), the 52-amino acid deletion mutant of EGF which lacks the amino terminal residue of the native EGF (EGF-D), the EGF deletion mutant in which the N-terminal residue as well as the two C-terminal residues (Arg-Leu) are deleted (EGF-B), the EGF-D in which the Met residue at position 21 is oxidized (EGF-C), the EGF-B in which the Met residue at position 21 is oxidized (EGF-A), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin, neuregulin, or a fusion protein comprising any of the above. Other useful EGF analogs or variants are described in U.S. Patent Application Publication No. 20020098178A1, and U.S. Pat. Nos. 6,191,106 and 5,547,935.

As another example, useful PACAP analogs and variants include, without being limited to, the 38 amino acid and the 27 amino acid variants of PACAP (PACAP38 and PACAP27, respectively), and the analogs and variants disclosed in, e.g., U.S. Pat. Nos. 5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,623,050; 5,801,147 and 6,242,563.

Erythropoietin analogs and variants are disclosed, for example, in U.S. Pat. Nos. 6,048,971 and 5,614,184.

Further contemplated in the present invention are functional agonists of prolactin or additional factors useful in the present invention. These functional agonists bind to and activate the receptor of the native factor, although they do not necessarily share a substantial sequence similarity with the native factor. For example, maxadilan is a polypeptide that acts as a specific agonist of the PACAP type-1 receptor (Moro et al., 1997).

Functional agonists of EPO have been extensively studied. EMP1 (EPO mimetic peptide 1) is one of the EPO mimetics described in Johnson et al., 2000. Short peptide mimetics of EPO are described in, e.g., Wrighton et al., 1996 and U.S. Pat. No. 5,773,569. Small molecular EPO mimetics are disclosed in, e.g., Kaushansky, 2001. Antibodies that activate the EPO receptor are described in, e.g., U.S. Pat. No. 5,885,574; WO 96/40231 and WO 97/48729).

Antibodies that have agonist activities for the EGF receptor are described, e.g., in Fernandez-Pol, 1985 and U.S. Pat. No. 5,723,115. In addition, activating amino acid sequences are also disclosed in U.S. Pat. No. 6,333,031 for the EPO receptor, EGF receptor, prolactin receptor and many other cell surface receptors; metal complexed receptor ligands with agonist activities for the prolactin and EPO receptors can be found in U.S. Pat. No. 6,413,952. Other methods of identifying and preparing ligands for receptors, e.g., EPO and prolactin receptors, are described, for example, in U.S. Pat. Nos. 5,506,107 and 5,837,460.

It should be noted that the effective amount of each analog, variant or functional agonist may be different from that for the native factor or compound, and the effective amount in each case can be determined by a person of ordinary skill in the art according to the disclosure herein. Preferably, the native factors, or analogs and variants that share substantial sequence similarity with the native factors, are used in the present invention.

Pharmaceutical compositions are also provided, comprising the factors as described above, and a pharmaceutically acceptable excipient and/or carrier.

The pharmaceutical compositions can be delivered via any route known in the art, such as parenterally, intrathecally, intravascularly, intravenously, intramuscularly, transdermally, intradermally, subcutaneously, intranasally, topically, orally, rectally, vaginally, pulmonarily or intraperitoneally. Preferably, the composition is delivered into the central nervous system by injection or infusion. More preferably it is delivered into a ventricle of the brain, particularly the lateral ventricle. Alternatively, the composition is preferably delivered by systemic routes, such as subcutaneous administration. For example, we have discovered that prolactin, growth hormone, IGF-1, PACAP and EPO can be effectively delivered by subcutaneous administration to modulate the number of neural stem cells in the subventricular zone.

When the composition is not directly delivered into the brain, and factors in the composition do not readily cross the blood brain barrier, a blood brain barrier permeabilizer can be optionally included to facilitate entry into the brain. Blood brain barrier permeabilizers are known in the art and include, by way of example, bradykinin and the bradykinin agonists described in U.S. Pat. Nos. 5,686,416; 5,506,206 and 5,268,164 (such as NH.sub.2-arginine-proline-hydroxyproxyproline-glycine-thienylala- nine-serine-proline-4-Me-tyrosine.psi.(CH.sub.2NH)-arginine-COOH). Alternatively, the factors can be conjugated to the transferrin receptor antibodies as described in U.S. Pat. No. 6,329,508; 6,015,555; 5,833,988 or 5,527,527. The factors can also be delivered as a fusion protein comprising the factor and a ligand that is reactive with a brain capillary endothelial cell receptor, such as the transferrin receptor (see, e.g., U.S. Pat. No. 5,977,307).

The pharmaceutical compositions can be prepared by mixing the desired therapeutic agents with an appropriate vehicle suitable for the intended route of administration. In making the pharmaceutical compositions of this invention, the therapeutic agents are usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the pharmaceutically acceptable excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the therapeutic agent. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the therapeutic agents, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable excipients include artificial cerebral spinal fluid, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the therapeutic agents after administration to the patient by employing procedures known in the art.

For preparing solid compositions such as tablets, the therapeutic agent is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the therapeutic agents are dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. The compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

Another formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the therapeutic agent of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences.
 

Claim 1 of 30 Claims

1. A method for producing neuronal precursor cells or glial precursor cells, comprising: (a) providing at least one neural stem cell; (b) contacting the neural stem cell with prolactin in an amount sufficient to increase the number of neural stem cells; and (c) contacting the neural stem cells from step (b) with at least one additional factor selected from the group consisting of erythropoietin (EPO), pituitary adenylate cyclase activating polypeptide (PACAP), serotonin, bone morphogenetic protein (BMP) and cAMP in an amount sufficient to enhance the formation of neuronal precursor cells or glial precursor cells from the neural stem cells.

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