WO2007016099A2 - Protection of transplanted stem cells with hmg-coa reductase inhibitors - Google Patents

Abstract

Impaired remyeliiiation due to degeneration of both postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) is the major hallmark of inflammatory demyelination in multiple sclerosis (MS) lesions and other inflammatory diseases. HMG-CoA reductase inhibitors also are proposed to stimulate and protect stems cells, such as OPs, for example in the context of transplantation.

Description

DESCRIPTION

PROTECTION OF TRANSPLANTED STEM CELLS WITH HMG-COA

REDUCTASE INHIBITORS

CROSS REFERNECE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/704,129, filed July 29, 2005, the contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

A. Field of the Invention The present invention relates generally to the field of immunology and cell biology. More particularly, it concerns the use of HMG-CoA Reductase inhibitors to protect and expand stem cell populations.

B. Description of Related Art

1. Stem Cells and Pluripotent Progenitors A stem cell is a cell that has the ability to divide (self replicate) for indefinite periods — often throughout the life of the organism. Under the right conditions, or given the right signals, stem cells can give rise (differentiate) to the many different cell types that make up the organism. That is, stem cells have the potential to develop into mature cells that have characteristic shapes and specialized functions, such as heart cells, skin cells, or nerve cells. Not surprisingly, there has been considerable interest in the use of stem cells in various replacement therapies, for example, in neurodegenerative, cardiac and diseases of the immune system. However, one limitation on the use of such cells is their maintenance, expansion and propagation ex vivo. Human embryonic cells were first successfully derived using mouse embryonic fibroblast feeder cells and serum-containing medium late in the last decade, and that culture method that has since been widely used since. One group described the derivation of a human embryonic stem ("hES") cell line from a blastocyst developed through somatic cell nuclear transfer (Hwang et al, 2004), which may prove a useful technique in the generation of histocompatible ES cell lines. Technical advances have partially overcome some of the limitations of the original systems for culturing hES cells, such as the spontaneous differentiation of the cells and the need to mechanically dissect ES colonies for subculture. For example, a serum-free system based on combining a serum substitute and FGF2 enables the propagation of cultures with a higher proportion of stem cells. This system removes the need to mechanically isolate stem cells, which can instead be dissociated enzymatically (Amit et al, 2000). Though this technique has been widely adapted for the growth of hES cells, it has only modestly improved the cloning efficiency of hES cells.

Another modification involves replacing the feeder cell component with Matrigel, an extracellular matrix (ECM) preparation, and conditioned medium from the feeder cell layer (Xu et al, 2001). This system enables the long-term maintenance of the stem cell phenotype, with strong suppression of the spontaneous differentiation observed at high passage levels (Carpenter et al, 2004). Amit et al. (2004) reported that the combination of FGF2, TGFβ, LIF and a serum replacer can achieve serum- free, feeder- free maintenance of hES cells on a fibronectin ECM. Another report also suggests that Wnt signaling modulation can support the short-term maintenance of some stem cell markers in hES cell cultures in the absence of a feeder cell layer (Sato et al., 2004). However, ongoing concerns regarding the maintenance of key properties of pluripotent stem cells are maintained and karyotypic altering of cells (Draper et al, 2004) continue to hamper these advances. To date, no culture method enables high- efficiency clonal propagation of hES cells.

2. Inflammatory Disease

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) characterized by inflammation, gliosis, demyelination, and loss of neuronal axons and oligodendrocytes (Brack et al, 2003). In recent studies, investigators have concluded that the developing lesions in the MS brain are heterogeneous in nature (Lassmann, 2001; Lucchinetti, 2000). Generally, the infiltrating cells are distributed throughout the developing acute lesions, in contrast to being restricted to the edges in clinically silent lesions. These lesions also differ in deposition of immunologlobulin and activated complement molecules, degeneration of distal oligodendrocytes followed by demyelination, and loss of neuronal axons which correlates with permanent functional deficit (Lassmann, 2001; Lucchinetti, 2000). Overall, these studies suggest the existence of multiple mechanisms for both deniyelination and remyelination processes in MS lesions which are responsible for the observed heterogeneity in MS pathology.

The concept of remyelination is of interest since naturally occuπing remyelination is impaired in MS lesions. Active MS lesions are usually remyelinated by existing postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) unless this process is impaired by recurring demyelinating episodes. The remyelination process is completely disrupted in silent demyelinated lesions in MS due to the lack of postmitotic oligodendrocytes and OPs in the lesions except in the border region between the plaque and peri-plaque white matter (Prineas et ah, 1993; Mews et ah, 1998). Remyelination helps to preserve axons, restore conduction velocity, and clinically silence the MS lesions. This can be achieved either by promoting endogenous repair mechanisms or by providing an exogenous source of myelinating cells. Current therapies are essentially targeted to promote CNS repair which include application of growth factors (Carson et ah, 1993; Althaus, 2004), intravenous administration of remyelinating immunoglobulin auto-antibodies (Sorensen, 2003), and the transplantation of OPs or embryonic stem cells (Pluchino et ah, 2003; Brustle et ah, 1999). These therapies have potential to induce remyelination in animal models and clinical trials are currently underway on MS patients.

The recruitment of OPs to demyelinating lesions is critical for the induction of remyelination, which requires proliferation followed by recruitment and differentiation into myelinating matured oligodendrocytes (Back et ah, 2001; Gensert et ah, 1997). Neurotrophic factors such as insulin-like growth factor- 1 (IGF-I), platelet derived growth factor (PDGF), fibroblast growth factor (FGF)-2, glial derived neurotrophic factor (GDNF), neuregulin (glial growth factor-2; GGF-2), and ciliary neurotrophic factor (CNTF) are reported to be important for the proliferation and recruitment of OPs to the demyelinated lesions and subsequent differentiation into matured oligodendrocytes for the purpose of myelinating the demyelinated axons (Carson et ah, 1993; Althaus, 2004; Linker, 2002). Studies using animal models of MS and a human clinical trial suggest the clinical relevance of the HMG-CoA reductase inhibitors, statins, as potential therapeutic agent for the treatment of MS patients; statins attenuate the neuro inflammatory response in the CNS (Youssef et ah, 2002; Paintlia et ah, 2004; Stanislaus et ah, 2001; Vollmer et ah, 2004; Nath et ah, 2004). However, the mechanisms by which statins interfere with the neuro inflammatory response are well established, but the effects on the restoration of remyelination and neurological function remain to be elucidated.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method of protecting implanted stem cells from a recipient immune response comprising administering to a transplant recipient a protective dose of an HMG-CoA reductase inhibitor. The transplant recipient can be a human, animal (e.g., dog, cat, mouse, rat, pig, cow, chicken, etc.). The HMG-CoA reductase inhibitor can be a statin. Non- limiting examples of statins include lovastatin, atorvastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, or cerivastatin. The stem cells can be adult or embryonic stem cells. In certain non-limiting aspects, the stem cells can be hematopoietic, neuronal, endothelial, epithelial, muscle, hepatic, pancreatic, bone marrow derived, lung derived, ear derived, or eye derived stem cells. In other non-limiting aspects, the stem cell can differentiate into a nerve cell, a glial cell, an oligodendrocyte cell, a Schwann cell, an astrocyte, a myocyte, or an islet cell. The transplant recipient, in certain non-limiting aspects, may suffer from a neurodegenerative disease, such as a demyelinating disease like multiple sclerosis or EAE. In other non-limiting embodiments, the method may further comprise administering to said transplant recipient an immunosuppressive drug.

In certain non-limiting aspects, the implanted stem cell can be the transplant recipient's own stem cells. In other aspects, the implanted stem cell is not the transplant recipient's own stem cells. For example, the transplantation procedure can be an autologous transplantation (i.e. the recipient's own cells) or an allogeneic transplantation (not the recipient's own cells).

In other non-limiting embodiments, the HMG-CoA reductase inhibitor can be provided to said transplant recipient prior to transplant, hi certain embodiments, HMG-CoA reductase inhibitor is administered to the recipient, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 90 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48, 72, 96, 154, or more hours prior to transplantation. Alternatively, the HMG-CoA reductase inhibitor can be provided to said transplant recipient in a continuous manner prior to transplant. The HMG-CoA reductase inhibitor can also be provided to said transplant recipient at about the same time as transplant, or following transplant, hi certain embodiments, the HMG-CoA reductase inhibitor is administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 90 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48, 72, 96, 154, or more hours after to transplantation. Alternatively, the HMG-CoA reductase inhibitor can be provided to said transplant recipient in a continuous manner after transplant. The HMG-CoA reductase inhibitor can be provided to said transplant recipient prior to and at the time of transplant, prior to and following transplant, at the time of and following transplant, or prior to, at the time of and following transplant.

In another embodiment of the present invention there is provided a method of expanding a stem cell population comprising contacting a stem cell population with an HMG-CoA reductase inhibitor. The stem cell population can be cultured with the

HMG-CoA reductase inhibitor for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 90 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24,

48, 72, 96, 154, or more hours. The stem cell population can be obtained from a stem cell donor in certain non-limiting aspects. The stem cell population can be transplanted into a transplant recipient in other non-limiting aspects. The transplant recipient can be administered an immunosuppressive drug prior to, during, or after transplantation.

In yet another non-limiting aspect of the present invention, there is disclosed a method of protecting a stem cell in a subject from a disease condition or treatment comprising administering to the subject a protective dose of an HMG-CoA reductase inhibitor. As noted above and throughout this specification, the stem cell can be a non-transplanted stem cell or an implanted stem cell. The implanted stem cell can be an endogenous or exogenous stem cell. In non-limiting aspects, the disease condition can be an oxidative stress condition or an inflammatory condition. It is contemplated the stem cell can be protected by the present invention against any type of oxidative stress or inflammatory disease condition that is known in the art. In certain non- limiting embodiments, for example, the disease treatment can be chemotherapy, radiation therapy, or any drug therapeutic that can have a deleterious effect on a stem cell.

The inventors also contemplate that the methods and compositions disclosed throughout this specification can be used to protect cells that have already differentiated {i.e., differentiated cells). Non-limiting examples include oligodendrocytes, neurons, astrocytes, microglia, schwaan cells and their precursors, pancreatic beta cells and their precursors, myoblasts and myocytes and their precursors, bone marrow derived cells {e.g. chondrocytes and osteoblasts) and their precursors, lung, ear, eye, skin, and organ cells and their precursors, and skin grafts. In still another aspects, there is provided a method of treating, preventing, or attenuating the development of a neurodegenerative disease in a subject comprising administering an HMG-CoA reductase inhibitor to the subject. The HMG-CoA reductase inhibitor can be included in a pharmaceutically acceptable composition and/or can be administered in a therapeutically effective amount. The method can further include determining whether a patient is in need of the prevention or treatment. Determining whether a patient is in need of the prevention or treatment can comprise determining whether a patient is at risk for developing a neurodegenerative disease or condition. Determining whether a patient is at risk for developing a neurodegenerative disease or condition can include taking a family history or a patient history.

There is also provided a method of remyelination of a nerve cell in a subject through enhancing survival and/or differentiation of postmitotic oligodendrocytes and/or oligodendrocyte progenitor cells comprising administering an HMG-CoA reductase inhibitor to the subject.

It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention. "Analogs" may include structural equivalents or mimetics.

A "patient" or "subject" may be an animal. Preferred animals are mammals, including but not limited to humans, pigs, cats, dogs, rodents, horses, cattle, sheep, goats and cows. Preferred patients and subjects are humans.

The terms "inhibiting," "reducing," "treating," "prevention," "protective," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1E - Lovastatin treatment attenuates myelin breakdown and facilitates its restoration. Spinal cord homogenates were analyzed for myelin lipids associated with the demyelination/ remyelination process. The percentage change in the ratio of lipid and protein/group was computed and plotted. The levels of cholesterol ester (FIG. IA), cerebrosides (non-hydroxy and hydroxy) (FIG. IB), sulfatides (non-hydroxy and hydroxy) (FIG. 1C), sphingomyelin (FIG. ID) and cholesterol (FIG. IE) are shown in EAE, lovastatin-treated EAE (E+LOV), lovastatin-treated control (C+LOV) and normal control (CON) groups. The results are expressed as Mean ± SD for n= 3-5/group in three independent experiments. The asterisks denote ** p<0.01, *** pO.OOl and NS (non-significant) versus control, and # p<0.05 and ### pO.OOl versus EAE. FIGS. 2A-2J - Lovastatin treatment enhanced the survival and differentiation of oligodendrocytes in the spinal cord of EAE animals. The expression of myelin proteins and mRNA associated with differentiating oligodendrocytes was observed in the spinal cord of animals in each group. A representative immunoblot demonstrates the expression of MBP and CNPase including β-actin (FIG. 2A). The representative sections of the spinal cord were immunostained with anti-MBP (left panel) and anti-CNPase (right panel) antibodies as described under 'Materials & Methods' (FIG. 2B). The weak immunofluorescence in demyelinated regions in the white matter is indicated (arrowheads) in EAE and E+LOV at a magnification of 40Ox. The quantification of immunofluorescence for MBP and CNPase in immunostained sections was performed as described under 'Materials & Methods' (FIG. 2C). No demyelinated regions were observed in the spinal cord of animals from control (CON), lovastatin-treated control (C+LOV) or recovered E+LOV/R groups. Real-time PCR™ analysis demonstrated the mRNA expression of myelin-protein genes, i.e., MBP (FIG. 2D), PLP (FIG. 2E), MOG (FIG. 2F), and MAG (FIG. 2G) in each group as described under 'Materials & Methods'. Similarly, the mRNA expression of transcription factors, i.e., MyTl-L (FIG. 2H), GTX (FIG. 21), and PPAR-δ (FIG. 2J) was analyzed in each group by real-time PCR™ analyses. Data are expressed as mean + SD of three independent experiments run in triplicate each time. The asterisks denote * p<0.05, ** p<0.01, *** p<0.001 and NS (non significant) versus control, and # p<0.05, ## p<0.01 and ### O.001 versus EAE.

FIGS. 3A-3H - Lovastatin enhanced proliferation and recruitment of OPs in the spinal cord of treated-EAE animals. To determine the impaired recruitment and proliferation of OPs in the spinal cord, the sections were immunostained with anti-BrdU and anti-NG2 antibodies as described under 'Materials & Methods'. Representative sections from each group show the distribution of NG2⁺/BrdU^' cells (green; red arrowheads) in the white matter (WM) of spinal cord of each group of animals (40Ox) (FIG. 3A). A graph represents the average number of NG2⁺/BrDU^" cell counts in ten-fields/section from 4-5 animals/group (FIG. 3B). A representative section demonstrates the co-localization of NG2 and BrdU immunostaining in spinal cord of lovastatin-treated recovered E+LOV/R animals (60Ox) (FIG. 3C). Red arrowheads indicate the co-localization of NG2⁺ (green), BrdU⁺ (red) and NG2⁺/BrdU⁺ (yellow) in proliferating OPs, whereas a yellow arrowhead represents the migrated or resident OP. A graph represents the number of NG2⁺/BrdU⁺ cell counts in ten-fields/section from 4-5 animals/group (FIG. 3D). The spinal cord tissue homogenates from each group were analyzed for the expression of OP proteins, i.e., A2B5 and PDGF-αR including β-actin by immunoblotting as described under 'Material & Methods' (FIG. 3E). The plots demonstrate the real-time PCR™ analysis of mPvNA expression for PDGF-αR (FIG. 3F), SOXlO (FIG. 3G), and Shh (FIG. 3H) in the spinal cord of each group of animals. The asterisks indicate * p<0.05, ** p<0.01, *** pO.001, and NS (non significant) versus CON, and # p<0.05, ## ρθ.01, and ### pO.001 versus EAE. FIGS. 4A-4E - Lovastatin enhanced the survival and proliferation of OPs in mixed glial cell cultures. Cortical mixed glial cell cultures were treated with LOV and CM as described under 'Materials & Methods'. The cells were immunostained using anti-PDGF-αR and anti-NG2 antibodies after 24 hr (DIV2) and 48 hr (DIV3) post stimulation. A graph represents PDGF-CtR⁺ cell counts/field in 10- fields/slide from three independent experiments (FIG. 4A). The representative slides demonstrate PDGF-αR⁺ cells (yellow arrowheads) present in all groups (FIG. 4B). A graph represents NG2⁺ cell counts/field in 10-fields/slide from three independent experiments (FIG. 4C). The representative slides demonstrate NG2⁺ cells (yellow arrowheads) in all groups (FIG. 4D). Real-time PCR™ analyses demonstrate PDGF- αR mRNA expression in LOV- and CM-treated mixed glial cells after 12 hr of stimulation (FIG. 4E). [³H] Thymidine uptake analyses of purified OPs pretreated (PT) with/without LOV and incubated in conditioned media obtained from mixed glial cell cultures following stimulation with cytokine mixture (CM) and LOV as described under 'Materials & Methods'. Results in graphs are expressed as Mean ± SD. The asterisks indicate * ρ<0.05, ** ρθ.01, ***ρ<0.001, and NS (non significant) versus control, and ### p<0.001 versus CM. PT indicates OPs were pretreated with LOV (1 μM) for 24 hr.

FIGS. 5A-5D - Lovastatin enhanced the differentiation of maturing oligodendrocytes in mixed glial cell cultures. Cortical mixed glial cells were treated with lovastatin (LOV) in the presence/absence of CM and immunostained with anti- Ol and anti-MBP antibodies as described under 'Materials & Methods'. A graph depicts the count of 0I⁺ and MBP⁺ cells/field in each slide (n=6) from three independent experiments (FIG. 5A). Representative slides demonstrate 0I⁺ (red) and MBP⁺ (red) cells as indicated (arrowheads), present in all groups after 120 hr (DIV5) and 144 hr (DIV6) (FIG. 5B). Likewise, representative slides Of Ol⁺ZDAPl⁺ (blue) and MBP⁺/DAPI⁺ (blue) of the same field demonstrate the cell nuclei and cell numbers. An MBP⁺ oligodendrocytes indicated by arrowheads (green) are photographed at a higher magnification (1,00Ox) to compare the length and branches of processes. A graph depicts mRNA expression for MBP and PLP in similarly treated cells as described under 'Materials & Methods' (FIG. 5C). Immunoblot demonstrates CNPase and β-actin levels in similarly treated mixed glial cells at DIV4 as described under 'Materials & Methods' (FIG. 5D). Graphs results are expressed as Mean ± SD. The asterisks indicate the following: * <0.05, ** p<0.01 and *** pO.OOl versus control, and ### p<0.001 versus CM.

FIGS. 6A-6C - Lovastatin treatment attenuates reactive gliosis in mixed glial cell cultures. Cortical mixed glial cells were treated with LOV in the presence/absence of CM and immunostained with anti-GFAP and anti-MBP antibodies as described under 'Materials & Methods'. Representative slides show GFAP⁺ (green) and MBP⁺ (red) cells (arrowheads) present in all groups (upper panel) after 144 hr (DIV6) of stimulation (FIG. 6A). Likewise, representative slides GFAP⁺/DAPI⁺/Hoechst (blue) slides of the same field (lower panel) demonstrate the cell nuclei and cell numbers (FIG. 6A). Representative graphs depict the immunofluorescence intensities for GFAP (green curve) and MBP (red curve) in immunostained slides at 20 μm distance scale as described under 'Material & Methods' (FIG. 6B). A plot demonstrates the mRNA expression for GFAP in similarly treated cells as described under 'Materials & Methods' (FIG. 6C). Plot data are expressed as Mean + SD. Magnification was at 40Ox (GFAP and MBP). The asterisks indicate *** p<0.001 and NS (non-significant) versus control.

FIGS. 7A-7H - Lovastatin treatment enhances the release of neurotrophic factors in the spinal cord of treated-EAE animals and in mixed Glial cell cultures. The mRNA expression of neurotrophic factors was determined by real-time PCR™ analysis in the spinal cord of LOV-treated/-untreated EAE animals and controls as well as CM-treated mixed glial cell cultures as described under 'Materials & Methods'. Graphs represent the mRNA expression of BDNF (FIG. 7A), CNTF (FIG. 7B), PDGF (FIG. 7C), GGF-2 (FIG. 7D), and LIF (FIG. 7E) in the spinal cord of animals from each group. Likewise, graphs represent the mRNA expression for BDNF (FIG. 7F), LIF (FIG. 7G), and GGF-2 (FIG. 7H) in LOV-and CM-treated mixed glial cells. The data in graphs are expressed as Mean + SD. The asterisks denote * p<0.05, ** p<0.01, *** p<0.001, and NS (non-significant) versus control, # p<0.05, ## pO.Ol, and ### pO.OOl versus EAE, and $$$ pO.OOl versus CM. FIG. 8 - Possible mechanism of action of lovastatin mediated OP survival and their differentiation into matured oligodendrocytes in the CNS. Lovastatin inhibits the synthesis of isoprenoids required for isoprenylation of small G-proteins (Rho family GTPase viz. RhoA, CDC42 and Racl). These isoprenylated small G- proteins regulate the expression of inflammatory mediators (CM and iNOS) by trans- activation of NF-κB and AP-I in immune cells (Peron et al, 1997; Dubreuil et al, 2003) and CNS resident immune cells, i.e., microglia (MC) and astrocytes (AS). The inactivation of small G-proteins (e.g., RhoA) has been associated with differentiation of maturing oligodendrocytes (Liang et al, 2004). Lovastatin treatment attenuates the inflammatory response mediated reactive gliosis and induces a pro-remyelinating environment, i.e., release of neurotrophic factors in the CNS.

FIGS. 9A-E - Lovastatin treatment attenuates inflammatory demyelination in the spinal cord of EAE animals. EAE induction and lovastatin treatment was performed as described under 'Materials & Methods'. Data are shown as average clinical disease scores. Age matched lovastatin-treated EAE (E+LOV) and control (C+LOV), and untreated EAE (EAE) and control animals were sacrificed on peak clinical day (13^th dpi) and on remission (20^th dpi). Graphs represent the clinical scores and body weight profile in animals from each group after days of post immunization (FIGS. 9A-B). Representative spinal cord tissues sections stained with LFB from control (FIG. 9C) and EAE (FIG. 9D) demonstrating severe demyelination (arrowheads) in white matter region (4Ox). Representative sections, stained with both LFB and H&E stains from each group animals are depicting a typical inflammatory infiltration (arrowheads) and demyelination in the white matter (40Ox) (FIG. 9E). The asterisks denote *** p<0.01 versus E+LOV.

DETAILED DESCRIPTION OF THE INVENTION Recently, the inventors and others have demonstrated the anti-inflammatory properties of HMG-CoA reductase inhibitors, i.e., statins in acute and remitting- relapsing EAE (Youssef et al, 2002; Paintlia et al, 2004; Stanislaus et al, 2001; Nath et al, 2004). The present study reveals a further correlation between attenuated mononuclear infiltration and decreased demyelination in the spinal cord, and early restoration of neurological functions during remission (within a week) mediated through the increased survival and differentiation of OPs in lovastatin-treated EAE animals.

The inventors provide evidence that HMG-CoA reductase inhibitor treatment attenuates the development of inflammatory demyelination similar to that observed within MS chronic lesions and restores remyelination process in the animal model of MS. In support of this, in a recent clinical trial with simvastatin conducted on 30 patients with remitting-relapsing MS for 6 months, the investigators reported a 45% reduction in the number and volume of Gd-positive lesions in the MS brain (Vollmer et al, 2004). Lovastatin provides protection to OPs against the inflammatory response and promotes their proliferation and differentiation through attenuation of reactive gliosis and induction of a pro-remyelinating environment in CNS. The pro- remyelinating properties of statins make them a viable option for using in CNS demyelination diseases such as, for example, MS, Alzheimer's disease, stroke, X- ALD, and HIV dementia.

These data also suggest a more general use of such inhibitors in the protection and propagation of progenitor cells, for example, in ex vivo culturing and transplantation. Thus, the present invention provides for the use of HMG CoA reductase inhibitors in the treatment of stem cells to improve their survival, propagation, expansion and reimplantation.

A. Stem Cells and Stem Cell Populations

A stem cell is a cell that has the ability to divide (self replicate) for indefinite periods — often throughout the life of the organism. Under the right conditions, or given the right signals, stem cells can give rise (differentiate) to the many different cell types that make up the organism. That is, stem cells have the potential to develop into mature cells that have characteristic shapes and specialized functions, such as heart cells, skin cells, or nerve cells. Much of the information that follows can be found at the NIH website "//stemcells.nih.gov/info/scireport/," which is hereby incorporated by reference.

Many of the terms used to define stem cells depend on the behavior of the cells in the intact organism (in vivo), under specific laboratory conditions (in vitro), or after transplantation in vivo, often to a tissue that is different from the one from which the stem cells were derived.

For example, the fertilized egg is said to be totipotent — from the Latin totus, meaning entire — because it has the potential to generate all the cells and tissues that make up an embryo and that support its development in utero. The fertilized egg divides and differentiates until it produces a mature organism. Adult mammals, including humans, consist of more than 200 kinds of cells. These include nerve cells (neurons), muscle cells (myocytes), skin (epithelial) cells, blood cells (erythrocytes, monocytes, lymphocytes, etc), bone cells (osteocytes), and cartilage cells (chondrocytes). Other cells, which are essential for embryonic development but are not incorporated into the body of the embryo, include the extraembryonic tissues, placenta, and umbilical cord. AU of these cells are generated from a single, totipotent cell — the zygote, or fertilized egg.

Most scientists use the term pluripotent to describe stem cells that can give rise to cells derived from all three embryonic genu layers — mesoderm, endoderm, and ectoderm. These three germ layers are the embryonic source of all cells of the body. All of the many different kinds of specialized cells that make up the body are derived from one of these germ layers. "Pluri" — derived from the Latin plures — means several or many. Thus, pluripotent cells have the potential to give rise to any type of cell, a property observed in the natural course of embryonic development and under certain laboratory conditions.

Unipotent stem cell, a term that is usually applied to a cell in adult organisms, means that the cells in question are capable of differentiating along only one lineage. "Uni" is derived from the Latin word unus, which means one. Also, it may be that the adult stem cells in many differentiated, undamaged tissues are typically unipotent and give rise to just one cell type under normal conditions. This process would allow for a steady state of self-renewal for the tissue. However, if the tissue becomes damaged and the replacement of multiple cell types is required, pluripotent stem cells may become activated to repair the damage. 1. Embryonic Stem Cells

The embryonic stem cell is defined by its origin — that is from one of the earliest stages of the development of the embryo, called the blastocyst. Specifically, embryonic stem cells are derived from the inner cell mass of the blastocyst at a stage before it would implant in the uterine wall. The embryonic stem cell can self-replicate and is pluripotent — it can give rise to cells derived from all three germ layers.

Some scientists argue that ES cells do not occur in the embryo as such. ES cells closely resemble the cells of the preimplantation embryo, but are not in fact the same. An alternative perspective is that the embryos of many animal species contain stem cells. These cells proliferate extensively in the embryo, are capable of differentiating into all the types of cells that occur in the adult, and can be isolated and grown ex vivo (outside the organism), where they continue to replicate and show the potential to differentiate. i. Defining Properties of an Embryonic Stem Cell

For research purposes, the definition of an ES cell is more than a self- replicating stem cell derived from the embryo that can differentiate into almost all of the cells of the body. Scientists have found it necessary to develop specific criteria that help them better define the ES cell. Austin Smith, whose studies of mouse ES cells have contributed significantly to the field, has offered a list of essential characteristics that define ES cells. The following attributes define the characteristics of ES cells.

First, ES cells are derived from the inner cell mass/epiblast of the blastocyst and are capable of undergoing an unlimited number of symmetrical divisions without differentiating (long-term self-renewal). Second, they exhibit and maintain a stable, full (diploid), normal complement of chromosomes (karyotype). Third, Pluripotent ES cells can give rise to differentiated cell types that are derived from all three primary germ layers of the embryo (endoderm, mesoderm, and ectoderm). Four, they are capable of integrating into all fetal tissues during development. Fifth, they are capable of colonizing the germ line and giving rise to egg or sperm cells. Sixth, they are clono genie, that is, a single ES cell can give rise to a colony of genetically identical cells, or clones, which have the same properties as the original cell. Seventh, they express the transcription factor Oct-4, which then activates or inhibits a host of target genes and maintains ES cells in a proliferative, non-differentiating state. Eighth, they can be induced to continue proliferating or to differentiate. Ninth, they lack the Gl checkpoint in the cell cycle. ES cells spend most of their time in the S phase of the cell cycle, during which they synthesize DNA. Unlike differentiated somatic cells, ES cells do not require any external stimulus to initiate DNA replication. And tenth, they do not show X inactivation. In every somatic cell of a female mammal, one of the two X chromosomes becomes permanently inactivated. X inactivation does not occur in undifferentiated ES cells.

Pluripotency — that is the ability to give rise to differentiated cell types that are derived from all three primary germ layers of the embryo, endoderm, mesoderm, and ectoderm — is what makes ES cells unique. Laboratory-based criteria for testing the pluripotent nature of ES cells derived from mice include three kinds of experiments. One test is conducted by injecting ES cells derived from the inner cell mass of one blastocyst into the cavity of another blastocyst. The "combination" embryos are then transferred to the uterus of a pseudopregnant female mouse, and the progeny that result are chimeras. Chimeras are a mixture of tissues and organs of cells derived from both donor ES cells and the recipient blastocyst.

This test has been extended in studies designed to test whether cultured ES cells can be used to replace the inner cell mass of a mouse blastocyst and produce a normal embryo. They can, but the process is far less efficient than that of using cells taken directly from the inner cell mass. Apparently, the ability of ES cells to generate a complete embryo depends on the number of times they have been passaged in vitro. A passage is the process of removing cells from one culture dish and replating them into fresh culture dishes. Whether the number of passages affects the differentiation potential of human ES cells remains to be determined. A second method for determining the pluripotency of ES cells is to inject the cells into adults (using mice, injected under the skin or the kidney capsule) that are either genetically identical or are immune-deficient, so the tissue will not be rejected. In the host animal, the injected ES cells develop into benign tumors called teratomas. When examined under a microscope, it was noted that these tumors contain cell types derived from all three primary germ layers of the embiyo — endoderm, mesoderm, and ectoderm. Teratomas typically contain gut-like structures such as layers of epithelial cells and smooth muscle; skeletal or cardiac muscle (which may contract spontaneously); neural tissue; cartilage or bone; and sometimes hair. Thus, ES cells that have been maintained for a long period in vitro can behave as pluripotent cells in vivo. They can participate in normal embryogenesis by differentiating into any cell type in the body, and they can also differentiate into a wide range of cell types in an adult animal. However, normal mouse ES cells do not generate trophoblast tissues in vivo. A third technique for demonstrating pluripotency is to allow mouse ES cells in vitro to differentiate spontaneously or to direct their differentiation along specific pathways. The former is usually accomplished by removing feeder layers and adding leukemia inhibitory factor (LIF) to the growth medium. Within a few days after changing the culture conditions, ES cells aggregate and may form embryoid bodies (EBs). In many ways, EBs in the culture dish resemble teratomas that are observed in the animal. EBs consist of a disorganized array of differentiated or partially differentiated cell types that are derived from the three primary germ layers of the embryo — the endoderm, mesoderm, and ectoderm. The techniques for culturing mouse ES cells from the inner cell mass of the preimplantation blastocyst were first reported 20 years ago, and versions of these standard procedures are used today in laboratories throughout the world. It is striking that, to date, only three species of mammals have yielded long-term cultures of self- renewing ES cells: mice, monkeys, and humans. 2. Adult Stems Cells

The adult stem cell is an undifferentiated (unspecialized) cell that is found in a differentiated (specialized) tissue; it can renew itself and become specialized to yield all of the specialized cell types of the tissue from which it originated. Adult stem cells are capable of self-renewal for the lifetime of the organism. Sources of adult stem cells have been found in the bone marrow, blood stream, cornea and retina of the eye, the dental pulp of the tooth, liver, skin, gastrointestinal tract, and pancreas. Unlike embryonic stem cells, at this point in time, there are no isolated adult stem cells that are capable of forming all cells of the body. That is, there is no evidence, at this time, of an adult stem cell that is pluripotent. Adult stem cells, like all stem cells, share at least two characteristics. First, they can make identical copies of themselves for long periods of time; this ability to proliferate is referred to as long-term self-renewal. Second, they can give rise to mature cell types that have characteristic morphologies (shapes) and specialized functions. Typically, stem cells generate an intermediate cell type or types before they achieve their fully differentiated state. The intermediate cell is called a precursor or progenitor cell. Progenitor or precursor cells in fetal or adult tissues are partly differentiated cells that divide and give rise to differentiated cells. Such cells are usually regarded as "committed" to differentiating along a particular cellular development pathway, although this characteristic may not be as definitive as once thought (Robey, 2000).

Adult stem cells are rare. Their primary functions are to maintain the steady state functioning of a cell — called homeostasis — and, with limitations, to replace cells that die because of injury or disease (Holtzer, 1978; Leblond, 1964). For example, only an estimated 1 in 10,000 to 15,000 cells in the bone marrow is a hematopoietic (bloodforming) stem cell (Weissman, 2000). Furthermore, adult stem cells are dispersed in tissues throughout the mature animal and behave very differently, depending on their local environment. For example, HSCs are constantly being generated in the bone marrow where they differentiate into mature types of blood cells. Indeed, the primary role of HSCs is to replace blood cells (Domen and Weissman, 1999). In contrast, stem cells in the small intestine are stationary, and are physically separated from the mature cell types they generate. Gut epithelial stem cells (or precursors) occur at the bases of crypts — deep invaginations between the mature, differentiated epithelial cells that line the lumen of the intestine. These epithelial crypt cells divide fairly often, but remain part of the stationary group of cells they generate (Slack, 2000).

Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), adult stem cells share no such definitive means of characterization. In fact, no one knows the origin of adult stem cells in any mature tissue. Some have proposed that stem cells are somehow set aside during fetal development and restrained from differentiating. Definitions of adult stem cells vary in the scientific literature range from a simple description of the cells to a rigorous set of experimental criteria that must be met before characterizing a particular cell as an adult stem cell. Most of the information about adult stem cells comes from studies of mice. The list of adult tissues reported to contain stem cells is growing and includes bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, retina, liver, and pancreas. In order to be classified as an adult stem cell, the cell should be capable of self- renewal for the lifetime of the organism.

Ideally, adult stem cells should also be clonogeriic. In other words, a single adult stem cell should be able to generate a line of genetically identical cells, which then gives rise to all the appropriate, differentiated cell types of the tissue in which it resides. This property is difficult to demonstrate in vivo; in practice, scientists show either that a stem cell is clonogenic in vitro, or that a purified population of candidate stem cells can repopulate the tissue.

An adult stem cell should also be able to give rise to fully differentiated cells that have mature phenotypes, are fully integrated into the tissue, and are capable of specialized functions that are appropriate for the tissue. The term phenotype refers to all the observable characteristics of a cell (or organism); its shape (morphology); interactions with other cells and the non-cellular environment (also called the extracellular matrix); proteins that appear on the cell surface (surface markers); and the cell's behavior {e.g., secretion, contraction, synaptic transmission). The majority of researchers who lay claim to having identified adult stem cells rely on two of these characteristics — appropriate cell morphology, and the demonstration that the resulting, differentiated cell types display surface markers that identify them as belonging to the tissue. Some studies demonstrate that the differentiated cells that are derived from adult stem cells are truly functional, and a few studies show that cells are integrated into the differentiated tissue in vivo and that they interact appropriately with neighboring cells. At present, there is, however, a paucity of research, with a few notable exceptions, in which researchers were able to conduct studies of genetically identical (clonal) stem cells. In order to fully characterize the regenerating and self-renewal capabilities of the adult stem cell, and therefore to truly harness its potential, it will be important to demonstrate that a single adult stem cell can, indeed, generate a line of genetically identical cells, which then gives rise to all the appropriate, differentiated cell types of the tissue in which it resides. i. Evidence for the Presence of Adult Stem Cells Adult stem cells have been identified in many animal and human tissues. In general, three methods are used to determine whether candidate adult stem cells give rise to specialized cells. Adult stem cells can be labeled in vivo and then they can be tracked. Candidate adult stem cells can also be isolated and labeled and then transplanted back into the organism to determine what becomes of them. Finally, candidate adult stem cells can be isolated, grown in vitro and manipulated, by adding growth factors or introducing genes that help determine what differentiated cells types they will yield. For example, currently, scientists believe that stem cells in the fetal and adult brain divide and give rise to more stem cells or to several types of precursor cells, which give rise to nerve cells (neurons), of which there are many types.

It is often difficult — if not impossible — to distinguish adult, tissue-specific stem cells from progenitor cells, which are found in fetal or adult tissues and are partly differentiated cells that divide and give rise to differentiated cells. These are cells found in many organs that are generally thought to be present to replace cells and maintain the integrity of the tissue. Progenitor cells give rise to certain types of cells — such as the blood cells known as T lymphocytes, B lymphocytes, and natural killer cells — but are not thought to be capable of developing into all the cell types of a tissue and as such are not truly stem cells. The current wave of excitement over the existence of stem cells in many adult tissues is perhaps fueling claims that progenitor or precursor cells in those tissues are instead stem cells. Thus, there are reports of endothelial progenitor cells, skeletal muscle stem cells, epithelial precursors in the skin and digestive system, as well as some reports of progenitors or stem cells in the pancreas and liver. A detailed summary of some of the evidence for the existence of stem cells in various tissues and organs is presented later in the chapter. ii. Adult Stem Cell Plasticity

It was not until recently that anyone seriously considered the possibility that stem cells in adult tissues could generate the specialized cell types of another type of tissue from which they normally reside — either a tissue derived from the same embryonic germ layer or from a different germ layer. For example, studies have shown that blood stem cells (derived from mesoderm) may be able to generate both skeletal muscle (also derived from mesoderm) and neurons (derived from ectoderm). That realization has been triggered by a flurry of papers reporting that stem cells derived from one adult tissue can change their appearance and assume characteristics that resemble those of differentiated cells from other tissues.

The term plasticity, as used in this report, means that a stem cell from one adult tissue can generate the differentiated cell types of another tissue. At this time, there is no formally accepted name for this phenomenon in the scientific literature. It is variously referred to as "plasticity"( Brazelton et ah, 2000; Krause et ah, 2001), "unorthodox differentiation" (Bianco and Cossu, 1999) or "transdifferentiation" (Anderson et al, 2001; Lagasse et ah, 2000). Ui. Adult Stem Cells of the Nervous System

More than 30 years ago, Altman and Das showed that two regions of the postnatal rat brain, the hippocampus and the olfactory bulb, contain dividing cells that become neurons (Altman and Das, 1965; Altman, 1969). Despite these reports, the prevailing view at the time was that nerve cells in the adult brain do not divide. In fact, the notion that stem cells in the adult brain can generate its three major cell types — astrocytes and oligodendrocytes, as well as neurons — was not accepted until far more recently. Within the past five years, a series of studies has shown that stem cells occur in the adult mammalian brain and that these cells can generate its three major cell lineages (Gage et al, 1995b; Joh et al, 1996; McKay, 1997; Shihabuddin et al, 1999; Temple and Alvarez-Buylla, 1999; Weiss and van der Kooy, 1998).

Today, scientists believe that stem cells in the fetal and adult brain divide and give rise to more stem cells or to several types of precursor cells. Neuronal precursors (also called neuroblasts) divide and give rise to nerve cells (neurons), of which there are many types. Glial precursors give rise to astrocytes or oligodendrocytes. Astrocytes are a kind of glial cell, which lend both mechanical and metabolic support for neurons; they make up 70 to 80 percent of the cells of the adult brain. Oligodendrocytes make myelin, the fatty material that ensheathes nerve cell axons and speeds nerve transmission. Under normal, in vivo conditions, neuronal precursors do not give rise to glial cells, and glial precursors do not give rise to neurons. In contrast, a fetal or adult CNS (central nervous system — the brain and spinal cord) stem cell may give rise to neurons, astrocytes, or oligodendrocytes, depending on the signals it receives and its three-dimensional environment within the brain tissue. There is now widespread consensus that the adult mammalian brain does contain stem cells. However, there is no consensus about how many populations of CNS stem cells exist, how they may be related, and how they function in vivo. Because there are no markers currently available to identify the cells in vivo, the only method for testing whether a given population of CNS cells contains stem cells is to isolate the cells and manipulate them in vitro, a process that may change their intrinsic properties (Morrison et al, 1999).

Despite these barriers, three groups of CNS stem cells have been reported to date. All occur in the adult rodent brain and preliminary evidence indicates they also occur in the adult human brain. One group occupies the brain tissue next to the ventricles, regions known as the ventricular zone and the sub-ventricular zone (see discussion below). The ventricles are spaces in the brain filled with cerebrospinal fluid. During fetal development, the tissue adjacent to the ventricles is a prominent region of actively dividing cells. By adulthood, however, this tissue is much smaller, although it still appears to contain stem cells (Morshead and van der Kooy, 2001).

A second group of adult CNS stem cells, described in mice but not in humans, occurs in a streak of tissue that connects the lateral ventricle and the olfactory bulb, which receives odor signals from the nose. In rodents, olfactory bulb neurons are constantly being replenished via this pathway (Lois and Alvarez-Buylla, 1994; Luskin, 1993). A third possible location for stem cells in adult mouse and human brain occurs in the hippocampus, a part of the brain thought to play a role in the formation of certain kinds of memory (Eriksson et al, 1998; Gage et al, 1995a).

Central Nervous System Stem Cells in the Sub ventricular Zone. CNS stem cells found in the forebrain that surrounds the lateral ventricles are heterogeneous and can be distinguished morphologically. Ependymal cells, which are ciliated, line the ventricles. Adjacent to the ependymal cell layer, in a region sometimes designated as the subependymal or subventricular zone, is a mixed cell population that consists of neuroblasts (immature neurons) that migrate to the olfactory bulb, precursor cells, and astrocytes. Some of the cells divide rapidly, while others divide slowly. The astrocyte- like cells can be identified because they contain glial fibrillary acidic protein (GFAP), whereas the ependymal cells stain positive for nestin, which is regarded as a marker of neural stem cells. Which of these cells best qualifies as a CNS stem cell is a matter of debate (Panicker and Rao, 2001).

A recent report indicates that the astrocytes that occur in the subventricular zone of the rodent brain act as neural stem cells. The cells with astrocyte markers appear to generate neurons in vivo, as identified by their expression of specific neuronal markers. The in vitro assay to demonstrate that these astrocytes are, in fact, stem cells involves their ability to form neurospheres — groupings of undifferentiated cells that can be dissociated and coaxed to differentiate into neurons or glial cells (Doetsch et al, 1999). Traditionally, these astrocytes have been regarded as differentiated cells, not as stem cells and so their designation as stem cells is not universally accepted.

A series of similar in vitro studies based on the formation of neurospheres was used to identify the subependymal zone as a source of adult rodent CNS stem cells. In these experiments, single, candidate stem cells derived from the subependymal zone are induced to give rise to neurospheres in the presence of mitogens — either epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2). The neurospheres are dissociated and passaged. As long as a mitogen is present in the culture medium, the cells continue forming neurospheres without differentiating. Some populations of CNS cells are more responsive to EGF, others to FGF (Tropepe et al, 1999). To induce differentiation into neurons or glia, cells are dissociated from the neurospheres and grown on an adherent surface in serum-free medium that contains specific growth factors. Collectively, the studies demonstrate that a population of cells derived from the adult rodent brain can self-renew and differentiate to yield the three major cell types of the CNS cells (Gritti et al, 1996; Morshead et al, 1994; Palme et al, 1997; Vescovi et al, 1993).

Central Nervous System Stem Cells in the Ventricular Zone. Another group of potential CNS stem cells in the adult rodent brain may consist of the ependymal cells themselves (Johansson et al, 1999). Ependymal cells, which are ciliated, line the lateral ventricles. They have been described as non-dividing cells (Doetsch et al, 1997) that function as part of the blood-brain barrier (Del Bigio, 1995 ). The suggestion that ependymal cells from the ventricular zone of the adult rodent CNS may be stem cells is therefore unexpected. However, in a recent study, in which two molecular tags — the fluorescent marker DiI, and an adenovirus vector carrying lacZ tags — were used to label the ependymal cells that line the entire CNS ventricular system of adult rats, it was shown that these cells could, indeed, act as stem cells. A few days after labeling, fluorescent or lacZΛ- cells were observed in the rostral migratory stream (which leads from the lateral ventricle to the olfactory bulb), and then in the olfactory bulb itself. The labeled cells in the olfactory bulb also stained for the neuronal markers βlll tubulin and Map2, which indicated that ependymal cells from the ventricular zone of the adult rat brain had migrated along the rostral migratory stream to generate olfactory bulb neurons in vivo (Johansson et al, 1999).

To show that DiI cells were neural stem cells and could generate astrocytes and oligodendrocytes as well as neurons, a neurosphere assay was performed in vitro. Dil-labeled cells were dissociated from the ventricular system and cultured in the presence of mitogen to generate neurospheres. Most of the neurospheres were DiI⁺; they could self-renew and generate neurons, astrocytes, and oligodendrocytes when induced to differentiate. Single, DiI ependymal cells isolated from the ventricular zone could also generate self-renewing neurospheres and differentiate into neurons and glia.

To show that ependymal cells can also divide in vivo, bromodeoxyuridine (BrdU) was administered in the drinking water to rats for a 2- to 6-week period. Bromodeoxyuridine (BrdU) is a DNA precursor that is only incorporated into dividing cells. Through a series of experiments, it was shown that ependymal cells divide slowly in vivo and give rise to a population of progenitor cells in the subventricular zone (Johansson et ah, 1999). A different pattern of scattered BrdU- labeled cells was observed in the spinal cord, which suggested that ependymal cells along the central canal of the cord occasionally divide and give rise to nearby ependymal cells, but do not migrate away from the canal.

Collectively, the data suggest that CNS ependymal cells in adult rodents can function as stem cells. The cells can self-renew, and most proliferate via asymmetrical division. Many of the CNS ependymal cells are not actively dividing (quiescent), but they can be stimulated to do so in vitro (with mitogens) or in vivo (in response to injury). After injury, the ependymal cells in the spinal cord only give rise to astrocytes, not to neurons. How and whether ependymal cells from the ventricular zone are related to other candidate populations of CNS stem cells, such as those identified in the hippocampus (Gage et al., 1995a), is not known. These studies and other leave open the question of whether cells that directly line the ventricles — those in the ventricular zone — or cells that are at least a layer removed from this zone — in the subventricular zone are the same population of CNS stem cells. A new study, based on the finding that they express different genes, confirms earlier reports that the ventricular and subventricular zone cell populations are distinct. The new research utilizes a technique called representational difference analysis, together with cDNA microarray analysis, to monitor the patterns of gene expression in the complex tissue of the developing and postnatal mouse brain. The study revealed the expression of a panel of genes known to be important in CNS development, such as L3-PSP (which encodes a phosphoserine phosphatase important in cell signaling), cyclin D2 (a cell cycle gene), and ERCC-I (which is important in DNA excision repair). All of these genes in the recent study were expressed in cultured neurospheres, as well as the ventricular zone, the subventricular zone, and a brain area outside those germinal zones. This analysis also revealed the expression of novel genes such as Al 6F10, which is similar to a gene in an embryonic cancer cell line. Al 6F10 was expressed in neurospheres and at high levels in the sub ventricular zone, but not significantly in the ventricular zone. Interestingly, several of the genes identified in cultured neurospheres were also expressed in hematopoietic cells, suggesting that neural stem cells and blood-forming cells may share aspects of their genetic programs or signaling systems (Geschwind et al, 2001). This finding may help explain recent reports that CNS stem cells derived from mouse brain can give rise to hematopoietic cells after injection into irradiated mice (Bjornson et al, 1999).

Central Nervous System Stem Cells in the Hippocampus. The hippocampus is one of the oldest parts of the cerebral cortex, in evolutionary terms, and is thought to play an important role in certain forms of memory. The region of the hippocampus in which stem cells apparently exist in mouse and human brains is the subgranular zone of the dentate gyrus. In mice, when BrdU is used to label dividing cells in this region, about 50% of the labeled cells differentiate into cells that appear to be dentate gyrus granule neurons, and 15% become glial cells. The rest of the BrdU -labeled cells do not have a recognizable phenotype (Shihabuddin et al, 1999). Interestingly, many, if not all the BrdU-labeled cells in the adult rodent hippocampus occur next to blood vessels.

In the human dentate gyrus, some BrdU-labeled cells express NeuN, neuron- specific enolase, or calbindin, all of which are neuronal markers. The labeled neuron- like cells resemble dentate gyrus granule cells, in terms of their morphology (as they did in mice). Other BrdU-labeled cells express glial fibrillary acidic protein (GFAP) an astrocyte marker. The study involved autopsy material, obtained with family consent, from five cancer patients who had been injected with BrdU dissolved in saline prior to their death for diagnostic purposes. The patients ranged in age from 57 to 72 years. The greatest number of BrdU-labeled cells were identified in the oldest patient, suggesting that new neuron formation in the hippocampus can continue late in life (Eriksson et al, 1998).

Fetal Central Nervous System Stem Cells. Not surprisingly, fetal stem cells are numerous in fetal tissues, where they are assumed to play an important role in the expansion and differentiation of all tissues of the developing organism. Depending on the developmental stage of an animal, fetal stem cells and precursor cells — which arise from stem cells — may make up the bulk of a tissue. This is certainly true in the brain (Johe et al, 1996), although it has not been demonstrated experimentally in many tissues. It may seem obvious that the fetal brain contains stem cells that can generate all the types of neurons in the brain as well as astrocytes and oligodendrocytes, but it was not until fairly recently that the concept was proven experimentally. There has been a long-standing question as to whether or not the same cell type gives rise to both neurons and glia. In studies of the developing rodent brain, it has now been shown that all the major cell types in the fetal brain arise from a common population of progenitor cells (Davis and Temple, 1994; Gage et al, 1995a; Johe et al, 1996; Reynolds and Weiss, 1992; Williams et al, 1991).

Neural tems cells in the mammalian fetal brain are concentrated in seven major areas: olfactory bulb, ependymal (ventricular) zone of the lateral ventricles (which lie in the forebrain), subventricular zone (next to the ependymal zone), hippocampus, spinal cord, cerebellum (part of the hindbrain), and the cerebral cortex. Their number and pattern of development vary in different species. These cells appear to represent different stem cell populations, rather than a single population of stem cells that is dispersed in multiple sites. The normal development of the brain depends not only on the proliferation and differentiation of these fetal stem cells, but also on a genetically programmed process of selective cell death called apoptosis (Panicker and Rao, 2001).

Little is known about stem cells in the human fetal brain. In one study, however, investigators derived clonal cell lines from CNS stem cells isolated from the diencephalon and cortex of human fetuses, 10.5 weeks post-conception (Vescovi et al, 1999). The study is unusual, not only because it involves human CNS stem cells obtained from fetal tissue, but also because the cells were used to generate clonal cell lines of CNS stem cells that generated neurons, astrocytes, and oligodendrocytes, as determined on the basis of expressed markers. In a few experiments described as "preliminary," the human CNS stem cells were injected into the brains of immunosuppressed rats where they apparently differentiated into neuron-like cells or glial cells.

In a 1999 study, a serum-free growth medium that included EGF and FGF2 was devised to grow the human fetal CNS stem cells. Although most of the cells died, occasionally, single CNS stem cells survived, divided, and ultimately formed neurospheres after one to two weeks in culture. The neurospheres could be dissociated and individual cells replated. The cells resumed proliferation and formed new neurospheres, thus establishing an in vitro system that (like the system established for mouse CNS neurospheres) could be maintained up to 2 years. Depending on the culture conditions, the cells in the neurospheres could be maintained in an undifferentiated dividing state (in the presence of mitogen), or dissociated and induced to differentiate (after the removal of mitogen and the addition of specific growth factors to the culture medium). The differentiated cells consisted mostly of astrocytes (75%), some neurons (13%) and rare oligodendrocytes (1.2%). The neurons generated under these conditions expressed markers indicating they were GABAergic, [the major type of inhibitory neuron in the mammalian CNS responsive to the amino acid neurotransmitter, gammaaminobutyric acid (GABA)]. However, catecholamine-like cells that express tyrosine hydroxylase (TH, a critical enzyme in the dopamine-synthesis pathway) could be generated, if the culture conditions were altered to include different medium conditioned by a rat glioma line (BB49). Thus, the report indicates that human CNS stem cells obtained from early fetuses can be maintained in vitro for a long time without differentiating, induced to differentiate into the three major lineages of the CNS (and possibly two kinds of neurons, GABAergic and TH-positive), and engraft (in rats) in vivo (Vescovi et ah, 1999).

Central Nervous System Neural Crest Stem Cells. Neural crest cells differ markedly from fetal or adult neural stem cells. During fetal development, neural crest cells migrate from the sides of the neural tube as it closes. The cells differentiate into a range of tissues, not all of which are part of the nervous system (Le Douarin, 1980; Le Douarin and Kalcheim, 1999; Sieber-Blum, 2000), Neural crest cells form the sympathetic and parasympathetic components of the peripheral nervous system (PNS), including the network of nerves that innervate the heart and the gut, all the sensory ganglia (groups of neurons that occur in pairs along the dorsal surface of the spinal cord), and Schwann cells, which (like oligodendrocytes in the CNS) make myelin in the PNS. The non-neural tissues that arise from the neural crest are diverse. They populate certain hormone-secreting glands — including the adrenal medulla and Type I cells in the carotid body — pigment cells of the skin (melanocytes), cartilage and bone in the face and skull, and connective tissue in many parts of the body (Panicker and Rao, 2001).

Thus, neural crest cells migrate far more extensively than other fetal neural stem cells during development, form mesenchymal tissues, most of which develop from embryonic mesoderm as well as the components of the CNS and PNS which arises from embryonic ectoderm. This close link, in neural crest development, between ectodermally derived tissues and mesodermally derived tissues accounts in part for the interest in neural crest cells as a kind of stem cell. In fact, neural crest cells meet several criteria of stem cells. They can self-renew (at least in the fetus) and can differentiate into multiple cells types, which include cells derived from two of the three embryonic germ layers (Panicker and Rao, 2001).

Recent studies indicate that neural crest cells persist late into gestation and can be isolated from E14.5 rat sciatic nerve, a peripheral nerve in the hindlimb. The cells incorporate BrdU, indicating that they are dividing in vivo. When transplanted into chick embryos, the rat neural crest cells develop into neurons and glia, an indication of their stem cell-like properties (Morrison et al, 1999). However, the ability of rat E14.5 neural crest cells taken from sciatic nerve to generate nerve and glial cells in chick is more limited than neural crest cells derived from younger, El 0.5 rat embryos. At the earlier stage of development, the neural tube has formed, but neural crest cells have not yet migrated to their final destinations. Neural crest cells from early developmental stages are more sensitive to bone morphogenetic protein 2 (BMP2) signaling, which may help explain their greater differentiation potential (White et al, 2001). iv. Stem Cells in the Bone Marrow and Blood

One population of bone marrow cells, the hematopoietic stem cells (HSCs), is responsible for forming all of the types of blood cells in the body. HSCs were recognized as a stem cells more than 40 years ago (Becker et al, 1963; Till and

McCullough, 1961). Bone marrow stromal cells — a mixed cell population that generates bone, cartilage, fat, fibrous connective tissue, and the reticular network that supports blood cell formation — were described shortly after the discovery of HSCs (Friedenstein et al, 1966; Friedenstein et al, 1970; Owen, 1988). The mesenchymal stem cells of the bone marrow also give rise to these tissues, and may constitute the same population of cells as the bone marrow stromal cells (Pittenger et al, 2001).

Recently, a population of progenitor cells that differentiates into endothelial cells, a type of cell that lines the blood vessels, was isolated from circulating blood (Asahara et al, 1997) and identified as originating in bone marrow (Shi et al, 1998). Whether these endothelial progenitor cells, which resemble the angioblasts that give rise to blood vessels during embryonic development, represent a bona fide population of adult bone marrow stem cells remains uncertain. Thus, the bone marrow appears to contain three stem cell populations — hematopoietic stem cells, stromal cells, and (possibly) endothelial progenitor cells.

Two more apparent stem cell types have been reported in circulating blood, but have not been shown to originate from the bone marrow. One population, called pericytes, may be closely related to bone marrow stromal cells, although their origin remains elusive (Bianco et al., 2001). The second population of blood-born stem cells, which occur in four species of animals tested — guinea pigs, mice, rabbits, and humans — resemble stromal cells in that they can generate bone and fat (Kuznetsov et ah, 2001). Hematopoietic Stem Cells. Of all the cell types in the body, those that survive for the shortest period of time are blood cells and certain kinds of epithelial cells. For example, red blood cells (erythrocytes), which lack a nucleus, live for approximately 120 days in the bloodstream. The life of an animal literally depends on the ability of these and other blood cells to be replenished continuously. This replenishment process occurs largely in the bone marrow, where HSCs reside, divide, and differentiate into all the blood cell types. Both HSCs and differentiated blood cells cycle from the bone marrow to the blood and back again, under the influence of a barrage of secreted factors that regulate cell proliferation, differentiation, and migration. HSCs can reconstitute the hematopoietic system of mice that have been subjected to lethal doses of radiation to destroy their own hematopoietic systems. This test, the rescue of lethally irradiated mice, has become a standard by which other candidate stem cells are measured because it shows, without question, that HSCs can regenerate an entire tissue system — in this case, the blood (Becker et al., 1963; Till and McCullough, 1961). HSCs were first proven to be blood-forming stem cells in a series of experiments in mice; similar blood-forming stem cells occur in humans. HSCs are defined by their ability to self-renew and to give rise to all the kinds of blood cells in the body. This means that a single HSC is capable of regenerating the entire hematopoietic system, although this has been demonstrated only a few times in mice (Osawa et al, 1996).

Over the years, many combinations of surface markers have been used to identify, isolate, and purify HSCs derived from bone marrow and blood. Undifferentiated HSCs and hematopoietic progenitor cells express c-kit, CD34, and H-2K. These cells usually lack the lineage marker Lin, or express it at very low levels (Lin^"/low). And for transplant purposes, cells that are CD34⁺ Thyl⁺ Lin^" are most likely to contain stem cells and result in engraftment.

Two kinds of HSCs have been defined. Long-term HSCs proliferate for the lifetime of an animal. In young adult mice, an estimated 8 to 10 % of long-term HSCs enter the cell cycle and divide each day. Short-term HSCs proliferate for a limited time, possibly a few months. Long-term HSCs have high levels of telomerase activity. Telomerase is an enzyme that helps maintain the length of the ends of chromosomes, called telomeres, by adding on nucleotides. Active telomerase is a characteristic of undifferentiated, dividing cells and cancer cells. Differentiated, human somatic cells do not show telomerase activity. In adult humans, HSCs occur in the bone marrow, blood, liver, and spleen, but are extremely rare in any of these tissues. In mice, only 1 in 10,000 to 15,000 bone marrow cells is a long-term HSC (Weissman, 2000).

Short-term HSCs differentiate into lymphoid and myeloid precursors, the two classes of precursors for the two major lineages of blood cells. Lymphoid precursors differentiate into T cells, B cells and natural killer cells. The mechanisms and pathways that lead to their differentiation are still being investigated (Akashi et al, 1999a; Akashi et al, 1999b). Myeloid precursors differentiate into monocytes and macrophages, neutrophils, eosinophils, basophils, megakaryocytes, and erythrocytes (Akashi et al., 2000). In vivo, bone marrow HSCs differentiate into mature, specialized blood cells that cycle constantly from the bone marrow to the blood, and back to the bone marrow (Domen and Weissman, 1999). A recent study showed that short-term HSCs are a heterogeneous population that differ significantly in terms of their ability to self-renew and repopulate the hematopoietic system (Guenechea et al., 2001). Attempts to induce HSC to proliferate in vitro — on many substrates, including those intended to mimic conditions in the stroma — have frustrated scientists for many years. Although HSCs proliferate readily in vivo, they usually differentiate or die in vitro (Domen and Weissman, 1999). Thus, much of the research on HSCs has been focused on understanding the factors, cell-cell interactions, and cell-matrix interactions that control their proliferation and differentiation in vivo, with the hope that similar conditions could be replicated in vitro. Many of the soluble factors that regulate HSC differentiation in vivo are cytokines, which are made by different cell types and are then concentrated in the bone marrow by the extracellular matrix of stromal cells — the sites of blood formation (Hunt et al, 1987; Whitlock et al, 1987). Two of the most-studied cytokines are granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) (Gordon et al, 1987; Roberts et al, 1988).

Also important to HSC proliferation and differentiation are interactions of the cells with adhesion molecules in the extracellular matrix of the bone marrow stroma (Roy and Verfaillie, 1999; Verfaillie, 1998; Zandstra et al. , 2000).

Bone Marrow Stromal Cells. Bone marrow (BM) stromal cells have long been recognized for playing an important role in the differentiation of mature blood cells from HSCs. But stromal cells also have other important functions (Friedenstein et al, 1966; Friedenstein et al, 1968). In addition to providing the physical environment in which HSCs differentiate, BM stromal cells generate cartilage, bone, and fat. Whether stromal cells are best classified as stem cells or progenitor cells for these tissues is still in question. There is also a question as to whether BM stromal cells and so-called mesenchymal stem cells are the same population (Pittenger and Marshak, 2001). BM stromal cells have many features that distinguish them from HSCs. The two cell types are easy to separate in vitro. When bone marrow is dissociated, and the mixture of cells it contains is plated at low density, the stromal cells adhere to the surface of the culture dish, and the HSCs do not. Given specific in vitro conditions, BM stromal cells form colonies from a single cell called the colony forming unit-F (CFU-F). These colonies may then differentiate as adipocytes or myelosupportive stroma, a clonal assay that indicates the stem cell -like nature of stromal cells. Unlike HSCs, which do not divide in vitro (or proliferate only to a limited extent), BM stromal cells can proliferate for up to 35 population doublings in vitro (Bruder et al, 1997). They grow rapidly under the influence of such mitogens as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-I) (Bianco et al, 2001).

To date, it has not been possible to isolate a population of pure stromal cells from bone marrow. Panels of markers used to identify the cells include receptors for certain cytokines (interleukin-1, 3, 4, 6, and 7) receptors for proteins in the extracellular matrix, (ICAM-I and 2, VCAM-I, the alpha-1, 2, and 3 integrins, and the beta-1, 2, 3 and 4 integrins), etc.. Despite the use of these markers and another stromal cell marker called Stro-1, the origin and specific identity of stromal cells have remained elusive. Like HSCs, BM stromal cells arise from embryonic mesoderm during development, although no specific precursor or stem cell for stromal cells has been isolated and identified. One theory about their origin is that a common kind of progenitor cell — perhaps a primordial endothelial cell that lines embryonic blood vessels — gives rise to both HSCs and to mesodermal precursors. The latter may then differentiate into myogenic precursors (the satellite cells that are thought to function as stem cells in skeletal muscle), and the BM stromal cells (Bianco and Cossu, 1999)..

In vivo, the differentiation of stromal cells into fat and bone is not straightforward. Bone marrow adipocytes and myelosupportive stromal cells — both of which are derived from BM stromal cells — may be regarded as interchangeable phenotypes (Bianco and Cossu, 1999; Bianco et al, 1999). Adipocytes do not develop until postnatal life, as the bones enlarge and the marrow space increases to accommodate enhanced hematopoiesis. When the skeleton stops growing, and the mass of HSCs decreases in a normal, age-dependent fashion, BM stromal cells differentiate into adipocytes, which fill the extra space. New bone formation is obviously greater during skeletal growth, although bone "turns over" throughout life. Bone forming cells are osteoblasts, but their relationship to BM stromal cells is not clear. New trabecular bone, which is the inner region of bone next to the marrow, could logically develop from the action of BM stromal cells. But the outside surface of bone also turns over, as does bone next to the Haversian system (small canals that form concentric rings within bone). And neither of these surfaces is in contact with BM stromal cells (Bianco and Cossu, 1999; Bianco et al. , 1999). v. Adult Stem Cells in Other Tissues

It is often difficult — if not impossible — to distinguish adult, tissue-specific stem cells from progenitor cells. With that caveat in mind, the following summary identifies reports of stem cells in various adult tissues. Endothelial Progenitor Cells. Endothelial cells line the inner surfaces of blood vessels throughout the body, and it has been difficult to identify specific endothelial stem cells in either the embryonic or the adult mammal. During embryonic development, just after gastrulation, a kind of cell called the hemangioblast, which is derived from mesoderm, is presumed to be the precursor of both the hematopoietic and endothelial cell lineages. The embryonic vasculature formed at this stage is transient and consists of blood islands in the yolk sac. But hemangioblasts, per se, have not been isolated from the embryo and their existence remains in question. The process of forming new blood vessels in the embryo is called vasculogenesis. In the adult, the process of forming blood vessels from pre-existing blood vessels is called angiogenesis (Keller, 2001).

Evidence that hemangioblasts do exist comes from studies of mouse embryonic stem cells that are directed to differentiate in vitro. These studies have shown that a precursor cell derived from mouse ES cells that express FIk-I [the receptor for vascular endothelial growth factor (VEGF) in mice] can give rise to both blood cells and blood vessel cells (Shalaby et al, 1995; Yamashita et al, 2000). Both VEGF and fibroblast growth factor-2 (FGF-2) play critical roles in endothelial cell differentiation in vivo (Pittenger and Marshak, 2001). Several recent reports indicate that the bone marrow contains cells that can give rise to new blood vessels in tissues that are ischemic (damaged due to the deprivation of blood and oxygen) (Asahara et al, 1997; Ferrari et al, 1998; Kalka et al, 2000; Takahashi et al, 1999). But it is unclear from these studies what cell type(s) in the bone marrow induced angiogenesis. In a study which sought to address that question, researchers found that adult human bone marrow contains cells that resemble embryonic hemangioblasts, and may therefore be called endothelial stem cells.

In more recent experiments, human bone marrow-derived cells were injected into the tail veins of rats with induced cardiac ischemia. The human cells migrated to the rat heart where they generated new blood vessels in the infarcted muscle (a process akin to vasculogenesis), and also induced angiogenesis. The candidate endothelial stem cells are CD34⁺(a marker for HSCs), and they express the transcription factor GATA-2 (Kocher et al, 2001). A similar study using transgenic mice that express the gene for enhanced green fluorescent protein (which allows the cells to be tracked), showed that bone-marrow-derived cells could repopulate an area of infarcted heart muscle in mice, and generate not only blood vessels, but also cardiomyocytes that integrated into the host tissue (Orlic et al, 2001).

And, in a series of experiments in adult mammals, progenitor endothelial cells were isolated from peripheral blood (of mice and humans) by using antibodies against CD34 and FIk-I, the receptor for VEGF. The cells were mononuclear blood cells (meaning they have a nucleus) and are referred to as MB^CD34+ cells and MB^Flkl+ cells. When plated in tissue-culture dishes, the cells attached to the substrate, became spindle-shaped, and formed tube-like structures that resemble blood vessels. When transplanted into mice of the same species (autologous transplants) with induced ischemia in one limb, the MB^CD34+ cells promoted the formation of new blood vessels (Asahara et aL, 1997). Although the adult MB^CD34+ and MB^Flkl+ cells function in some ways like stem cells, they are usually regarded as progenitor cells.

Skeletal Muscle Stem Cells. Skeletal muscle, like the cardiac muscle of the heart and the smooth muscle in the walls of blood vessels, the digestive system, and the respiratory system, is derived from embryonic mesoderm. To date, at least three populations of skeletal muscle stem cells have been identified: satellite cells, cells in the wall of the dorsal aorta, and so-called "side population" cells.

Satellite cells in skeletal muscle were identified 40 years ago in frogs by electron microscopy (Mauro, 1961), and thereafter in mammals (Schultz, 1976). Satellite cells occur on the surface of the basal lamina of a mature muscle cell, or myofϊber. In adult mammals, satellite cells mediate muscle growth (Schultz, 1996). Although satellite cells are normally non-dividing, they can be triggered to proliferate as a result of injury, or weight-bearing exercise. Under either of these circumstances, muscle satellite cells give rise to myogenic precursor cells, which then differentiate into the myofibrils that typify skeletal muscle. A group of transcription factors called myogenic regulatory factors (MRPs) play important roles in these differentiation events. The so-called primary MRFs, MyoD and Myf5, help regulate myoblast formation during embryogenesis. The secondary MRFs, myogenin and MRF4, regulate the terminal differentiation of myofibrils (Seale and Rudnicki, 2000).

With regard to satellite cells, scientists have been addressing two questions. Are skeletal muscle satellite cells true adult stem cells or are they instead precursor cells? Are satellite cells the only cell type that can regenerate skeletal muscle. For example, a recent report indicates that muscle stem cells may also occur in the dorsal aorta of mouse embryos, and constitute a cell type that gives rise both to muscle satellite cells and endothelial cells. Whether the dorsal aorta cells meet the criteria of a self-renewing muscle stem cell is a matter of debate 9 De Angelis et aL, 1999).

Another report indicates that a different kind of stem cell, called an SP cell, can also regenerate skeletal muscle may be present in muscle and bone marrow. SP stands for a side population of cells that can be separated by fluorescence-activated cell sorting analysis. Intravenously injecting these muscle-derived stem cells restored the expression of dystrophin in mdx mice. Dystrophin is the protein that is defective in people with Duchenne's muscular dystrophy; mdx mice provide a model for the human disease. Dystrophin expression in the SP cell-treated mice was lower than would be needed for clinical benefit. Injection of bone marrow- or muscle-derived SP cells into the dystrophic muscle of the mice yielded equivocal results that the transplanted cells had integrated into the host tissue. The authors conclude that a similar population of SP stem cells can be derived from either adult mouse bone marrow or skeletal muscle, and suggest "there may be some direct relationship between bone marrow-derived stem cells and other tissue- or organ-specific cells" (Gussoni et al, 1999). Thus, stem cell or progenitor cell types from various mesodermally-derived tissues may be able to generate skeletal muscle.

Epithelial Cell Precursors in the Skin and Digestive System. Epithelial cells, which constitute 60 percent of the differentiated cells in the body are responsible for covering the internal and external surfaces of the body, including the lining of vessels and other cavities. The epithelial cells in skin and the digestive tract are replaced constantly. Other epithelial cell populations — in the ducts of the liver or pancreas, for example — turn over more slowly. The cell population that renews the epithelium of the small intestine occurs in the intestinal crypts, deep invaginations in the lining of the gut. The crypt cells are often regarded as stem cells; one of them can give rise to an organized cluster of cells called a structural-proliferative unit (Slack, 2000)..

The skin of mammals contains at least three populations of epithelial cells: epidermal cells, hair follicle cells, and glandular epithelial cells, such as those that make up the sweat glands. The replacement patterns for epithelial cells in these three compartments differ, and in all the compartments, a stem cell population has been postulated. For example, stem cells in the bulge region of the hair follicle appear to give rise to multiple cell types. Their progeny can migrate down to the base of the follicle where they become matrix cells, which may then give rise to different cell types in the hair follicle, of which there are seven (Ghazizadeh and Taichman, 2001). The bulge stem cells of the follicle may also give rise to the epidermis of the skin(Taylor et al, 2000).

Another population of stem cells in skin occurs in the basal layer of the epidermis. These stem cells proliferate in the basal region, and then differentiate as they move toward the outer surface of the skin. The keratinocytes in the outermost layer lack nuclei and act as a protective barrier. A dividing skin stem cell can divide asymmetrically to produce two kinds of daughter cells. One is another self-renewing stem cell. The second kind of daughter cell is an intermediate precursor cell which is then committed to replicate a few times before differentiating into keratinocytes. Self- renewing stem cells can be distinguished from this intermediate precusor cell by their higher level of βl integrin expression, which signals keratinocytes to proliferate via a mitogen-activated protein (MAP) kinase (Zhu et al, 1999). Other signaling pathways include that triggered by -catenin, which helps maintain the stem-cell state (Zhu and Watt, 1999), and the pathway regulated by the oncoprotein c-Myc, which triggers stem cells to give rise to transit amplifying cells (Gandarillas and Watt, 1997).

Stem Cells in the Pancreas and Liver. The status of stem cells in the adult pancreas and liver is unclear. During embryonic development, both tissues arise from endoderm. A recent study indicates that a single precursor cell derived from embryonic endoderm may generate both the ventral pancreas and the liver (Deutsch et al, 2001). In adult mammals, however, both the pancreas and the liver contain multiple kinds of differentiated cells that may be repopulated or regenerated by multiple types of stem cells. In the pancreas, endocrine (hormone-producing) cells occur in the islets of Langerhans. They include the beta cells (which produce insulin), the alpha cells (which secrete glucagon), and cells that release the peptide hormones somatostatin and pancreatic polypeptide. Stem cells in the adult pancreas are postulated to occur in the pancreatic ducts or in the islets themselves. Several recent reports indicate that stem cells that express nestin — which is usually regarded as a marker of neural stem cells — can generate all of the cell types in the islets (Lumelsky et al, 2001; Zulewski et al;., 2001).

The identity of stem cells that can repopulate the liver of adult mammals is also in question. Recent studies in rodents indicate that HSCs (derived from mesoderm) may be able to home to liver after it is damaged, and demonstrate plasticity in becoming into hepatocytes (usually derived from endoderm) (Lagasse et al, 2000; Petersen et al, 1999; Temple and Alvarez-Buylla, 1999). But the question remains as to whether cells from the bone marrow normally generate hepatocytes in vivo. It is not known whether this kind of plasticity occurs without severe damage to the liver or whether HSCs from the bone marrow generate oval cells of the liver (Crosby and Strain, 2001). Although hepatic oval cells exist in the liver, it is not clear whether they actually generate new hepatocytes (Sell, S. (1990; Thorgeirsson, 1993). Oval cells may arise from the portal tracts in liver and may give rise to either hepatocytes 9 Dabeva, M.D. and Shafritz, 1993; Lazaro et al, 1998) and to the epithelium of the bile ducts (Germain et al, 1988; Sirica et al; 1990). Indeed, hepatocytes themselves, may be responsible for the well-know regenerative capacity of liver.

B. HMG-CoA Reductase Inhibitors

HMG-CoA reductase catalyzes the conversion of hydroxymethylglutaryl-CoA to mevalonic acid, an early rate-limiting step in cholesterol biosynthesis. Particular HMG-CoA reductase inhibitors that can be used to improve the survival and to facilitate the expansion of stem cells include statins. Statins that are contemplated as being useful with the present invention include, but are not limited to, atorvastatin, lovastatin, rosuvastatin, fluvastatin, pravastatin, simvastatin, and cerivastatin. The chemical formulas for these statins are illustrated below:

Simvastatin (ZOCOR)

Lovastatin (MEVACOR)

Pravastatin sodium (PRAVACHOL)

Fluvastatin sodium (LESCOL)

Cerivastatin sodium (BAYCOL)

Atorvastatin calcium (LIPITOR)

It is contemplated that HMG-CoA reductase inhibitors can be used alone, or in combination with, the other compounds to use the methods and compositions of the present invention. Additionally, it is also contemplated that derivative of HMG-CoA reductase inhibitors can be used with the methods and compositions disclosed in this specification.

1. Second Generation Compounds

In addition to the compounds described above, the inventor also contemplates that other sterically similar compounds may be formulated to mimic the key portions of HMG-CoA reductase inhibitors. The generation of further structural equivalents or mimetics may be achieved by the techniques of modeling and chemical design known to those of skill in the art. The art of computer-based chemical modeling is now well known. Using such methods, a chemical compounds acting in a similar manner as an HMG-CoA reductase inhibitor can be designed and synthesized. It will be understood that all such sterically similar constructs and second generation molecules fall within the scope of the present invention.

2. Combination Therapy

In order to increase the effectiveness of a treatment with the compositions of the present invention, it may be desirable to combine these compositions with other therapies effective in the treatment or prevention of the methods and compositions disclosed in this specification.

For example, one may use HMG-CoA reductase inhibitors in combination with other known agents, compounds, or drugs for use in protecting implanted stem cells or expanding a stem cell population. For instance, it may prove beneficial to deliver an immunosuppressive therapy to a transplant recipient to prevent rejection of implanted stem cells. A general approach to transplant immunosuppression is to combine agents in small doses so as to get an added immunosuppressive effect, but without individual side effects of the different drugs. Commonly used agents include azathioprine, corticosteroids and cyclosporin are combined in a variety of protocols.

In other non-limiting embodiments, HMG-CoA reductase inhibitors can be used in combination with other known agents, compounds, or drugs that are used to treat neuro inflammatory diseases such as, for example, MS, Alzheimer's disease, Parkinson's disease, Landry-Guillain-Barre-Strohl syndrome, multiple sclerosis, stroke, viral encephalitis, acquired immunodeficiency disease (AIDS)-related dementia amyotrophic lateral sclerosis, brain trauma, or spinal cord disorders.

The compositions of the present invention can precede or follow the other agent, drug, or compound treatment by intervals ranging from minutes to weeks. It is contemplated that one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed where a compositions including a composition contemplated by the present invention is "A" and the HMG-CoA reductase inhibitor, and is "B" is the other known agent, compound, or drug:

A/B/A B/A/B B/B/A AIAIB AIBIB B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B AIBIAJB AJBIBIA B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A. C. Expansion and Protection of Stem Cells

1. Obtaining Stem Cells

Depending on the source of stem cells, one will employ different methods to obtain them. For example, blastocyst embryonic stem cells, are considered to be the most versatile type of stem cell because they can become almost any type of cell in the body. The first success at culturing human embryonic stem cells was reported in 1998. Fetal stem cells are pluripotent cells found in fetal brain tissue. Umbilical cord blood stem cells are multipotent stem cells from umbilical cord blood and have the potential to turn into many different types of cells, but their natural fate is to become blood and immune cells. Adult stem cells come in many different types, each of which is responsible for developing into the cells of a certain type of tissue.

Stem cells may come from the patient (autologous), an identical twin (syngeneic), or someone other than the patient (allogeneic). Allogeneic stem cells are further classified by whether the individual donating the stem cells is related or unrelated to the patient.

For autologous or syngeneic transplants in adults, one will simply identify the most relevant source for the stem cell and obtain those cells. A common scenario for autologous transplants involves obtaining bone marrow cells which can repopulate a patient's blood cells. In ablative chemo- or radiotherapy, the hematopoietic system can be damaged or destroyed. Thus, in order to ensure that the patient survives the treatment, BM stems cells are removed prior to treatment, and then returned to the patient following treatment. In other cases, one can perform allogenic stem cell transplants, with cells of one subject being transferred to another. As with organ transplants, stem cell transplants can be rejected by the recipient's immune system. Therefore, the transplanted stem cells must match the recipient closely enough that they won't be recognized as intruders. Immunosuppressive agents also may be used To determine whether the donor is a good immunological match with the recipient, a tissue typing test is performed using blood samples from both individuals. This test identifies certain proteins, called HLA antigens, which reside on the surfaces of specific immune cells. If the donor and the recipient have identical HLA antigens, they are a good match. One of the most common stem cell transplants is a bone marrow transplant (BMT) or peripheral blood stem cell transplant (PBSCT).

Bone marrow harvesting involves collecting stem cells with a needle placed into the soft center of the bone, the marrow. Most sites used for bone marrow harvesting are located in the hip bones and the sternum. The procedure takes place in the operating room. In PBSC harvesting, the donor will receive a growth factor, usually, G-CSF, a few days before collecting the stem cells. This forces the stem cells from the bone marrow into the general circulation. A catheter is placed (or an IV lines are placed into veins in each arm) and the donor's blood is removed. The stems cells are removed and the blood is returned to the donor in a process called apheresis. 2. Culturing and Expanding Stem Cell Populations

A variety of protocols for culturing stem cells have been described. The following U.S. Patents which describe such methods are hereby incorporated by reference: 6,887,706, 6,875,608, 6,863,900, 6,833,269, 6,800,480, 6,777,233, 6,777,231, 6,787,353, 6,759,039, 6,667,034, 6,642,048, 6,589,728, 6,562,619,

6,541,024, 6,506,574, 6,498,018, 6,495,365, 6,458,589, 6,410,320, 6,331,406,

6,326,198, 6,261,549, 6,245,566, 6,184,035, 6,165,785, 6,117,675, 6,107,543,

6,090,622, 6,060,270, 6,040,180, 6,033,906, 6,001,654, 5,922,597, 5,908,782,

5,851,832, 5,843,780, 5,817,773, 5,763,266, 5,753,506, 5,750,376, 5,736,396, 5,728,581, 5,670,372, 5,670,351, 5,670,147 and 5,668,104.

3. Transplants

A variety of transplants are currently performed and may involve or be improved by the use of stem cells. For example, bone marrow transplants (following chemo- or radiotherapy), corneal transplant, kidney islet cell transplants (for the treatment of diabetes), neuronal cells (for the treatment of neurodegenerative disease or spinal cord injury), skin cells (in burn grafts), and cardiac cells (to treat ischemic heart disease).

D. Treatment of Neurodegenerative Diseases

In another aspect of the present invention, there are provided methods of treating neurodegenerative diseases. The methods comprise administering to the patient a therapeutically effective amount of an HMG-CoA reductase inhibitor, or a derivative thereof. The HMG-CoA reductase inhibitor can be a statin. In non- limiting aspects, the statin can be atorvastatin, lovastatin, rosuvastatin, fluvastatin, pravastatin, simvastatin, or cerivastatin. Non-limiting examples of these types of treatment methods are disclosed in U.S. Patent No. 6,511,800, and PCT Application No. PCT/US04/43432, both of which are incorporated into this document by reference.

1. Multiple Sclerosis

Multiple Sclerosis (MS) is one of the most common diseases of the central nervous system (brain and spinal cord). It is an inflammatory condition associated with demyelination, or loss of the myelin sheath. Myelin, a fatty material that insulates nerves, acts as insulator in allowing nerves to transmit impulses from one point to another. In MS, the loss of myelin is accompanied by a disruption in the ability of the nerves to conduct electrical impulses to and from the brain and this produces the vaiious symptoms of MS, such as impairments in vision, muscle coordination, strength, sensation, speech and swallowing, bladder control, sexuality and cognitive function. The plaques or lesions where myelin is lost appear as hardened, scar-like areas. These scars appear at different times and in different areas of the brain and spinal cord, hence the term "multiple" sclerosis, literally meaning many scars.

Currently, there is no single laboratory test, symptom, or physical finding that provides a conclusive diagnosis of MS. To complicate matters, symptoms of MS can easily be confused with a wide variety of other diseases such as acute disseminated encephalomyelitis, Lyme disease, HIV-associated myelopathy, HTLV-I-associated myelopathy, neurosyphilis, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, polyarteritis nodosa, Sjogren's syndrome, Behcet's disease, sarcoidosis, paraneoplastic syndromes, subacute combined degeneration of cord, subacute myelo-optic neuropathy, adrenomyeloneuropathy, spinocerebellar syndromes, hereditary spastic paraparesis/primary lateral sclerosis, strokes, tumors, arteriovenous malformations, arachnoid cysts, Arnold-Chiari malformations, and cervical spondylosis. Consequently, the diagnosis of MS must be made by a process that demonstrates findings that are consistent with MS, and also rules out other causes.

Generally, the diagnosis of MS relies on two criteria. First, there must have been two attacks at least one month apart. An attack, also known as an exacerbation, flare, or relapse, is a sudden appearance of or worsening of an MS symptom or symptoms which lasts at least 24 hours. Second, there must be more than one area of damage to central nervous system myelin sheath. Damage to sheath must have occurred at more than one point in time and not have been caused by any other disease that can cause demyelination or similar neurologic symptoms. MRI (magnetic resonance imaging) currently is the preferred method of imaging the brain to detect the presence of plaques or scarring caused by MS.

The diagnosis of MS cannot be made, however, solely on the basis of MRI. Other diseases can cause comparable lesions in the brain that resemble those caused by MS. Furthermore, the appearance of brain lesions by MRI can be quite heterogeneous in different patients, even resembling brain or spinal cord tumors in some. In addition, a normal MRI scan does not rule out a diagnosis of MS, as a small number of patients with confirmed MS do not show any lesions in the brain on MRI. These individuals often have spinal cord lesions or lesions which cannot be detected by MRI. As a result, it is critical that a thorough clinical exam also include a patient history and functional testing. This should cover mental, emotional, and language functions, movement and coordination, vision, balance, and the functions of the five senses. Sex, birthplace, family history, and age of the person when symptoms first began are also important considerations. Other tests, including evoked potentials (electrical diagnostic studies that may reveal delays in central nervous system conduction times), cerebrospinal fluid (seeking the presence of clonally-expanded immunoglobulin genes, referred to as oligoclonal bands), and blood (to rule out other causes), may be required in certain cases.

2. Other Neurodegenerative Diseases

Other neurodegenerative diseases or conditions that are contemplated as being treatable and/or preventable with the methods and compositions disclosed throughout this specification include, but are not limited to, Alzheimer's disease, Parkinson's disease, Landry-Guillain-Barre-Strohl syndrome, stroke, viral encephalitis, acquired immunodeficiency disease (AIDS)-related dementia, amyotrophic lateral sclerosis, brain trauma, spinal cord disorders, adrenoleukodystrophy, Alexander disease, Canavan disease, Diffuse Cerebral Sclerosis of Schilder, Leukodystrophy — Globoid Cell, Leukodystrophy — Metachromatic, Neuromyelitis Optica, post infectious encephalomyelitis (including those that follow both virus infections and vaccination), progressive multifocal leukoencephalopathy, disorders affecting the peripheral nervous system, and transverse myelitis. Other inflammatory diseases discussed throughout the present specification and those known to a person of ordinary skill in the art are also contemplated as being treatable or preventable with the disclosed methods and compositions of the present invention.

E. Screening Methods Involving the Protection of Stem Cells In certain aspects, a statin can inhibit HMG-CoA Reductase which can result in a reduction of intermediates of the cholesterol pathway (e.g., isoprenoids). Isoprenoids include a large and diverse class of naturally occurring organic chemicals similar to terpenes. They are derived from five-carbon isoprene units that can be assembled and modified in thousands of different ways. A majority of isoprenoids are multicyclic structures which can differ from one another not only in functional groups, but also in their basic carbon skeletons. Isoprenoids can be used to modify proteins (e.g., small G-proteins (e.g., Ras, Rho, Rab, Ran and Arf) that are used in the cholesterol synthesis pathway.

It is contemplated by the inventors for identifying new compounds that can be used to protect stem cells by inhibiting or reducing the transfer of isoprenoids to proteins involved in the cholesterol synthesis pathway. A. Screening for Modulators of Isoprenoid Modification of Proteins

The present invention further comprises methods for identifying modulators of isoprenoid modification of proteins. These assays may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of isoprenoid modification of proteins.

By function, it is meant that one may assay for a measurable effect on isoprenoid modification of proteins activity. To identify a modulator, one generally will determine the activity or level of inhibition of isoprenoid modification of proteins in the presence and absence of the candidate substance, wherein a modulator is defined as any substance that alters these characteristics. For example, a method can generally include:

(a) providing a candidate modulator;

(b) admixing the candidate modulator with an isolated compound(s) (e.g., isoprenoids or proteins) or cell(s) expressing the compound(s);

(c) measuring one or more characteristics of the compound(s) (e.g., interaction between isoprenoids and proteins or modification of protein) or cell(s) in step (b); and

(d) comparing the characteristic measured in step (c) with the characteristic of the compound(s) or cell(s) in the absence of said candidate modulator, wherein a difference between the measured characteristics indicates that said candidate modulator is, indeed, a modulator of the compound(s) or cell(s). Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals.

It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

1. Modulators

"Candidate substance" includes any molecule that may be a "modulator" of isoprenoid/protein interaction or isoprenoid modification of a protein. A modulator may be a compound that overall effects the isoprenoid/protein interaction or isoprenoid modification of a protein (e.g. isoprenylation). This may be accomplished by inhibiting isoprenoid or protein synthesis, activity, expression, translocation or transport, function, expression, post-translational modification, location, half-life, or more directly by preventing its activity, such as by binding to the isoprenoid or the protein or both. A modulator may be an enhancer, which enhances or increases the interaction between isoprenoids and proteins or the modification of proteins, by increasing, for example, expression, translocation or transport, function, expression, post-translational modification, location, half-life, or more directly its activity. Any modulator described in methods and compositions herein may be an inhibitor or an enhancer.

The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. An example of pharmacological compounds include compounds that are structurally related to isoprenoids or the targeted protein. Using lead compounds to help develop improved compounds is know as "rational drug design" and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs, which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches. It also is possible to use antibodies to ascertain the structure of a target compound activator or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorial generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.

Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man- made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators. Other suitable modulators include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. Such compounds are well known to those of skill in the art. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors. In addition to the modulating compounds initially identified, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators. Such compounds, which may include peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators.

2. In vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.

One example of a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.

A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.

B. Diagnostic Methods

In some embodiments of the present invention, methods of screening for isoprenoid/protein interaction or isoprenoid modification of a protein maybe employed as a diagnostic method to identify subjects who need or are at the risk of needing stem cell protection. Isoprenoid/protein interaction or isoprenoid modification of a protein may be evaluated using any of the methods and compositions disclosed herein. Any other the compounds or methods described herein may be employed to implement these diagnostic methods.

F. Pharmaceutical Compositions

One embodiment of this invention includes methods of treating stem cells or progenitor cells using an HMG-COA reductase inhibitor, either ex vivo or in vivo. The invention optionally includes treating individuals with an immunosuppressive compound. An effective amount of the pharmaceutical compounds and compositions of the present invention, generally, is defined as that amount sufficient to detectably and repeatedly to improve stem cell survival, increase the number or quality of stem cell populations, and to inhibit graft rejection following transplant.

Pharmaceutical compositions of the present invention can include an HMG- COA reductase inhibitor. The phrases "pharmaceutical" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human. The preparation of a pharmaceutical composition including an HMG-COA reductase inhibitor will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, for animal {e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

'Therapeutically effective amounts" are those amounts effective to produce beneficial results in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 micro gram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The compositions of the present invention may comprise different types of earners depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.

The compositions may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments, the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

In certain embodiments, the compositions are prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

In certain embodiments, an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

The composition should be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrauterinely, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g.. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington's, 1990). EXAMPLES

Specific embodiments of the invention will now be further described by the following, nonlimiting examples which will serve to illustrate in some detail various features. The following examples are included to facilitate an understanding of ways in which the invention may be practiced. It should be appreciated that the examples which follow represent embodiments discovered to function well in the practice of the invention, and thus can be considered to constitute preferred modes for the practice of the invention. However, it should be appreciated that many changes can be made in the exemplary embodiments which are disclosed while still obtaining like or similar result without departing from the spirit and scope of the invention. Accordingly, the examples should not be construed as limiting the scope of the invention.

Example 1 - Materials and Methods

Chemicals and Antibodies. Guinea pig myelin basic protein (MBP), complete Freund 's adjuvant (CFA), DAPI and Hoechst 33342 stains, and murine anti- 2'3 '-cyclic nucleotide phosphodiesterase (CNP ase) (clone 11-5B) and anti-mouse myelin basic protein (MBP, clone 1:129-138), antibodies were purchased from Sigma (St. Louis, MO). DMEM (4.5-g/L glucose), and FBS were purchased from Invitrogen (Carlsbad, CA). Recombinant rat IFN -γ, TNF-α, and IL- lβ proteins were purchased from R&D Systems (Minneapolis, MN). Lovastatin was purchased from Calbiochem (San Diego, CA). Anti-mouse PDGF-αR antibodies were purchased from Research Diagnostic, Inc (Flanders, NJ). Rabbit anti-NG2 chondroitin sulfate proteoglycan, mouse anti-A2B5 and rabbit anti-β-actin antibodies were purchased from Chemicon International (Temecula, CA). Anti-rabbit glial fibrillary acidic protein (GFAP) antibodies were purchased from DAKO (Carpentaria, CA). Secondary antibodies; Texas red-X-conjugated goat anti-mouse IgG (for PDGF-αR, NG2, CNPase and MBP) and FITC-conjugated goat anti-rabbit IgG (for GFAP), and anti-isolectin B4 antibodies were purchased from Vector Lab Inc (Burlingame, CA).

Induction and clinical assessment of EAE. Female Lewis rats (Harlan

Laboratory, Harlan, IN) weighing 225-300 g were housed in the animal care facility at the Medical University of South Carolina (MUSC) throughout the experiment and were provided with food and water ad lib. AU experimental protocols were reviewed and approved by MUSCs Animal Ethics Committee. The procedures used for the induction of EAE and for lovastatin (LOV) treatment were the same as those described earlier (Paintlia et al, 2004). Four experimental groups were used: EAE with placebo treatment (EAE), EAE plus LOV treatment (E+LOV), control placebo treatment (Control), and control LOV treatment (C+LOV). In brief, EAE was induced via administration of 50 μg of MBP (dissolved in PBS; pH 7.4) emulsified in an equal volume of CFA (Sigma) in hind limb footpads (on the 1^st and 7^th day), and received an injection of vehicle (ip, placebo, 0.1% Triton X-IOO in PBS), each day. E+LOV animals were immunized similarly with MBP antigen and received LOV (ip, 2-mg/ kg-body-wt, dissolved in 0.1% Triton X-100/PBS) immediately prior to MBP administration. Thereafter, LOV was administered for the duration of experiment based upon the past experiences and studies which suggest that LOV pretreatment before the onset of EAE disease is required to exert its protective effects (Stanislaus et al, 2001). For control groups, an emulsion of CF A/PBS was injected in hind limb foot pads in animals. Then, control and C+LOV groups received daily injections (ip) of placebo and LOV, respectively. Two injections (one on each day) of pertussis toxin (ip, 200 ng) were given to each animal in each group on the 1^st and 2" day of immunization. Clinical signs in EAE animals manifested as ascending paralysis starting on the 9^th day of post immunization (dpi) onwards, resulting in death in most animals by the 13^th dpi. The clinical signs of EAE animals were scored by an experimentally blinded investigator as 0=normal; l=piloerection; 2=loss in tail tonicity; 3=hind leg paralysis; 4=paraplegia, and 5=moribund or dead. Five- animals/group in each experiment were sacrificed to collect their spinal cords (lumbar region) on peak clinical day (13^th dpi) and on remission (20^th dpi) because obvious and marked pathological changes related to the demyelination/remyelination process in the spinal cord are known to occur in diseased animals during these time points. Each experiment was repeated four times.

BrdU administration. Cellular proliferation was examined during the development of acute EAE and remission using the thymidine analog BrdU (50- mg/kg, ip, Sigma). Animals (n=3) from each group received a daily injection of BrdU for two days before being sacrificed; thus, the proliferation response was determined at the 13^th dpi and 20^th dpi.

Assessment of myelin breakdown and its restoration. Lumbar spinal cord tissues were dissected out carefully from each group animals as most of the pathological changes are detected in this region of the spinal cord in EAE. Lipids were extracted from ~50 mg of frozen spinal cord tissue from LOV-treated and untreated EAE animals and controls as described earlier (Khan et al, 1998). Sphingomyelin, free cholesterol, and cholesterol esters were quantitated by high performance thin layer chromatography followed by densitometry as described earlier (Khan et al, 2000). Both non-hydroxy and hydroxy forms of cerebrosides and sulfatides were analyzed and quantitated as described by Ganser et al (1988).

Histology, immunohistochemistry/iminunocytocliemistry assessments. For histological examination of the tissue from experimental animals, lumbar spinal cord tissues were fixed in 10% buffered formalin (Stephens Scientific, Riverdale, NJ) followed by sectioning of tissue. The sections were then stained with H&E and luxol fast blue using standard protocols. For single-label immunohistochemistry, standard methodology was used whereby sections were incubated with primary antibodies (1:100) followed by incubation with secondary antibodies (1 :100). Mouse IgG and rabbit polyclonal IgG were used as control antibodies. The sections were incubated with Texas red-conjugated IgG and FITC-conjugated IgG without primary antibodies for negative controls. The sections were analyzed by immunofluorescence microscopy (Olympus BX-60) with an Olympus digital camera (Optronics; Goleta, CA) using a dual-band pass filter. Images were captured and processed with Adobe PhotoShop 7.0 and were adjusted using the brightness and contrast to enhance image clarity.

For double-labeling with anti-NG2/anti-BrdU antibodies, the sections were incubated in IN HCl at 37⁰C for 30 min followed by PBS washings. The sections were incubated with anti-NG2 antibodies (1 :100) at 4⁰C overnight. After washing three times in PBS, the sections were incubated with secondary antibodies such as FITC-conjugated rabbit anti-IgGs antibodies for NG2 and mouse anti-BrdU Cy5- conjugated IgGs for BrdU followed by their analyses as described above. Total numbers of both NG2⁺/BrdU^" and NG2⁺/BrdU⁺ cells/field were determined by manual counting at magnifications 40Ox and 60Ox respectively in 10-fields/slide using tissue sections from 3 animals/group in blinded fashion. AU NG2⁺/BrdU^" and NG2⁺/BrdU⁺ cells within the white and grey matter were counted. Mean numbers of NG2⁺/BrdU^" and NG2⁺/BrdU⁺ cells/field were computed for statistical analysis among groups and plotted. Likewise, for immunocytochemistry analysis, live mixed glial cells obtained after treatment were immunostained with primary antibodies in slide chambers followed by secondary antibodies or were stained/treated for double labeling and analysis as described above.

Preparation of total RNA, GeneChip hybridization, and data analysis.

Total RNA from spinal cord tissues or cells was purified using "TRIZOL Reagent" (Invitrogen; Carlsbad, CA) and RNA cleaning kits (Qiagen; Valencia, CA) as described earlier (Paintlia et al, 2003). RNA from similarly treated animals (n= 5) was pooled in equal proportions to increase the sample size and to reduce individual disease variability among animals. RNA was converted to double stranded cDNA and then to biotinylated cRNA as described earlier (Paintlia et al, 2004). After confirming the quality of labeled cRNA with Affymetrix Test 2 arrays, it was hybridized to Affymetrix Rat U34A GeneChip^® arrays. These arrays had 8,800 gene transcripts including appropriate transcripts for housekeeping genes as well as negative and positive control genes. Hybridization, washing, staining with streptavidin- phycoerythrin, and scanning with a probe array scanner (Gene Array Scanner; Affymetrix) were performed at the UCI DNA Microarray facility (University of California, Irvine CA). GeneChip^® data were analyzed with Affymetrix Microarray Suite MAS 5.0 software and a one-sided wilcoxon's signed rank test was used to generate a detection p-value (<0.05) to determine statistical significance of transcript expression on the chip. The software generated, based on the p-value, a present (p<0.04), marginal (p<0.04 to p<0.05), or absent (p>0.05) call for each transcript. For comparison analysis, each probe set on the experimental array was compared with its counterpart on the baseline array to calculate the change in p-value that was used to generate the difference call of increase (I: p<0.04); marginal increase (MI: p<0.04 to p<0.06); decrease (D: p>0.997); marginal decrease (MD: ρ>0.992 to p>0.997); or no change (NC: p>0.06 to pθ.997). Comparison analysis generated a signal-log ratio algorithm for each probe-pair on the experimental array to the corresponding probe pair across the baseline array. This strategy cancels out differences due to different probe-binding coefficients. Software computed the signal log ratio number by using a one-step Tukey's Biweight method by taking a mean of the log-ratio of probe-pair intensities across the two arrays. Two GeneChips^c /group were used to repeat microarray analyses in two independent experiments. Fold-changes were computed compared to control for each expressed transcript using standard formulae as described earlier (Paintlia et al, 2004). The changes were considered significant if the magnitude of change was at least 1.5-fold or greater and with p-value <0.05 between experimental groups. cDNA synthesis and real-time PCR™ analyses. Single stranded cDNA was synthesized from pooled RNA samples from similarly treated animals/cells using a superscript pre-amplification system for first strand cDNA synthesis (Invitrogen;

Carlsbad, CA) as described earlier (Paintlia et al, 2003). Real-time PCR™ was performed for some of the genes listed in Table 1 using IQ™ SYBR Green Supermix and BIO-RAD Laboratories iCycler iQ RT-PCR (Bio-Rad; Hercules, CA). Thermal cycling conditions were as follows: activation of iTaq™ DNA polymerase at 95⁰C for 10 min, followed by 40 cycles of amplification at 95⁰C for 30s and 55-58⁰C for 30-

60s. The normalized mRNA expressions were computed with GAPDH expression as described previously (Paintlia et al., 2003).

Table 1: List of primer sequences used for real-time PCR analysis

Name Primer Sequence SEQ ID NO:

Glyceraldehyde-3 -phosphate FP: 5'-cctacccccaatgtatccgttgtg-3' 1 dehydrogenase (GAPDH) RP: 5'-ggaggaatgggagttgctgttgaa-3 2

Myelin basic protein (MBP) FP: 5'-ctctggcaaggactcacacac-3' 3

RP: 5'-tctgctgagggacaggcctctc-3' 4

Proteolipid protein (PLP) FP: 5'-gccttccctagcaagacctctgag-3' 5

RP: 5'-gaacttggtgcctcggcccatgag-3' 6

Myelin oligodendrocyte FP: 5'-cagagaccactcctaccaag-3' 7 glycoprotein (MOG) RP: 5 '- ttctgcacggagttttcctct-3 ' 8

Myelin associated glycoprotein FP: 5'-tgccatcctgattgccattg-3' 9 (MAG) RP: 5'-ctcatacttatcaggtgctcc-3^! 10 α-platelet derived growth factor FP : 5 ' -cagacattgaccctgttccagagg-3 ' 11 receptor (PDGF-αR) RP: 5'-gaatctatgccaatatcatccatc-3^! 12

Brain derived growth factor FP: 5'-tacacgaaggaaggctgca-3' 13 (BDNF) RP: 5'-cgaacatacgattgggtagtt-3' 14

Ciliary Neurotrophic Factor FP: 5'-cttcaagagctctcacagtg-3' 15 (CNTF) RP: 5'-tgcttatctttggccccataat-3' 16

Leukemia inhibitory factor (LIF) FP: 5'-tctgtgcaacaagtaccatgt-3' 17

RP: 5'-gcagcccaacttcttcctt-3' IS

Platelet derived growth factor- α FP: 5'-gatgccttggagacaaacctgaga-3' 19 (PDGF) RP: 5'-atacttctcttcctgcgaatgggc-3' 20

Glial derived growth factor- FP: 5'-gagagtatatgtgcaaagtgatc-3' 21 (GGF-2) RP: 5'-ctcagtcgaggctggca-3' 22

SOXlO FP: 5'-tctacacggccatctctgacc-3' 23

RP: 5'-gtcgtatatactggctgttcccagtg-3' 24

Sonic hedgehog homolog (Shh) FP: 5'-ggtggcaccaagttagtgaaggat-3' 25

RP: 5'-cgtagaagaccttcttggcacctt-3' 26

Myelin transcription factor 1 -like FP: 5'-ggtgcccaagagcaaagaa-3' 27 (MyTl-L) RP: 5'-atcacagccaggtaccgga-3' 28

GTX FP: 5'-gataaggatggcaagaagaaaca-3' 29

RP: 5'-cagagagtaggcaagccg-3' 30

Glial Fibrillary Acidic Protein FP: 5 ' -ccaagccagacctcacagc-3 ' 31 (GFAP) RP: 5'-ccgataccactcttctgtttcttg-3' 32

Peroxisome Proliferation FP: 5'-agctggtcactgaacacg-3' 33 Activator Receptor-δ (PPAR-δ) RP: 5'-gccttagtacatgtccttgta-3' 34

Note: FP; Forward primer and RP; Reverse primer

Immunoblotting. Immunoblotting of spinal cord tissue and cells was carried out as described earlier (Paintlia et al, 2004). Briefly, tissues were homogenized/or cells were lysed in ice cold lysis buffer (50 mm Tris-HCl, pH 7.4, containing 5OmM NaCl, ImM EDTA, 0.5mM EGTA, 10% glycerol, and protease inhibitors mixture). Twenty microgram of protein/lane was separated by 10% SDS-PAGE and blotted to nitrocellulose (Amersham, UK). Immunoblots were incubated with primary antibodies (1 :1,000) followed by incubation with secondary peroxidase-conjugated antibodies (1 :10,000; Sigma). Immunoreactivity was detected using the enhanced chemiluminescence detection method according to the manufacturer's instructions with subsequent exposure of immunblot to X-Ray films (Amersham, UK), followed by autoradiography.

Cortical mixed glial cell cultures and treatment. Rat cortical mixed glial cell cultures were generated from P1-P2 SD rat brains (Charles River, Wilmington, MA) and OPs were purified from mixed glial cultures as described earlier (McCarthy and de Vellis, 1980). Purity of OPs was determined by FACS analysis using anti- A2B5 (OPs), anti-GFAP (astrocytes), and anti-isolectin B4 (microglia) antibodies using standard protocols, which showed -95% purity. Mixed glial cells at a density of 1x10 -cells/slide were plated on glass chamber slides precoated with poly-D-lysine. After 24 hr, the fresh DMEM without FBS was changed and cells were pretreated with LOV (1 μM) for 24 hr prior to addition of cytokine mixture (CM: TNF-α, IL-I β, and IFN -γ; each 10 ng/ml). The proliferation of OPs was determined by immunostaining for PDGF-αR and NG2 antigens expressed by dividing OPs at days in vitro (DIV) 2 and DIV3 respectively. The differentiation of oligodendrocytes was determined by immunostaining for Ol and MBP at DIV5 and DIV6 respectively. The number of positive cells was counted manually in 1 O-fields/slide as described above. Similarly, the double-immunostaining for GFAP and MBP was performed to quantify reactive gliosis and survival of differentiating oligodendrocytes at DIV6. For mRNA and protein expression analysis, cells were treated with CM and LOV in 100-mm plates, and harvested after 12 hr (mRNA) or 48 hr (protein) of post-treatment. All experiments were repeated 3-4 times.

Quantification of immunofluorescence intensity. The fluorescence in the different areas of slides immunostained with anti-MBP, anti-CNPase, or anti-GFAP antibodies was measured by using Image-Pro Plus (Media Cybernetics, Silver Springs, MD). The fluorescence intensity signals were plotted directly or classified as "weak" if the maximum peak level was below 130, "intermediate" if peak level was between 130-200, "moderate" if levels of half of the peaks were greater than 200 and "strong" if all peak levels were greater than 200. A distance scale of 20 μm was chosen for measurement in all groups.

Thymidine uptake analysis. The purified primary OPs (lxlO⁴-cells/ml) were pre-incubated with/without LOV (1 μM) for 24 hr in 96-well plates followed by incubation in conditioned media, obtained from mixed glial cell cultures treated similarly with LOV and CM for 24 hr as described above and then 0.5 μCi of methyl-

[³H] thymidine was added into each well. Media was removed after 16-18 hr later and cells were detached with the addition of 100-μl of 0.25% trypsin in Ca²⁺/Mg²⁺-free

PBS containing 5.5-mM glucose. Cells were harvested onto glass fiber filters using a cell harvester (InfoTech AG; Switzerland) and the filters were washed thoroughly with water and counted in a MicroBeta system (Wallac; Finland).

Statistical analysis. Using the Student's unpaired t-test and ANOVA

(Student-Newman-Keuls: comparison of all pairs of columns), p-values were determined for respective experiments from three independent experiments using GraphPad software (GraphPad Software Inc., San Diego, CA). A p<0.05 was used as the criterion for statistical significance.

Example 2 - Results

Lovastatin treatment attenuates myelin breakdown and facilitates its restoration. LOV-treated EAE animals had lower clinical scores (3.5 + 0.47) compared to EAE (4.6 + 0.57) animals on peak clinical day (E+LOV) and restored neurological functions by the 20^th dpi (E+LOV/R) in addition to having by the 45^th dpi a body weight similar to healthy controls (FIGS. 9A-B). However, a relapse with small clinical scores was also observed between the 21^st to 26* dpi in 10% of LOV- treated recovering animals. Histological examinations of the spinal cord sections showed less demyelination and inflammatory cell infiltration in the white matter region of E+LOV animals when compared with EAE animals (Supplementary FIGS. 9 C-E). Consistent with the previous observations, recovered E+LOV animals showed no cellular infiltration or demyelination in the white matter region of spinal cord (Paintlia et al, 2004). To correlate these histological changes with myelin breakdown and its repair, the ratio of myelin lipid: protein was determined in each group of animals. There was a significant (p<0.001) increase in cholesterol ester in EAE animals when compared with controls (FIG. IA). Cerebrosides, sulfatides, and sphingomyelin lipids were significantly decreased in EAE animals compared with controls (FIGS. IB-D). Conversely, E+LOV animals showed no significant change in myelin lipids when compared with controls (Fig. IA-D). Notably, no significant change in the levels of cholesterol was observed in animals from each group (FIG. ID). The levels of myelin lipids were close to healthy controls in recovered E+LOV animals (data not shown). Together, these data demonstrate that LOV limits the breakdown of the myelin sheath and improves its restoration in treated EAE animals.

Lovastatin treatment enhances the survival and differentiation of oligodendrocytes. Axonal loss and demyelination are the major hallmarks of EAE/MS lesions as a result of mononuclear infiltration into the CNS, which is responsible for multiplicity of neurological deficits in sick animals (Brack et cil., 2003). Because, LOV attenuates the progression of disease and restores the levels of myelin lipids in treated EAE animals, the inventors next determined the expression of myelin proteins associated with remyelination. First, the inventors performed microarray GeneChip analysis which revealed the differential expression of mRNA for myelin-proteins in LOV-treated and untreated EAE animals relative to controls (Table 2). These observations were further confirmed by immunoblotting, immunohistochemistry, and real-time PCR™ analyses. Corresponding to microarray analysis, there were low levels of MBP and CNPase proteins in EAE animals compared to controls (FIG. 2A). The levels of these myelin proteins were similar to controls in LOV-treated EAE animals. Consistent with these findings, the immunohistochemistry analysis also showed more demyelinated regions (arrowheads) as indicated by weak immunofluorescence for both MBP (left panel) and CNPase (right panel) in the white matter of EAE animals compared to controls (FIG. 2B). Relatively fewer demyelinated regions were seen in the white matter of E+LOV animals when compared with recovered E+LOV animals or controls. Furthermore, quantification of the immunofluorescence intensity signal also showed a significant decrease in signal for MBP and CNPase in EAE animals when compared with controls (FIG. 2C). Although, the intensity of the signal for these myelin proteins was significantly less in E+LOV animals compared to controls, it was significantly greater compared to EAE animals. These results were in accordance with the mRNA expression for MBP, PLP, MOG, and MAG proteins which was significantly less in EAE animals compared with controls (FIG. 2D-G). Parallel to immunohistochemistry studies, the mRNA expression for these proteins was significantly less in E+LOV animals compared with controls, but was significantly higher than EAE animals (FIG. 2D-G). There was no significant change in the mRNA expression for MOG and MAG proteins between recovered E+LOV animals and controls, except for MBP and PLP mRNA expressions. Corresponding with these results, the expression of transcription factors such as MyTl-L, GTX, and PPAR-δ, associated with the differentiating oligodendrocytes was also determined. Microarray analysis demonstrated the increase in expression of MyT2 mRNA, a transcription factor similar to MyTl-L in LOV- treated EAE animals compared to EAE animals corresponding to controls (Table 2). Real-time PCR™ analysis further corroborated these data and demonstrated a significant increase in expression of MyTl-L in LOV-treated EAE animals compared to EAE and control animals (FIG. 2H). In addition, the expression of GTX mRNA was significantly elevated in LOV-treated EAE animals compared to EAE animals (FIG. 21). Likewise, the expression of PPAR-δ mRNA was also significantly elevated in E+LOV animals compared to EAE animals (FIG. 2J and Table 2). However, the expression for PPAR-δ mRNA was significantly higher in E+LOV and EAE animals when compared with controls. Collectively, these changes in expression for myelin- proteins and transcription factors are indicative of enhanced remyelination mediated through increased survival and differentiation of postmitotic oligodendrocytes in the spinal cord of LOV-treated EAE animals. Table 2: Changes in expression of message for myelin proteins associated with proliferation and differentiation of oligodendrocyte progenitors in CNS of lovastatin-treated/-ιmtreated EAE and control animals

Gene Bank Gene Name 'C+LOV vs. 'EAE' vs. 'E+LOV VS. Ε+LOV/R'

Accession 'CON' 'CON' 'CON' vs. 'CON'

No. Fold Change Fold change Fold Change Fold Change

(«=2) («=2) (n=2) («=2)

D28111 Myelin-associated NC -6.4^a -2.0^a -1.5^a oligodendrocytic basic protein (MOBP)

M11185 Proteolipid protein (PLP) NC -6.2^a -3.0^a -1.6

M99485 Myelin oligodendrocyte NC -3.0^a -1.5^a NC glycoprotein (MOG)

M22357 Myelin-associated NC -6.0^a -2.0^a NC glycoprotein (MAG) K03242 Myelin protein zero NC -8.0^a -3.0^a NC

(Charcot-Marie-Tooth neuropathy IB)

U31367 Myelin protein MVP 17 NC -6.4'¹ -2.2^a NC

U67081 C2-HC type zinc finger NC -1.8^a 6.0^a 5.0^a protein r-MyT2 (MyT2)

U40064 Peroxisome proliferators 2.4^a 2.6^a 2.8^a NC activated receptor, δ

(PPAR-δ)

Data are expressed as average mean of fold change in expression compared with controls for each gene transcript from two independent experiments. a: Change in expression was significant with fold change >1.5. NC: No change in expression.

Lovastatin treatment enhances the proliferation and recruitment of OPs;

Furthermore, to understand the effect of LOV on proliferation and recruitment of OPs in the spinal cord of treated EAE animals, the inventors performed immunohistochemistry analysis using anti-NG2 and anti-BrdU antibodies. The inventors observed the scattered distribution of NG2⁺/BrdU^" cells (arrowheads) in both white and grey matter of spinal cord in each group of animals (FIG. 3A). Manual counting revealed a significant decrease in the number of NG2⁺/BrdU^" cells/field in EAE animals when compared with controls (FIG. 2B). Interestingly, LOV treatment significantly increased the number of NG2⁺/BrdU^" cells/field in recovered E+LOV animals compared with controls. Likewise, NG2⁺/BrdU⁺ cells were also found to be scattered throughout the grey and white matter of spinal cord in each group of animals (FIG. 3C). There was a significant decrease in NG2⁺/BrdU⁺ cells/field in EAE animals compared to controls (FIG. 3D). Interestingly, similar to the NG2⁺/BrdU^" cell counts, NG2⁺/BrdU⁺ cells/field were also increased significantly in recovered E+LOV animals when compared with controls (FIG. 3D).

The expression of NG2 is also reported in other cell types such as endothelial cell and smooth muscle cell progenitors (Espinosa-Heidmann et al, 2003), therefore, the inventors next validated these data by immunoblotting of spinal cord tissue homogenates with anti-A2B5 and anti-PDGF-αR antibodies, which recognize gangliosides (Asakura et al. , 1998) or glycoprotein (Bolot et al. , 2003), and PDGF-αR (Baron et al, 2002) proteins, respectively, expressed in dividing OPs. There was a relative decrease in both A2B5 glycoprotein (a major band of 70 kDa was observed in addition to several minor bands of greater than 120 kDa and between 30 to 40 kDa) and PDGF-αR (150 IcDa) proteins in EAE animals compared to LOV-treated EAE animals and controls (Fig. 3E). The protein level of PDGF-αR was increased (~4- fold) in recovered E+LOV animals compared to controls. Lysates of purified OPs and matured oligodendrocytes (cultured OPs for 6 days in vitro) were included as positive and negative controls, respectively (data not shown). Corresponding with this, the expression of PDGF-αR mRNA was also significantly increased in recovered E+LOV animals compared with controls (FIG. 3F). In contrast, the expression of PDGF-αR mRNA was significantly decreased in EAE animals (FIG. 3F). Parallel to these findings, the expression of SOXlO, a transcription factor which is expressed exclusively in dividing OPs was significantly decreased in EAE animals as compared to controls (FIG. 3G). Although, the expression of SOXlO mRNA was slightly lower in E+LOV animals compared to controls, it was significantly more when compared with EAE animals. Corresponding with PDGF-αR expression, the SOXlO mRNA expression was significantly higher in recovered E+LOV animals compared to controls. Furthermore, the mRNA expression of Shh, a pre-OP specific transcription factor also followed the same trend in EAE animals versus controls except in E+LOV animals in which no significant difference was observed when compared with controls (FIG. 3H). Altogether, these data are suggestive of a significant increase in both proliferation and recruitment of OPs in the spinal cord of LOV-treated EAE animals, especially on remission.

Lovastatin enhances the survival and proliferation of OPs in cultures of activated mixed glial cells. The in vivo studies described above indicate that LOV improves the proliferation, recruitment, and differentiation of OPs in treated EAE animals. These findings were further evaluated by in vitro studies with primary rat mixed glial cell cultures. These cells were treated with a cytokine mixture (CM; IFN- γ, TNF-α, and IL-I β) in the presence/absence of LOV for different time points. Mixed glial cells were preferred for this study because cell-to-cell interactions are needed for OPs with astrocytes for their proliferation/differentiation, and treatment with CM mimics inflammatory disease states akin to that observed in EAE/MS brain. Immunocytochemical analysis revealed that the majority of OPs were PDGF-αR⁺ as compared to differentiating CNPaSe⁺ or MBP⁺ oligodendrocytes at 0 hr in mixed glial cell cultures (data not shown). There was a significant decrease in PDGF-CtR⁺ cells/field in CM-treated cells after 24 hr (DIV2) of stimulation when compared with control cells, whereas LOV pretreatment significantly increased the number of PDGF- OtR⁺ cells in CM+LOV-treated cells as compared to CM-treated and control cells (FIG. 4A). PDGF-αR⁺ cells were bipolar (arrowheads) and clustered in all groups except in the CM-treated group as demonstrated by immunocytochemistry (FIG. 4B). A similar trend was observed when cells were immunostained with anti-NG2 antibodies followed by manual counting. NG2⁺ cells/field were significantly decreased in CM-treated cells after 48 hr (DIV3) of stimulation when compared with controls, whereas LOV pretreatment significantly increased the number of NG2⁺ cells in CM+LOV-treated cells as compared to CM-treated and control cells (FIG. 4C). Like PDGF-OtR⁺ cells, NG2⁺ cells demonstrated an increase in the number of processes (arrowheads) in all groups except those in the CM-treated group (FIG. 4D). Consistent with protein expression for PDGF-αR and NG2, PDGF-αR mRNA expression was also decreased significantly in CM-treated cells, but increased significantly in CM+LOV-treated cells when compared with both CM-treated and control cells (FIG. 4E). Interestingly, both of these qualitative and quantitative analyses revealed the attenuation of cytokine mediated loss of OPs and in turn an increase in proliferation of OPs in CM+LOV-treated cells. To further confirm these findings, the inventors examined OPs proliferation by using thymidine uptake analysis. The inventors observed an increase in OP proliferation in mixed glial cells; therefore, the inventors preferred to use conditioned media obtained from similarly treated mixed glial cells for thymidine uptake analysis. Thymidine uptake was increased significantly in primary OPs cultured in CM+LOV-treated cell culture conditioned media, but this was significantly lower in primary OPs cultured in CM- treated culture conditioned media when compared with control glial cell culture conditioned media (FIG. 4F). No significant change was observed for thymidine uptake in OPs cultured in conditioned media obtained from LOV-treated mixed glial cell cultures or controls (FIG. 4F). Notably, LOV pretreatment did not protect purified OPs against the cytotoxic effects of mediators released in cultures of CM-treated mixed glial cells. Altogether, these data demonstrate that LOV treatment attenuates the inflammatory cytokine induced release of cytotoxic mediators and augments the survival and proliferation of OPs in activated mixed glial cell cultures. Lovastatin enhances the differentiation of maturing oligodendrocytes in primary culture of activated mixed glial cells. To determine the effect of LOV treatment on differentiation of oligodendrocytes in activated mixed glial cells, immunocytochemistry was performed using anti-01 and anti-MBP antibodies at DIV5 (120 lor) and DIV6 (144 hr) respectively. There was significantly fewer 0I⁺ and MBP⁺ cells in CM-treated cells when compared with controls (FIG. 5A). Immunostaining revealed 0I⁺ cells, characteristically stained around the cell body (arrowheads) and weakly stained on the processes, whereas MBP⁺ cells stained all over the cell body including extended processes and branches (FIG. 5B). Notably, there was no significant change in 0I⁺ and MBP⁺ cell counts among Cont+LOV- and CM+LOV-treated cells and controls, but there was an increase in the length and number of processes in Cont+LOV-treated cells compared to controls. Similarly, the mRNA expression for MBP and PLP was also decreased in CM-treated cells compared with controls (FIG. 5C). Conversely, the expression for these proteins was significantly increased in CM+LOV- and Cont+LOV-treated cells as compared to controls. Furthermore, the immunoblotting also revealed a decrease in CNP ase protein levels in CM-treated cells compared with controls and this was restored by LOV treatment in CM+LOV-treated cells (FIG. 5D). Together, these data show the increase in differentiation of oligodendrocytes by LOV in activated mixed glial cell cultures. Lovastatin treatment attenuates reactive gliosis and induces a pro- remyelinating environment in the CNS and activated mixed glial cell cultures.

Microarray analysis demonstrated an increase in the expression of GFAP in EAE animals compared with controls and its attenuation with LOV treatment (Table 2). To determine the effect of LOV treatment on cytokine-induced reactive gliosis, double- immunostaining was performed with anti-GFAP and anti-MBP antibodies. Immunocytochemistry analysis revealed a characteristically bushy appearance of reactive hypertrophic astrocytes in CM-treated cells compared with controls (FIGS. 6A-B). Small and poorly differentiated oligodendrocytes (arrowheads) were observed in CM-treated cells, whereas LOV treatment demonstrated fully differentiating matured oligodendrocytes in Cont+LOV- and CM+LOV-treated cells. The quantitative analysis of GFAP immunofluorescence demonstrated a strong intensity for GFAP, but weak intensity for MBP in CM-treated cells (FIG. 6B). Conversely, CM+LOV-treated cells had moderate GFAP and strong MBP intensities similar to controls. No significant change in intensities for GFAP and MBP was observed between Cont+LOV-treated cells and controls. In accordance with these results, the expression of GFAP mRNA was also increased significantly in CM-treated cells compared to controls (FIG. 6C). However, no significant change in GFAP mRNA was observed among Cont+LOV- and CM+LOV-treated cells and controls.

Neurotrophic factors are known as important pro-remyelinating growth factors for the induction of proliferation/differentiation of OPs (Althaus, 2004). Microarray analysis revealed the up-regulation of mRNA expression for various neurotrophic factor proteins {i.e., CNTF, GDNF, BDNF, IGF-I, FGF-9, and LIF) in LOV-treated EAE animals when compared to EAE animals and controls (Table 3). Corresponding to this, the real-time PCR™ analysis also demonstrated a increase in expression of BDNF in E+LOV animals when compared with EAE animals and controls (FIG. 7A). Although, the expression of BDNF in E+LOV animals was significantly less when compared with controls, but it was significantly increased in recovered E+LOV animals. CNTF expression was not remarkably altered in EAE animals, but was significantly increased in LOV-treated EAE animals when compared with controls (FIG. 7B). Furthermore, the expression for PDGF, GGF-2, and LIF was significantly decreased in EAE animals compared to controls (FIGS. 6C-E). There expression was significantly lower (PDGF), close to control (GGF-2) or elevated (LIF) in E+LOV animals, but was significantly elevated in recovered E+LOV animals when compared with controls with the exception of PDGF and GGF-2 mRNA. Next, the inventors determined the expression of these neurotrophic factors in LOV- and CM-treated mixed glial cells. In support of the in vivo data, the inventors observed a significant increase in mRNA expression of GGF-2, BDNF, and LIF in LOV+CM-treated cells compared to controls (FIGS. 6F- H). There was no expression of these neurotrophic factors in CM-treated cells. Notably, Cont+LOV-treated cells also demonstrated a significant increase in expression of BDNF and GGF-2 when compared with controls. Together, these data show that LOV treatment attenuates the reactive gliosis which in turn helps to induce a pro-remyelinating environment in the CNS. Table 3. Changes in expression of message for neurotrophic factors required for proliferation and differentiation of oligodendrocyte progenitors in CNS of lovastatin-treatedΛ-untreated EAE and control animals

Gene Bank Gene Name 'C+LOV vs. 'EAE' vs. Ε+LOV VS. 'E+LOV/R'

Accession 'CON' 'CON' 'CON' vs. 'CON'

No. Fold Change Fold change Fold Change Fold Change

0=2) (rc=2) (n=2) (*=2)

X17457 Ciliary neurotrophic factor NC NC 4.0^a 6.5^a

(CNTF)

L15305 Glial Cell line derived NC -3.6^a 6.4^a 4.6^a neurotrophic factor (GDNF)

X67108 Brain derived neurotrophic NC -5.6^a -1.5^a NC factor (BDNF)

D12498 FGF receptor-1 (FGFR-I) NC -4.2^a -2.0^a NC

D14839 Fibroblast growth factor 9 NC -5.6^a -2.0^a -1.5^a

(FGF-9)

D13966 Insulin receptor-related NC -4.2^a -2.32^a NC receptor-2

0RR-R2)

L29232 Insulin-like growth factor 1 NC -3.2^a NC NC

. receptor

M15481 Insulin-like growth factorl NC -1.5" 5.4^a 5.6^a

(IGF-I)

J04486 Insulin-like growth factor NC 3.4^a NC NC binding protein 2

M62781 Insulin-like growth factor- NC -3.2^a NC NC binding protein 5

ABO 10275 Leukemia inhibitory factor NC -6.4^a 3.9^a 4.2

(LIF)

AF028784 Glial fibrillary acidic protein- NC 5.0^a 1.5^a NC and δ (GFAP)

Data are expressed as average mean of fold change in expression compared with controls for each gene transcript from two independent experiments. a: Change in expression was significant with fold change >1.5. NC: No change in expression.

Ht H: * ^ * ^ ^ ^ All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. A method of protecting implanted stem cells from a recipient immune response comprising administering to a transplant recipient a protective dose of an HMG-CoA reductase inhibitor.

2. The method of claim 1 , wherein said transplant recipient is a human.

3. The method of claim 1 , wherein said HMG-CoA reductase inhibitor is a statin.

4. The method of claim 3, wherein said statin is lovastatin, atorvastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, or cerivastatin.

5. The method of claim 1, wherein said stem cell is an embryonic stem cell or an adult stem cell.

6. The method of claim 1, wherein the stem cell differentiates into a nerve cell, a glial cell, an oligodendrocyte cell, a Schwann cell, an astrocyte, a myocyte, or an islet cell.

7. The method of claim 6, wherein the differentiated cell is an oligodendrocyte cell.

8. The method of claim 6, wherein the differentiated cell is a glial cell and wherein the glial cell is a microglia cell.

9. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient prior to transplant.

10. The method of claim 9, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient at about 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, 96 or 154 hours prior to transplant.

11. The method of claim 9, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient in a continuous manner prior to transplant.

12. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient at about the same time as transplant.

13. The method of claim 1, wherein said HMG-CoA reductase inhibitor provided to said transplant recipient following transplant.

14. The method of claim 13, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient at about 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, 96 or 154 hours following transplant.

15. The method of claim 13, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient in a continuous manner following transplant.

16. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient prior to and at the time of transplant.

17. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient prior to and following transplant.

18. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient at the time of and following transplant.

19. The method of claim 1, wherein said HMG-CoA reductase inhibitor is provided to said transplant recipient prior to, at the time of and following transplant.

20. The method of claim 1, wherein said transplant recipient suffers from a neurodegenerative disease.

21. The method of claim 20, wherein said neurodegenerative disease is a demyelinating disease.

22. The method of claim 21, wherein said demyelinating disease is multiple sclerosis or EAE.

23. The method of claim 22, wherein said demyelinating disease is multiple sclerosis.

24. The method of claim 1, further comprising administering to said transplant recipient an immunosuppressive drug.

25. The method of claim 1, wherein the implanted stem cell is an endogenous stem cell.

26. The method of claim 1, wherein the implanted stem cell is an exogenous stem cell.

27. A method of expanding a stem cell population comprising contacting a stem cell population with an HMG-CoA reductase inhibitor.

28. The method of claim 27, wherein said stem cell population is a human stem cell population.

29. The method of claim 27, wherein said HMG-CoA reductase inhibitor is a statin.

30. The method of claim 29, wherein said statin is lovastatin, atorvastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, or cerivastatin.

31. The method of claim 27, wherein said stem cell is an embryonic stem cell or an adult stem cell.

32. The method of claim 27, wherein the stem cell differentiates into a nerve cell, a glial cell, an oligodendrocyte cell, a Schwann cell, an astrocyte, a myocyte, or an islet cell.

33. The method of claim 32, wherein the differentiated cell is an oligodendrocyte cell.

34. The method of claim 32, wherein the differentiated cell is a glial cell and wherein the glial cell is a microglia cell.

35. The method of claim 27, wherein said stem cell population is cultured with said HMG-CoA reductase inhibitor for at least one hour.

36. The method of claim 35, wherein said stem cell population is cultured with said HMG-CoA reductase inhibitor for about 2, 4, 6, 8, 12, 18, 24, 48, 72, 96 or 154 hours.

37. The method of claim 27, further comprising the step of obtaining said stem cell population from a stem cell donor.

38. The method of claim 27, further comprising the step of transplanting said stem cell population to a transplant recipient.

39. The method of claim 38, further comprising administering to said transplant recipient an immunosuppressive drug.

40. A method of protecting a stem cell in a subject from a disease condition or treatment comprising administering to the subject a protective dose of an HMG-CoA reductase inhibitor.

41. The method of claim 40, wherein the stem cell is a non-transplanted stem cell.

42. The method of claim 40, wherein the stem cell is an implanted stem cell.

43. The method of claim 42, wherein the implanted stem cell is an endogenous stem cell.

44. The method of claim 43, wherein the implanted stem cell is an exogenous stem cell.

45. The method of claim 40, wherein the disease condition is oxidative stress/inflamated disease conditions.

46. The method of claim 40, wherein the disease treatment is chemotherapy or radiation therapy.

47. The method of claim 40, wherein the subject is a human.

48. The method of claim 40, wherein the HMG-CoA reductase inhibitor is a statin.

49. The method of claim 40, wherein the statin is lovastatin.

50. The method of claim 49, wherein the stem cell is an oligodendrocyte progenitor cell.