CN1961068A - Compositions and methods to culturing neural stem cells with bone marrow stromal cells - Google Patents

Abstract

The present invention encompasses methods and compositions for enhancing the growth of neural stem cells. Methods for modulating MHC molecule expression on a neural stem cell (NSC) are also included in the invention. Figure 1 is a graph depicting the proliferation of BMSCs in BMSC medium (DMEM-low glucose, 10% lot tested fetal bovine serum) or NSC medium (DMEM-F12, N2-Supplement, EGF 20 ng/ml, bFGF 10 ng/ml, heparin 8 ug/ml, penicillin-streptomycin).

Description

Method and composition for culturing neural stem cells from bone marrow stromal cells

Background technique

Bone marrow includes at least two types of stem cells: hematopoietic stem cells and non-hematopoietic tissue stem cells, the latter referred to variously: mesenchymal stem cells, spinal cord stromal cells (MSCs) or bone marrow stromal cells (BMSCs), these terms are used in this document synonymous. Spinal stromal cells are of interest because they can be easily isolated from small bone marrow aspirate and readily generate single-cell-derived colonies. Single cell-derived colonies can have 50 population doublings within 10 weeks and differentiate into osteoblasts, adipocytes, and chondrocytes (A.J.Friedenstein et al. 1970 Cell Tissue Kinet.3:393-403; H.Castro-Malaspina People such as.1980 Blood 56:289-301; People such as N.N.Beresford.1992 J.Cell Sci.102:341-351; D.J.Prockop 1997 Science 276:71-74), myocyte (people such as S.Wakitani.1995 Muscle Nerve 18:1417-1426), astrocytes, oligodendrocytes and neurons (S.A.Azizi et al. 1998 Proc.Natl.Acad.Sci.USA 95:3908-3913; G.C.Kopen et al. 1999 Proc USA 96: 10711-10716; M. Chopp et al. 2000 Neuroreport 11, 3001-3005; D. Woodbury et al. 2000 Neuroscience Res. 61: 364-370).

Furthermore, spinal cord stromal cells (MSCs) give rise to cells of all three germ layers (Kopen, G C. et al. 1999 Proc. Natl. Acad. Sci. 96:10711-10716; Liechty, K.W. et al. 2000 Nature Med.6 : 1282-1286); Kotton, D.N. et al. 2001 Development 128: 5181-5188; Toma, C. et al. 20002 Circulation 105: 93-98; Jiang, Y. et al. 2002 Nature 418: 41-49). In vivo evidence suggests that undifferentiated bone marrow-derived cells, like pure cell populations of MSCs, give rise to epithelial cells, including those of the lung (Krause, et al. 2001 Cell 105:369-377; Petersen, et al. 1999 Science 284:1168-1170 ). However, several recent studies have shown that transplantation of MSCs is increased due to tissue damage (Ferrari, G. et al. 1998 Science 279: 1528-1530; Okamoto, R. et al. 2002 Nature Med. 8: 1101-1017). For these reasons, the application of spinal cord stromal cells in cell therapy and gene therapy of various human diseases is currently being investigated (Horwitz et al., 1999 Nat.Med.5:309-313; Caplan, et al. 2000 Clin.Orthoped .379:567-570).

Spinal cord stromal cells constitute a source of choice for pluripotent stem cells. Under physiological conditions, spinal cord stromal cells maintain bone marrow tissue structure and regulate the production of hematopoietic cells with the help of different cell adhesion molecules and cytokines (Clark, B.R. & Keating, A.1995 Ann NY Acad Sci 770: 70-78 ). Spinal cord stromal cells grown outside the bone marrow by selective adsorption to tissue culture plastic can be efficiently expanded (Azizi, S.A., et al. 1998 Proc Natl Acad Sci USA, 95:3908-3913; Colter, D.C., et al. 2000Proc Natl Acad Sci USA 97:3213-218), and it can be genetically manipulated (Schwarz, E.J., et al. 1999 Hum Gene Ther 10:2539-2549).

Spinal cord stromal cells are also known as mesenchymal stem cells because of their ability to differentiate into multiple mesoderm tissues, including bone (Beresford, J.N., et al. 1992 J Cell Sci 102:341-351), cartilage (Lennon, D.P, et al. 1995 Exp Cell Res 219:211-222), fat (Beresford, J.N., et al. 1992 J Cell Sci 102:341-351) and muscle (Wakitani, et al. 1995 Muscle Nerve 18:1417-1426). In addition, differentiation into neuron-like cells expressing neuronal markers has been reported (Woodbury, D., et al. 2000 J Neurosci Res 61:364-370; Sanchez-Ramos, J., et al. 2000 Exp Neurol 164:247 -256; Deng, W., et al. 2001 Biochem Biophys Res Commun 282:148-152), suggesting that spinal cord stromal cells may be able to overcome germ layer commitment.

Stem cells are self-renewing multifunctional progenitor cells with the broadest developmental potential in specific tissues at specific times (Morrison et al. 1997 Cell 88:287-298). Recently, research on stem cells in the nervous system has attracted widespread interest due to Their importance for understanding neurodevelopment and their therapeutic potential in treating neurodegenerative diseases.

Existing methods for isolating and maintaining embryonic and other stem cell lines rely on the use of murine embryonic fibroblasts as trophoblasts. It has been found that the frequency of embryonic stem cell clones is increased several-fold by using a serum replacement in the culture medium (Amit et al. 2000 Dev Biol 227:271-278). Persistent, undifferentiated propagation of clonal embryonic stem cells requires the presence of essential fibroblast growth factors. Current methods for isolating, culturing, and expanding hESCs are limited by their reliance on trophoblasts of murine embryonic fibroblasts. What remains to be demonstrated is that stem cells can remain in an undifferentiated state indefinitely in the absence of trophoblast cells.

To increase the growth rate of human fetal brain stem cells, many different research groups have used several different approaches and growth factors over the past decade. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been shown to be required for expansion and maintenance of human fetal neural stem cells (hNSCs). These human neural stem cell cultures grew normally as clusters of dissociated free-floating cells (neurospheres), but in the presence of only bFGF and EGF, the neurospheres could not proliferate indefinitely. Leukemia inhibitory factor (LIF) has been shown to increase growth rate and prolong the survival time of FGF- and EGF-responsive neural stem cells (Carpenter et al., 1999 and Wright et al., 2003). In addition to regulating growth rate, leukemia inhibitory factor also actively regulates several genes, including major histocompatibility complex molecules on the surface of neural stem cells (Wright et al., 2003).

Human fetal brain stem cells are considered attractive candidates for stem cell transplantation for regeneration of damaged tissues. Stem cells derived from embryonic or fetal donors require allogeneic transplantation. Cell transplantation between genetically distinct individuals is often accompanied by the risk of host rejection. Almost all cells express the products of the major histocompatibility complex, class I MHC molecules. Furthermore, many cell types express MHC class II molecules upon induction by inflammatory cytokines. Allograft rejection is mainly mediated by CD4 and CD8 subclasses of T cells. (Rosenberg et al. 1992 Annu. Rev. Immunol. 10:333). Alloreactive CD4 T cells produce cytokines that exacerbate cytolytic CD8 responses to alloantigens. In these subclasses, a competing cell subpopulation is formed following antigenic stimulation with characteristics conferred by the cytokines produced by the subclass. Th1 cells producing interleukin-2 (IL-2) and immunoreactive fibronectin-gamma (IFN-gamma) are primarily involved in allograft rejection (Mossmann et al., 1989 Annu. Rev. Immunol. 7:145). Th2 cells producing interleukin-4 (IL-4) and interleukin-10 (IL-10) can downregulate IL-10-mediated Th1 responses (Fiorentino et al. 1989 J. Exp. Med. 170:2081 ). In fact, many efforts have been made to divert unwanted Th1 responses to the Th2 pathway. Adverse allograft rejection of a graft by T cells in a patient is typically treated with immunosuppressant drugs such as prednisone, azathioprine, and cyclosporine A. Unfortunately, these drugs are often required for the patient's life, and they have many dangerous side effects, including systemic immunosuppression.

Neural stem cells express low (or negligible) levels of MHC class I and/or MHC class II antigens (McLaren et al. 2001 J. Neuroimmunol. 112:35), but these cells are often protected after transplantation into allogeneic recipients. Rejection, unless immunosuppressive drugs are used. Exposure to inflammatory cytokines such as IFN increases the level of MHC molecules on the cell membrane surface, which may trigger rejection. Therefore, standardization of culture conditions is highly desirable to maximize the proliferation and multiple potential of NSCs for therapeutic use. Additionally, it is currently believed that a successful NSC transplantation translates host rejection of NSCs by preventing and/or reducing unwanted immune effector cell-mediated NSC immune responses.

Accordingly, there has been a long-felt desire for methods of suppressing or preventing unwanted immune responses during nerve cell transplantation between genetically dissimilar individuals, and the present invention fulfills this need.

Contents of the invention

The invention includes methods and compositions for culturing neural stem cells. The present invention also includes cells produced using the compositions and methods described above.

The present invention includes a composition comprising an isolated bone marrow stromal cell (BMSC) and a chemically defined culture medium comprising neural stem cell (NSC) growth medium and said BMSC secreting factor.

In one aspect, the culture medium is free of exogenous leukemia inhibitory factor (LIF).

In another aspect, the factors secreted by BMSCs are selected from growth factors, trophic factors and cytokines.

In another aspect, the factor is selected from the group consisting of LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived Neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, trypsin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL -6, IL-8, monocyte chemoattractant protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein (BMP4), IL1- a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor beta (TGFβ-1 ) and TGFβ-3.

The invention also encompasses the co-cultivation of BMSCs and NSCs in a chemically defined culture medium comprising neural stem cell growth medium.

In one aspect, BMSCs and NSCs are cultured in a contact-dependent manner, wherein the BMSCs are in physical contact with the NSCs.

On the other hand, BMSCs and NSCs are cultured in a contact-independent manner, where BMSCs and NSCs are not in physical contact.

In another aspect, the NSC is derived from the human central nervous system.

In another aspect, the BMSCs are of human origin.

In another aspect, exogenous genetic material is introduced into the cells of the invention.

The present invention also includes a bone marrow stromal cell conditioned culture medium (BMSC-CM), comprising a chemically defined culture medium containing neural stem cell growth medium and factors secreted by isolated BMSCs.

In one aspect, the BMSC-CMs do not contain exogenous leukemia inhibitory factor (LIF).

In another aspect, BMSC-CMs are substantially free of BMSCs.

In another aspect of the present invention, the factors contained in BMSC-CM are selected from growth factors, nutritional factors and cytokines.

In another aspect, the factor is selected from the group consisting of leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, Glial cell-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, trypsin-like growth factor-binding protein (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, monocyte chemoattractant protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase ( TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein ( BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor Beta (TGF beta-1) and TGF beta-3.

The invention also includes a method for regulating the expression of major histocompatibility complex molecules on the surface of isolated NSCs.

In one aspect, co-culture of isolated BMSCs and isolated NSCs modulates the expression of MHC molecules on the surface of the NSCs.

In another aspect, the expression of MHC molecules on NSCs can be regulated by culturing NSCs with BMSC-CMs.

The invention includes an isolated NSC prepared by co-cultivating BMSC and NSC.

In one aspect of the invention, NSCs generated by the methods of the invention exhibit reduced expression of MHC class I molecules.

In another aspect, NSCs generated by the methods of the invention exhibit baseline levels of MHC class II molecules.

The present invention also includes a neural cell culture device comprising isolated NSCs, isolated BMSCs, NSC growth medium and a means for preventing physical contact between the NSCs and BMSCs.

In another aspect, the above device further includes a filter or membrane to prevent physical contact between the NSCs and BMSCs.

In another aspect, the above-mentioned filter or membrane has pores to allow the factors secreted by the BMSCs to pass through the filter or membrane.

Description of drawings

To illustrate the invention, certain embodiments of the invention are depicted in the drawings. However, the invention is not limited to the arrangements and instrumentalities of the specific embodiments depicted in the drawings.

Figure 1: depicts the growth of BMSC in BMSC culture medium (DMEM-low glucose, fetal bovine serum (FBS) of 10% sampling test) or NSC medium (DMEM-F12, N2-supplement, epidermal growth factor (EGF) 20ng/ ml, basic fibroblast growth factor (bFGF) 10 ng/ml, heparin 8 μg/ml, penicillin-streptomycin (P/S)).

Figure 2: Comprising Figures 2A-2C, is a series of images depicting NSCs growing into neurospheres. Figures 2B and 2C depict NSCs grown in the presence of exogenous leukemia inhibitory factor (LIF). Figure 2A depicts neurospheres grown in the absence of LIF.

Figure 3: Comprising Figures 3A-3D, is a series of images depicting co-cultured BMSCs and NSCs in the presence of exogenous LIF (Figures 3C and 3D) and in the absence of exogenous LIF (Figures 3A and 3B).

Figure 4: including Figures 4A-4F, is a series of images depicting the expression and pre-differentiation of nestin (nestin) and glial fibrillary acidic protein (GFAP) when NSCs and BMSCs were co-cultured (Figures 4A-4F). Figures 4G and 4H depict the lack of expression of nestin and GFAP when cultured by BMSCs alone.

Figure 5: Comprising Figures 5A-5D, is a series of images demonstrating that NSCs grown on BMSCs retain the potential to differentiate into neurons and astrocytes. Figures 5A-5D depict MAP2-GFAP-DAPI staining of differentiated co-cultures. MAP2 is a neurocytoskeletal protein. DAPI is 4',6'-diamidino-2-phenylindole hydrochloride for nuclear staining.

Figure 6: Images depicting nestin-GFAP staining of differentiated co-cultures showing minimal expression of nestin after differentiation.

Figure 7: Images depicting GFAP-DAPI staining of differentiated NSCs.

Figure 8: Comprising Figures 8A and 8B, is a series of images depicting MAP2-GFAP-DAPI staining of differentiated BMSCs showing minimal expression of neuronal and astrocytic markers.

Figure 9: including 9A and 9B, is a series of fluorescence-activated cell sorter analysis profiles (FACS), depicting the phenotype of NSC after BMSC deletion. Figure 9A illustrates that less than 2% of cells are positive for CD-105 (a marker for BMSCs). Figure 9B illustrates that more than 90% were positive for CD-133.

Figure 10: Comprising Figure 10A and Figure 10B, is a series of fluorescence activated cell sorter analysis profiles, depicting the loss of nestin expression of BMSC (Figure 10A) and the nestin expression of NSC isolated from BMSC co-culture (Figure 10B).

Figure 11: Illustrates that NSCs grown on BMSCs retain their pluripotency to differentiate into neurons and astrocytes.

Figure 12: Depicts the growth of NSCs in Transwell ^™ in the presence or absence of BMSCs.

Figure 13: Comprising Figures 13A and 13B, depicts images of NSC growth on uncoated plates in the presence (Figure 13A) or absence of BMSCs (Figure 13B) in Transwell.

Figure 14: Comprising Figures 14A-14D, depicting NSCs cultured with bone marrow stromal cell conditioned medium (BMSC-CM) (Figures 14A and 14B) and in the presence of exogenous LIF (Figure 14C) or in the absence of exogenous LIF ( Figure 14D) NSCs cultured in NSC-medium.

Figure 15: Comparison of the effect of BMSC-CM with standard NSC medium with or without exogenous LIF on NSC growth. BMSC-CM1 medium is derived from BMSCs cultured in NSC medium containing EGF and FGF. BMSC-CM2 medium is derived from BMSCs cultured in NSC medium without EGF and FGF.

Figure 16: Comprising Figures 16A-16D, is a series of FACS analysis profiles depicting phenotypic profiles of NSCs isolated from co-culture with two different BMSC donors (Figures 16A and 16B). Figures 16C and 16D depict the phenotype curves of NSCs cultured without the addition of BMSCs in NSC medium and NSC medium containing exogenous LIF, respectively.

Figure 17: Comprising Figures 17A-17D, is a series of FACS analysis profiles depicting the phenotypes of NSCs cultured in NSC medium containing and not containing exogenous LIF, and in BMSCs containing and not containing growth factors- Phenotype of NSCs cultured in CM medium. Figures 17A-17D depict curves for CD56, CD133, MHC class II molecules, and MHC class I molecules, respectively.

Figure 18: Comprising Figures 18A-18D, is a series of FACS analysis profiles depicting the phenotypes of NSCs cultured in NSC medium containing (Figure 18B) and without (Figure 18A) exogenous LIF, and in complete Phenotype of NSCs co-cultured in NSC medium (FIGS. 18C and 18D). Figure 18 depicts the expression of MHC class I and MHC class II molecules of NSCs cultured under various conditions (black = heterotypic control; gray = class I or class II).

Detailed ways

The present invention includes compositions and methods for inducing and/or enhancing proliferation of neural stem cells (NSCs) while retaining their pluripotency. The invention also includes compositions and methods for modulating the expression of MHC molecules in NSCs.

The present invention relates to the discovery that bone marrow stromal cells (BMSCs) can act as a trophoblast to support the proliferation of NSCs. Thus, the present invention includes compositions and methods for inducing and/or enhancing proliferation of NSCs while maintaining their pluripotency, culturing NSCs using BMSCs as feeder layers.

In addition, the present disclosure demonstrates that compared to otherwise identical NSCs cultured in neural stem cell culture media (NSC medium) without BMSC feeder layers but supplemented with exogenous LIF for enhanced expansion, the BMSC feeder layer Co-cultivating NSCs on NSCs reduces the upregulation and/or induction of expression of MHC molecules in NSCs. Accordingly, the present invention includes compositions and methods for reducing and/or preventing expression of MHC molecules by NSCs using BMSCs as feeder layers for culturing and expanding NSCs.

The data disclosed herein also demonstrate that BMSCs secrete growth factors, trophic factors and/or cytokines useful for NSCs while retaining their pluripotency. Factors secreted by BMSCs can be collected by culturing BMSCs in a medium for a period of time and collecting the conditioned medium for later use. Therefore, the present invention also includes a bone marrow stromal cell conditioned medium (BMSC-CM), which is useful for the proliferation of NSCs while maintaining pluripotency.

The present invention also relates to the discovery that factors secreted by BMSCs and present in BMSC-CMs reduce the upregulation and/or induction of expression of MHC molecules by NSCs. Accordingly, the present invention includes compositions and methods for expanding NSCs using BMSC-CMs in the absence and/or reduced upregulation of expression of MHC molecules on the NSC surface.

Accordingly, the present invention includes methods and compositions for generating NSCs for medical use. Accordingly, the present invention includes compositions and methods for generating NSCs useful for treating patients affected by central nervous system diseases, disorders or conditions. The method includes the steps of culturing, expanding the NSCs, and administering the NSCs to a patient.

definition

The following terms used herein have the meanings stated in this section.

The use of "a" herein means that the subject of its qualification is one or more than one (ie at least "one"). For example, "an element" means one or more elements.

The term "about" can be understood by those of ordinary skill in the art and will vary to some extent depending on its context.

The term "autologous" as used herein means any substance derived from the same individual and reintroduced into that individual.

The term "allogeneic" as used herein refers to any substance derived from the body of a different animal of the same species.

The terms "bone marrow stromal cells", "stromal cells", "mesenchymal stem cells" or "MSCs" are used interchangeably herein to refer to the small fraction of cells in the bone marrow that are capable of serving as bone cells, chondrocytes and adipose Stem cell-like precursors of cells and were isolated from bone marrow by their ability to adhere to plastic dishes. Spinal cord stromal cells can be derived from any animal. In some embodiments, the stromal cells are preferably derived from primates.

"Differentiated"is used herein to refer to a cell that has attained a mature terminal state that is fully developed and exhibits biological specificity and/or adaptation to a particular environment and/or function. Typically, a differentiated cell is characterized by the expression of genes encoding differentiation-associated proteins in the particular cell. For example, the expression of myelin lipoprotein and the formation of myelin in glial cells are typical examples of the terminal differentiation of glial cells. When referring to a cell being "differentiated," as the term is used herein, the cell is in the process of being differentiated.

"Differentiation medium" is used herein to mean a medium for the growth of cells, with or without an additive, such as: stem cells, embryonic stem cells, similar embryonic stem cells, neurospheres, NSC or other similar precursor cells, in culture A cell that is not fully differentiated when cultured in medium develops a cell that has some or all of the characteristics of a differentiated cell.

"Expansion capacity" is used herein to refer to the ability of a cell to reproduce, eg, expand in number or where a population of cells undergoes a population doubling.

"Feeder layer" is intended to mean cells that produce growth factors, cytokines, other cell-derived products, and provide physical support through contacts necessary in co-culture to enhance stem cell proliferation and maintain stem cell undifferentiated pluripotency .

"Feeder layer" is used herein to describe cells of a first tissue type that are co-cultured with cells of a second tissue type to provide an environment in which cells of the second tissue type can grow.

The term "growth medium" as used herein means a culture medium that promotes the growth of cells. Growth media will typically contain animal serum. In some cases, the growth medium can be free of animal serum.

The term "NSC medium" as used herein means a culture medium for culturing and expanding NSCs. A typical NSC medium includes DMEM/F12, N2 supplement, EGF, bFGF and heparin. In some cases, NSC medium may be free of growth factors (such as EGF and bFGF).

"Bone marrow stromal cell conditioned medium" (BMSC-CM) is used herein to refer to a culture medium conditioned by culturing BMSCs. Based on the present disclosure, BMSC-CM is obtained by culturing BMSC in NSC medium, therefore, by causing BMSC to secrete growth factors, nutritional factors and cytokines present in other compounds into NSC medium, BMSC-CM The conditions are defined by the BMSCs in culture. NSCs can be cultured with BMSC-CM to enhance NSC proliferation while maintaining NSC pluripotency. In addition, BMSC-CMs can be used to culture NSCs in a manner that regulates the expression of MHC molecules.

"Leukemia inhibitory factor" (LIF) is used herein to refer to a 22KDa protein in the interleukin-6 family, which has various biological functions. LIF has been shown to have the ability to induce terminal differentiation in leukemia cells, induce hematopoietic differentiation in normal and myeloid leukemia cells and stimulate the synthesis of acute phase proteins in hepatocytes. Here, LIF was also shown to enhance NSC proliferation in the undifferentiated state while maintaining NSC pluripotency.

"Exogenous LIF" refers to LIF introduced into or produced and excreted by an organism, cell or system.

The term "pluripotent" or "multipotency" as used herein means the ability of a stem cell of the central nervous system to differentiate into multiple cell types. For example, a pluripotent central nervous system stem cell is capable of differentiating into, but not limited to, neurons, astrocytes, and oligodendrocytes.

"Neurosphere" is used herein to refer to a neural stem/progenitor cell in which expression of nestin can be detected, including, inter alia, by immunostaining to detect nestin in the cells. Neurospheres reproduce aggregates of neural stem cells, and the formation of neurospheres is characteristic of neural stem cells cultured in vivo.

"Neural stem cell" is used herein to refer to an undifferentiated, multipotent, self-renewing nerve cell. Neural stem cells are a kind of clonal pluripotent stem cells that can differentiate and have self-renewal ability under suitable conditions, and can eventually differentiate into neurons, astrocytes and oligodendrocytes. Thus, neural stem cells are "pluripotent" in that the progeny of the stem cells have multiple differentiation pathways. The ability of neural stem cells to maintain themselves means that the daughter cells that result from each cell division will generally also be stem cells.

"Nerve cell" is used herein to refer to a cell that has similar morphological, functional and phenotypic characteristics to glial cells and neurons derived from the central nervous system and/or peripheral nervous system.

"Neuron-like cell" is used herein to refer to a cell that has a morphology similar to a neuron and that detectably expresses neuron-specific markers such as (but not limited to) MAP2, neurofilament 200kDa, neurofilament -L, neurofilament-M, synaptophysin, β-tubulin (TUJI), taurine (Tau), N-acetylneuraminic acid (NeuN), neurofilament and synaptic proteins (synaptic protein).

"Astrocyte-like cell" is used herein to refer to a cell having a phenotype similar to an astrocyte, which expresses an astrocyte-specific marker, such as, but not limited to, GFAP.

"Oligodendrocyte-like cell" is used herein to refer to an oligodendrocyte-specific marker that is similar to an oligodendrocyte, such as, but not limited to, 0-4.

"Cell cycle progression" is used herein to refer to the process by which a cell prepares for and/or enters mitosis and/or meiosis. The progression of the cell cycle includes G1 phase, S phase, G2 phase and M-phase.

"Reproduction" is used herein to refer to the duplication or multiplication of similar types, especially cells. That is, reproduction includes the generation of larger numbers of cells, which can be measured by simple counting of cell numbers, detection of incorporation of 3H-thymidine into cells, and the like.

The term "exogenous" as used herein refers to any substance introduced into or excreted by an organism, cell or system.

"Coding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or amino acid sequences and the resulting biological properties of polymers and macromolecules. Thus, a gene encodes a protein if its corresponding mRNA in a cell or other biological system is transcribed and translated to produce that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided as a sequence listing) and the noncoding strand (which serves as a template for the transcription of a gene or cDNA) can both be referred to as encoding the protein or other product of said gene or cDNA .

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all mutually denatured forms of nucleotide sequences that encode the same amino acid. Nucleotide sequences encoding proteins and RNA may include introns.

"Isolated nucleic acid" refers to a nucleic acid segment that is separated from naturally occurring sequences flanking it, e.g. a DNA segment removed from its normal adjacent sequences, e.g. natural separation in sequence. The term also refers to nucleic acids purified in large quantities from other naturally associated components, such as RNA, DNA or proteins, with which they are naturally associated in cells. The term thus includes, for example, recombinant DNA integrated into a vector, self-replicating plasmid or virus, or prokaryotic or eukaryotic genomic DNA, or present as a separate molecule independent of other sequences (e.g. by PCR or restriction enzymes). cDNA, genomic fragment or cDNA fragment produced by cutting). The term also includes a recombinant DNA that is part of a hybrid gene and encodes additional polypeptide sequences.

In the context of the present invention, the following abbreviations representing ubiquitous nucleic acid bases apply. "A" refers to adenine, "C" refers to cytosine, "G" refers to guanine, "T" refers to thymine, and "U" refers to uracil.

A "vector" is a composition of matter comprising an isolated nucleic acid and capable of delivering the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides conjugated to ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes a self-replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as polylysine compounds, liposomes and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

"Expression vector"refers to a vector comprising a recombinant polynucleotide containing regulatory sequences linked to a nucleotide sequence for which expression is desired. An expression vector includes sufficient cis-acting elements for expression, and other elements for expression can be provided by the host or an in vitro expression system. Expression vectors include all those known in the art, such as Cosmids, plasmids (eg, naked or contained in liposomes) and viruses that incorporate recombinant polynucleotides.

describe

The present invention includes a method of increasing the proliferation of NSCs while maintaining their pluripotency. The method comprises isolating NSCs by methods known in the prior art, and co-cultivating NSCs and BMSCs to increase the proliferation of NSCs while maintaining the pluripotency of NSCs. Both cell types can be cultured in a contact-dependent manner, where NSCs are in physical contact with BMSCs, or in a contact-independent manner, where NSCs are not in physical contact with BMSCs.

The present invention relates to the discovery that the expansion capacity of NSCs (the ability of NSCs to replicate themselves multiple times) can be enhanced by co-cultivation with BMSCs. That is, one embodiment of the present invention relates to the discovery that BMSCs can serve as supporting cells for the expansion of NSCs in a co-culture system. Those familiar with the prior art can recognize that according to the disclosure of the present invention, BMSC can serve as a trophoblast for NSC and provide factors including - but not limited to - growth factors, nutritional factors and cytokines, to support the cultivation of NSC , while maintaining the pluripotency of NSCs. The BMSC feeder layer can also act as a monolayer on which NSCs can grow.

Those skilled in the art will recognize, based on the present disclosure, that BMSCs and NSCs can also be co-cultured in the presence of other factors known in the art to enhance the proliferation of NSCs.

In the present invention, BMSCs and NSCs can be co-cultured in the absence of exogenous LIF to increase the proliferation of NSCs while maintaining the pluripotency of NSCs. The present disclosure demonstrates that the NSCs proliferate and expand at significantly higher levels than NSCs cultured alone on coated plates containing exogenous LIF (i.e., polyornithine/fibronectin coated plates). increase level. Thus, the present invention provides a method for culturing NSCs that does not require the use of coated plates and/or exogenous LIF.

Another embodiment of the invention includes a method of depleting or isolating BMSCs from a co-culture of BMSCs and NSCs. The present invention relates to the discovery that BMSCs can be isolated from co-cultures by incubating an antibody that binds BMSCs in the co-cultures, followed by an additional isolation step including, but not limited to, magnetic separation. An example of an antibody that binds BMSCs is an anti-CD13 antibody. The magnetic separation process is accompanied by the use of magnetic beads, including, but not limited to, Dynabeads (Dynal Biotech, Brown Deer, WI). Regarding the use of Dynabead, BMSCs can be removed from co-cultures using MACs separation solvent (Miltenyi Biotec, Auburn, CA). One result of the isolation step is that pure NSCs are obtained. FACS can also be used to remove BMSCs, or, to positively select NSCs.

In the NSC culture/expansion method of the present invention, the NSC maintains its pluripotency (the ability to differentiate into different cell types, such as neurons, astrocytes, oligodendrocytes and others). In another embodiment of the present invention, compared with NSCs expanded or cultured by prior art methods, the NSCs expanded by the method of the present invention maintain a greater degree (ie, a greater proportion) of differentiation ability.

The NSC culture/expansion method described here addresses an important issue in the application of NSCs to treat human diseases, namely, that prior to the present disclosure, it was difficult to isolate and expand NSCs from culture (i.e., it was difficult to induce them to proliferate in sufficient numbers). ). The disclosure herein demonstrates that NSCs can be cultured and isolated in large quantities for therapeutic purposes.

The present invention also relates to the discovery that expression of MHC molecules by NSCs can be regulated by co-culturing BMSCs and NSCs. The disclosure herein demonstrates that, in addition to enhancing the proliferation of NSCs and retaining their pluripotency, co-culturing BMSCs and NSCs also reduces the upregulation and expression of MHC molecules in NSCs compared to NSCs cultured using methods known in the art. / or induced. That is, the present invention provides a method of culturing NSCs that provides additional benefits over standard methods of increasing NSC proliferation in culture.

The co-culture system provides a method for culturing NSCs with significantly better benefits than culturing NSCs alone on coated plates or coated plates containing exogenous LIF. As discussed elsewhere in this paper, the co-culture system provides for the first time a method to generate large numbers of NSCs without the use of coated plates or exogenous LIF. In addition, the method of the co-culture system has additional benefits and/or greater benefits compared with the method of culturing NSCs alone using a standard NSC medium in the prior art. These benefits include (but are not limited to): increasing NSC proliferation and maintaining NSC pluripotency and modulating NSC expression of MHC molecules.

In another embodiment of the present invention, NSCs can be co-cultured with BMSCs in the absence of exogenous LIF to reduce the up-regulation and/or induction of the expression of MHC molecules of NSCs. That is, the present invention provides a method for culturing NSCs that does not require the use of exogenous LIF. The disclosure herein demonstrates that BMSCs in a co-culture system can serve as a trophoblast to provide factors to co-cultured NSCs, including (but not limited to) growth factors, nutritional factors and cytokines. It is believed that BMSCs provide factors that provide beneficial effects to co-cultured NSCs in terms of expansion and expression of MHC molecules that outweigh the benefits of culturing NSCs alone on spread plates of NSC medium.

The discovery that the expression of MHC molecules can be modulated using the methods disclosed herein provides a method for propagating NSCs that are useful in therapy, diagnosis, experimental use, and the like. For example, reduced expression of MHC molecules in NSCs using the methods disclosed herein provides a means of reducing the immunogenicity of NSCs compared to methods known in the art. Preferably, reduced expression of MHC molecules by NSCs provides a means of increasing the success rate of NSC transplantation into recipients.

Co-culture of NSCs and BMSCs provides a means to modulate the expression of MHC molecules in NSCs in a contact-dependent or -independent manner of both cells. In one embodiment of the present invention, NSCs can be co-cultured with BMSCs in a contact-dependent manner. Without wishing to be bound by any particular theory, the physical contact of BMSCs and NSCs confers beneficial effects on NSC propagation and expansion. The physical contact of the two cells also contributed to the regulation of the expression of MHC molecules in NSCs. Preferably, co-culturing NSCs and BMSCs in a two-cell contact-dependent manner reduces the MHC of NSCs compared to the same NSCs cultured on other spread plates using standard NSC medium supplemented with exogenous LIF but without BMSCs Up-regulation and/or induction of molecule expression.

In another embodiment of the present invention, NSCs can be co-cultured with BMSCs in a contact-independent manner to regulate the expression of MHC molecules of NSCs. The present invention relates to the discovery that NSCs and BMSCs can be co-cultured in Costar Transwell ^(TM) through a permeable membrane filter to separate the two cells and prevent physical contact between NSCs and BMSCs. The permeable membrane filter allows factors secreted by BMSCs to permeate the membrane and thus effectively contribute to the phenotype of NSCs, such as enhancing the proliferation of NSCs and maintaining their pluripotency and regulating the expression of MHC molecules of NSCs.

Experiments using Transwell ^TM demonstrated that the present disclosure shows that by supplementing BMSCs into the culture medium (factors secreted by BMSCs also enter the culture medium), BMSCs are able to support co-culture in the absence of direct contact between the two cells Growth and expansion of NSCs in the system. Without wishing to be bound by any particular theory, factors secreted by BMSCs contribute to the NSC phenotype. Therefore, the present invention provides a method for co-cultivating BMSCs and NSCs without requiring BMSCs as a feeder layer on which NSCs are directly grown. The present invention provides a method for co-cultivating BMSCs and NSCs in a contact-independent manner, wherein, NSCs obtain benefits from BMSCs by absorbing BMSCs secreted by BMSCs in the co-cultivation system.

Those skilled in the art will recognize, based on the present disclosure, that any method that prevents NSCs and BMSCs from forming physical contact can be used in a co-culture system. For example, any system/device other than Transwell( ^TM ) can be used to co-culture cells in a contact-independent manner. Such systems/devices include, but are not limited to, filters and membranes with a pore size that will prevent direct contact of the two cells, but allow some factors to pass through the filter/membrane. The benefit of using such a system/device to co-culture BMSCs and NSCs is to provide a method for culturing NSCs in a system with a continuous source of factors for NSC proliferation. Thus, the present invention includes a combination comprising: a neural stem cell culture device containing isolated NSCs, isolated BMSCs, NSC growth medium and factors secreted by said isolated BMSCs; devices in contact with each other.

Based on the finding that BMSCs are able to support NSC growth in a co-culture system in a contact-independent manner by secreting factors into the culture medium, it was evaluated whether BMSC-conditioned media can enhance NSC proliferation and maintain their pluripotency and regulate NSC growth. MHC molecules are expressed in a manner to support the growth of NSCs. The present disclosure demonstrates that BMSC conditioned media can support the growth of NSCs with significant beneficial effects, although co-cultivation in a less contact-dependent manner is more effective. Therefore, the present invention provides a method for culturing NSCs using bone marrow stromal cell conditioned medium (BMSC-CM) without using a co-culture system. The use of BMSC-CM to culture NSC also provides another method for propagating NSC, and the NSC produced by this method has the same properties as the NSC cultured by the co-culture system.

Additionally, the present disclosure demonstrates that BMSC-CM can replace NSC medium supplemented with exogenous LIF for culturing NSCs. The present disclosure demonstrates that the number of cells generated following the method of culturing NSCs in BMSC-CMs is comparable to that generated using NSC medium supplemented with exogenous LIF. Therefore, the present invention provides a method of using BMSC-CM as a source of factors beneficial to inducing NSC propagation, so that the propagation rate of NSC is equivalent to or greater than that of NSC culture medium supplemented with exogenous LIF. .

The present invention also demonstrates that BMSC-CM can replace coated plates, such as polyornithine/fibronectin coated plates, for culturing and expanding NSCs using NSC medium. The disclosure here demonstrates that NSCs can be expanded on uncoated plates using BMSC-CMs. It can be observed that the expansion of NSCs using BMSC-CM on uncoated plates is at least equivalent to or greater than that of NSCs cultured alone on coated plates of NSC medium, even NSC medium containing exogenous LIF. Amplify. Thus, the present invention includes a method for culturing NSCs using BMSC-CMs without the need for plated plates and exogenous LIF.

The use of BMSC-CMs also provides a method of culturing NSCs in a manner that provides benefits to NSCs without using BMSCs in the co-culture system. The present disclosure demonstrates that BMSC-CMs are able to support the culture of NSCs and provide them with benefits comparable to those observed using co-culture systems comprising BMSCs and NSCs. Thus, the present invention includes a method of culturing NSCs using BMSC-CMs to enhance proliferation and maintain pluripotency of NSCs and to modulate the expression of MHC molecules of NSCs. As demonstrated here, BMSCs can be used to generate bone marrow stromal cell conditioned medium (BMSC-CM). BMSC-CM is a culture medium conditioned by BMSCs in culture by culturing BMSCs in NSC medium and allowing BMSCs to secrete growth factors, nutritional factors and/or cytokines in NSC medium. BMSC-CMs include growth factors, trophic factors, and/or cytokines secreted by BMSCs, including (but not limited to): LIF, brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, trypsin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, monocyte chemoattractant protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), metalloproteinase Tissue inhibitor (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone Morphogenetic protein (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB) , transforming growth factor beta (TGF beta-1) and TGF beta-3.

BMSC-CMs are beneficial for the propagation and expansion of NSCs and maintaining their pluripotency. The use of BMSC-CM provides a way to introduce growth factors, nutritional factors and/or cytokines, etc. secreted by BMSCs into NSCs for the propagation and expansion of NSCs.

The use of BMSC-CM provides a way to induce NSCs to proliferate at a rate comparable to or greater than when cultured with NSC medium supplemented with exogenous LIF, even on uncoated plates. Accordingly, the present invention provides compositions and methods for using BMSC-CMs to generate large numbers of NSCs for therapeutic use.

In addition to the use of BMSC-CMs to support NSC propagation and maintain their pluripotency, the present disclosure also demonstrates that culturing NSCs in BMSC-CMs provides a means of modulating the expression of MHC molecules of NSCs. Preferably, culturing NSCs in BMSC-CM reduces expression of MHC molecules by NSCs compared to otherwise identical NSCs cultured in NSC medium containing exogenous LIF. The use of BMSC-CMs to modulate the expression of MHC molecules in NSCs is based on the finding that under the conditions used to detect MHC class II molecules, compared with other identical NSCs cultured in NSC medium containing exogenous LIF, NSCs grown in BMSC-CMs did not express MHC class II molecules and exhibited lower levels of MHC class I molecules. This finding is consistent with the finding that the expression of MHC molecules by NSCs is reduced according to the BMSC and NSC co-culture method (in a contact-dependent or contact-independent manner). Whether using BMSCs as feeder layers or culturing NSCs in BMSC-CMs, whether in a contact-dependent or contact-independent manner, provides a way to reduce the expression of MHC molecules in NSCs. The discovery that the expression of MHC molecules can be modulated using the methods disclosed herein provides a method of NSC propagation that facilitates therapeutic use. The reduced expression of HMC molecules in NSCs using the methods disclosed herein also provides a means of increasing the success rate of NSC transplantation into recipients.

Based on the present disclosure, the present invention includes the use of BMSC-CMs to culture and expand NSCs and reduce the expression of MHC molecules of NSCs. That is, the present invention is based on the discovery that expansion of NSCs using BMSC-CM reduces and/or prevents upregulation of expression of MHC molecules on the surface of NSCs compared to otherwise identical NSCs cultured in NSC medium containing exogenous LIF. Thus, the present invention includes a method of propagating cells that facilitates therapeutic use.

It has been widely established that cell transplantation between genetically different individuals (allogeneic) is often accompanied by a risk of transplant rejection. Almost all cells express histocompatibility complex (MHC) class I molecules. In addition, many cell types can be induced to express MHC class II molecules when exposed to inflammatory cytokines. Allograft rejection is primarily mediated by T cell CD4 and CD8 subsets that recognize MHC class I and class II molecules. The main goal in transplantation is permanent engraftment of the donor graft without inducing a graft-rejecting immune response in the recipient. Thus, the present invention includes methods of reducing and/or eliminating the immune response of recipient cells against NSCs transplanted into a recipient by culturing the NSCs prior to transplantation using the methods disclosed herein with the aim of reducing expression of MHC molecules by the NSCs. Without wishing to be bound by any particular theory, reducing the expression of MHC molecules in NSCs using the methods disclosed herein can reduce the amount of MHC molecules displayed on the NSC cell membrane, thereby reducing the immunogenicity of the NSCs in the recipient.

The present invention can also be used to obtain NSCs expressing foreign genes, so NSCs can be used in cell therapy or gene therapy. That is, the present invention contemplates mass production of NSCs expressing foreign genes. The exogenous gene can be an exogenous version of an endogenous gene (ie, a wild-type version of the same gene can be used to replace the defective allele containing the mutation). Exogenous genes may include trophic factors for central nervous system (CNS) regeneration or cytotoxic genes targeting cancer. An exogenous gene is usually, but not necessarily, covalently linked (ie, "fused with") to one or more other genes. Exemplary "other" genes include genes that should be used in a "positive" screen to select cells that have integrated the exogenous gene and genes that should be used in a "negative" screen to select cells that have integrated the exogenous gene into the same chromosomal locus as the endogenous gene , or both.

The NSCs obtained by the method of the present invention can be induced to differentiate into neurons, astrocytes, oligodendrocytes, etc., and the NSCs can be differentiated into a selected type by selective culture under the culture conditions known in the prior art Cell.

The cultured or expanded NSCs described in this disclosure, either before or after differentiation into a selected cell type, can be used to treat a variety of conditions known in the art to be treatable with NSCs. NSCs useful in these therapeutic methods may include NSCs containing an inserted foreign gene, and NSCs not containing an inserted foreign gene. Such conditions include, but are not limited to, traumatic brain injury, Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, stroke, multiple Sexual sclerosis, cancer, CNS lysosomal storage diseases, and head trauma.

Isolation of NSCs and BMSCs

NSC can be obtained from the central nervous system of mammals, preferably humans. These cells can be obtained from a variety of tissues including, but not limited to, forebrain, hindbrain, whole brain and spinal cord. NSCs can be isolated and cultured using methods detailed elsewhere herein or known in the art, for example using methods disclosed in US Pat. No. 5,958,767, the entire contents of which are hereby incorporated by reference. Other methods known in the art for isolating NSCs can be readily used by the skilled artisan, including methods developed in the future. The present invention is in no way limited to these or any other methods of obtaining related cells.

NSCs can be isolated from many different types of tissues, for example: donor tissue by isolating individual cells from the tissue's attached extracellular matrix; or from commercial sources of NSCs. In one embodiment, the brain-derived tissue is aseptically stripped, and then any method known in the art is used to dissociate the cells, including enzymatic treatment methods, such as trypsin, collagenase, etc., or physical dissociation methods, such as using Blunt grinding or handling. Dissociation of neural cells and other pluripotent stem cells can be performed in sterile tissue culture medium. The dissociated cells were centrifuged at low speed (between 200rpm and 2000rpm), the supernatant was aspirated, and the cells were then suspended in culture medium.

Sources of BMSCs and methods of obtaining BMSCs from these sources have been described in the prior art. BMSCs can be obtained in large quantities from any bone marrow, including, for example, bone marrow obtained by aspiration from the iliac crest of a human donor. Methods of obtaining bone marrow from donors are well known in the art and are described, for example, in US Patent 6653134 and WO96/30031, the entire contents of which are hereby incorporated by reference. Human mesenchymal stem cells can be purchased from Cambrex Corporation (Walkersville, MD.),

Applications of Isolated Neural Stem Cells

Isolated neural stem cells have applications in several ways. These cells can be used to reconstitute cells in mammals that have been lost to disease or injury. Genetic diseases can be treated by modifying genetic defects or defending against diseases through genetic modification of autologous or allogeneic neural stem cells. Diseases associated with a deficiency of specific secreted products (eg, hormones, enzymes, growth factors, etc.) can also be treated using NSCs. Central nervous system disorders include many diseases such as neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), acute brain injury (such as stroke, brain injury, cerebral palsy, tumor resection, chemotherapy and radiation supportive therapy) and Many central nervous system disorders (eg, depression, epilepsy, and schizophrenia). Diseases including (but not limited to) Alzheimer's disease, multiple sclerosis (MS), Huntington's Chorea, amyotrophic lateral sclerosis (ALS), and Parkinson's disease Associated with the degeneration of nerve cells in specific locations in the nervous system, resulting in the inability of these cells or the brain to perform their intended functions. The isolated and cultured NSCs described herein can be used as a source of progenitor cells for specific cells to treat these diseases. NSCs can be used as a source of trophic factors to stimulate endogenous stem cells and regeneration of the nervous system. The cultured NSCs described here can be frozen at liquid nitrogen temperature for long-term storage, and can be used again after thawing. The cells are usually maintained in a medium of 10% DMSO and 90% NSC. Once thawed, the cells can be expanded using methods described elsewhere herein.

genetic modification

The cells of the present invention can be genetically modified through the introduction of exogenous genetic material to produce trophic factors, growth factors, cytokines, neurotrophic factors and other similar factors, which is beneficial to the cultivation of NSCs. For example, BMSCs were genetically modified to express and secrete high levels of EGF compared to non-genetically modified BMSCs. Without wishing to be bound by any particular theory, BMSCs that have been genetically modified to express and secrete EGF will secrete the same factor at increased levels over the same BMSCs that have not been genetically modified.

The role of using engineered BMSCs in the co-culture system is to make the modified BMSCs continuously provide exogenous factors in the co-culture system. Exogenous factors can bring benefits to cultured BMSCs or NSCs or both. The exogenous genetic material introduced into BMSCs can also contribute to the secretion of other endogenous factors in engineered BMSCs. In addition, genetically modified BMSCs can facilitate the secretion of endogenous factors in neighboring cells. Thus, the present invention includes the use of genetically modified BMSCs to provide a continuous supply of endogenous factors to the co-culture system. In some instances, exogenous genetic material introduced into the BMSCs contributes to the genetically modified BMSCs and/or adjacent cells. Secretion of endogenous factors. Regardless, exogenous and/or endogenous factors secreted by BMSCs provide beneficial factors for the culture and proliferation of NSCs.

Furthermore, BMSCs genetically modified to express and secrete a factor such as EGF can also be used to generate BMSC-CMs with increased EGF levels. In addition to providing BMSC-CMs with increased levels of exogenous factors, genetically modified BMSCs also facilitate the secretion of endogenous factors in engineered BMSCs and/or adjacent cells. Such factors include (but are not limited to): LIF, brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived neurotrophic factor (GDNF), granulocyte Colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, trypsin-like growth factor-binding protein (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, mono Nuclear cell chemoattractant protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor ( TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein (BMP4), IL1-a, IL-3, Lep Leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor beta (TGFβ-1) and TGFβ-3.

The benefit of using genetically modified BMSCs to generate BMSC-CMs is to increase the level of exogenous factors, such as EGF, so that EGF is secreted into the medium from engineered BMSCs, but not from otherwise identical but non-genetically modified BMSCs. As the levels of EGF secreted from genetically modified cells increased, more EGF appeared in BMSC-CMs. Additionally, the increased levels of EGF secreted by engineered BMSCs may contribute to the secretion of other endogenous factors in engineered BMSCs and/or adjacent cells. BMSC-CMs with elevated levels of EGF and/or other factors can be used for the culture and expansion of NSCs.

In another aspect, BMSCs can be genetically modified to express proteins such as HSV-thymidine kinase or green fluorescent protein (GFP), which can be used for isolation of NSCs after expansion in BMSC co-cultures, respectively. Gancyclovir treatment or separation on flow cytometry.

In addition to genetically modified BMSCs, the present invention also includes genetically modified NSCs. Genetically modified NSCs can be used to replace defective cells in an individual. The present invention can also be used to express secreted desired proteins. That is, NSCs can be isolated and introduced with a gene for a target protein, and then introduced into an individual in which the target protein will be produced and exert or produce a therapeutic effect. This aspect of the invention relates to gene therapy, wherein the individual is infused with a therapeutic protein by introducing genetically modified NSCs into the individual. Genetically modified NSCs are cultured, isolated and implanted by the methods disclosed herein into an individual who will benefit from the expression and secretion of the protein by the NSCs in vivo.

According to the present invention, a constructed gene containing a nucleotide sequence encoding a heterologous protein is introduced into NSC. That is, NSC cells are genetically altered to introduce a gene whose expression has a therapeutic effect in the individual. According to some aspects of the invention, NSCs derived from one individual or another individual or non-human animal can be genetically altered to replace a defective gene and/or introduce a gene whose expression in the individual has a therapeutic effect.

In all cases, the construct gene is transfected into the cell, and the exogenous gene is controllably linked to the regulatory sequences required for gene expression in the cell. Such regulatory sequences include promoters and polyadenylation signals.

The constructed gene is preferably provided as an expression vector, including a coding sequence of a foreign protein controllably linked with basic regulatory sequences. When the vector is transfected into a cell, the coding sequence will be expressed by the cell. The coding sequence is linked to the regulatory elements necessary for expression of the sequence in the cell. The nucleotide sequence encoding the protein may be cDNA, genomic DNA, synthetic DNA or a hybrid thereof, or an RNA molecule such as mRNA.

The constructed gene includes a nucleotide sequence that encodes a beneficial protein and is controllably connected to a regulatory element. It can exist in a cell as a functional cytoplasmic molecule or a functional free molecule, and can also be integrated into the homologous chromosomal DNA of the cell. Exogenous genetic material can be introduced into cells and exist in the form of independent genetic material (such as plasmid). Alternatively, linear DNA capable of integrating into homologous chromosomes can be introduced into cells. When DNA is introduced into cells, some reagents that promote the integration of DNA with homologous chromosomes can be added. DNA sequences to facilitate integration may also be included in the DNA molecule. Alternatively, RNA can be introduced into cells.

Regulatory elements for gene expression include: promoters, start codons, stop codons, and polyadenylation signals. Preferably, these elements are operable in the cells of the invention. In addition, preferably, these elements are controllably linked to the nucleotide sequence encoding the protein, so that the nucleotide sequence can be expressed in the cell, thereby producing the protein. Start codons and stop codons are generally considered to be part of the nucleotide sequence encoding a protein. However, preferably, these elements are functional in the cell. Likewise, the promoter and polyadenylation signal used must be functional in the cells of the invention. Promoters useful in the practice of the present invention include, but are not limited to, promoters effective in many cells, such as cytomegalovirus promoters, SV40 promoters, and retrovirus promoters. Other promoters useful in practicing the invention include, but are not limited to: tissue-specific promoters, i.e. promoters that are functional in some tissues but not in others; promoters that are normally expressed in cells, With or without specific or general enhancer sequences. In some embodiments, a promoter is used with or without an enhancer sequence for sustained expression of the gene in the cell. Where appropriate or desired, enhancer sequences are provided in these embodiments.

The cells of the invention can be transfected by known techniques readily available to those of ordinary skill in the art. Foreign genes can be introduced into cells that will express the protein encoded by the gene by standard methods for introducing constructed genes into cells. In some implementations, cells are transfected by calcium phosphate precipitation transfection, diethylaminoethyl dextran transfection, electroporation, microinjection, liposome-mediated transfer, chemical-mediated transfer, ligand Gene-mediated transfer or recombinant viral vector transfer.

In some embodiments, recombinant adenoviral vectors are used to introduce DNA with a desired sequence into cells. In some embodiments, recombinant retroviral vectors are used to introduce DNA with a desired sequence into cells. In some embodiments, the desired DNA is incorporated into isolated cells using standard CaPO4, diethylaminoethyl dextran, or lipid carrier-mediated transfection techniques. Transfected cells can be identified and selected using standard antibiotic resistance screening techniques. In some embodiments, DNA is introduced directly into cells by microinjection. Similarly, known electroporation or particle bombardment techniques can also be used to introduce foreign DNA into cells. A second gene is usually co-transfected with or linked to the therapeutic gene. The second gene is often a selectable antibiotic resistance gene. Transfected cells can be selected by incubation with antibiotics that will kill cells that do not receive the selectable gene. In most cases, the two genes were separated and co-transfected, and cells that survived antibiotic treatment had both genes and expressed them.

The following examples are intended to more fully describe the preferred embodiments of the present invention. These examples should in no way be construed as limiting the scope of the invention as defined in the claims.

Example

Example 1: BMSCs are used as trophoblast cells for the cultivation and expansion of NSCs

The cultivation of NSCs is challenging because they require growth factors and special substrates to enhance their growth rate and expansion. For growth factors, the addition of exogenous LIF to serum-indeterminate medium containing FGF and/or EGF significantly enhanced NSC expansion. As discussed elsewhere herein, incorporation of exogenous LIF and coating of the culture dish further increased the level of expansion of NSCs while maintaining their pluripotency.

Bone marrow stromal cells (BMSCs) can be readily obtained and expanded in culture to a substantially homogeneous cell population. In addition, BMSCs also secrete several trophic factors that can promote the growth of NSCs. Therefore, this example illustrates that BMSCs can be used as supporting cells for the expansion of NSCs in a co-culture system.

Now, the materials and methods used in the experiment in this embodiment are described as follows:

Establishment, Maintenance and Characterization of Human Fetal Neural Stem Cells (NSCs)

Human brains (from 11-14 week old fetuses) were provided by Advanced Bioscience Resources (Alameda, CA). Brain tissue was ground in cold PBS. After centrifugation, the cells were suspended in 10ml NSC growth medium (DMEM/F12, 8mM glucose, glutaminase, 20mM sodium bicarbonate, 15mM HEPES, 8μg/ml heparin, N2 supplement, 10ng/ml bFGF, 20ng/ml EGF ). The above cells were placed on a T-25cm type ² box coated with polyornithine and fibronectin, and cultured at 37°C in a 5% CO ₂ incubator. The culture medium was replaced 50% with fresh medium every 2 days, and passaged by trypsinization every 14 days. Cells were frozen in liquid nitrogen in NSC medium containing 10% DMSO. Cells were thawed and initially cultured for passage 1-2 in the presence of bFGF and EGF, then placed in complete growth medium supplemented with 10 ng/ml LIF.

Other NSC growth media can be used in the methods disclosed herein. For example, cells can be cultured in Neurobasal medium supplemented with L-glutamine, bFGF, EGF, and B27, but without retinoic acid (Invitrogen, Carlsbad, CA). Another NSC medium is NeuroCult, to which growth factors are added (StemCell Technologies, Vancouver BC, Canada). Without wishing to be bound by any particular theory, any medium can be used to culture NSCs. However, a suitable medium enables the cells to grow and expand while maintaining their ability to differentiate into a variety of cell types.

For identification, NSCs at different passages were plated on coated cell culture chamber slides, fixed with 4% paraformaldehyde and stained for nestin and glial fibrillary acidic protein (GFAP). NSCs were differentiated for approximately 14 days by withdrawal of bFGF, EGF and LIF, and treatment with Neurobasal medium, B27 supplement and BDNF. Other differentiation conditions can be used to differentiate cells into specific lineages. Cells were fixed and stained for microtubule-associated protein (MAP2; producer of neurons) and GFAP (producer of astrocytes). To identify neuronal subtypes, cells were stained with anti-gamma aminobutyric acid (GABA) and anti-tyrosine hydroxylase (TH). The primary antibodies used were human-specific nestin, 1:10 (R&D Systems); MAP2, 1:500 (Sigma); GFAP, 1:1000 (DAKO); O4, 1:100; NG-2 (1:200 ) (Chemicon). The secondary antibodies used in these experiments were Alexa Fluor 488 chicken anti-mouse 1:500 (Molecular Probes) and Alexa Fluor 594 chicken anti-rabbit, 1:500 (Molecular Probes).

Expression of several markers in NSCs was analyzed by flow cytometry, including hematopoietic markers CD45 and CD14, stem cell markers CD34, CD133 and CD56, and immunogenicity/stimulatory markers CD80, CD86, MHC class I and MHC class II .

Quantification of nestin expressing cells was also determined by flow cytometry. This analysis was performed using anti-nestin (R&D) and goat anti-mouse Ig conjugated to Alexa-fluor488. NSCs were fixed using the Becton-Dickinson Cytofix/Cytoperm kit and instrument with minor modifications.

Establishment, maintenance and characterization of bone marrow stromal cells (BMSCs)

BMSCs are produced by methods known in the art. For example, human bone marrow is collected by needle aspiration. Count the number of nucleated cells in the bone marrow. Bone marrow was diluted with PBS and mixed with hydroxyethyl starch. The hydroxyethyl starch/cell suspension was allowed to stand for 45 minutes to 1 hour. During this time, erythrocytes settle, leaving nucleated cells present in the supernatant layer. After removal of red blood cells, nucleated cells were washed and counted.

Culture nucleated cells in tissue culture treated vessels (eg, polystyrene), DMEM-low glucose, 10% FBS. Primary cultures were continued for 12-17 days with medium changes every 3 or 4 days. When there are sufficient numbers of adhesin, spindle cells, trypsin subcultures are used to remove adherent cells. Cells were replated and cultured for approximately 1 week, with medium changes at each subsequent passage.

Flow cytometry uses specific markers to test cell purity. The first or second passage (P1 or P2) BMSCs that were CD45 negative and more than 90% CD90 and CD13 positive were used in the co-culture experiments.

Culturing BMSCs in NSC Growth Medium

BMSCs were placed in 6-well plates at different densities and allowed to adhere overnight in BMSC medium (DMEM-low glucose, 10% FBS for sampling). The next day, one plate's medium was replaced with NSC medium (DMEM-F12, N2-supplement, EGF, 20 ng/mL, bFGF, 10 ng/mL, 8 μg/mL heparin, P/S), and the other plate received Fresh BMSC medium. Supplement cells with fresh BMSC or NSC medium every three days. One set of cultures was trypsinized and cells were counted 7 days later. Another set of cells was harvested and counted 12 days later. As shown in Table 1 and Figure 1, BMSCs proliferate much more in BMSC medium than in NSC medium. In NSC medium, the initial increase in cell number was less than in BMSC medium. However, after 7 days of culture in NSC medium, the cell number of BMSCs did not increase significantly. In BMSC medium, cells continued to proliferate for 12 days and were highly confluent at this end. In NSC medium, BMSCs did not form any vortexes of confluent cells, while exhibiting normal morphology without visible cell death; the opposite was observed for cell morphology in BMSC medium.

Table 1 culture medium initial cell 7 day cell number 12 day cell number NSC medium 50,000 210,000 240,000 NSC medium 100,000 300,000 460,000 NSC medium 200,000 615,000 650,000 BMSC medium 50,000 310,000 1,300,000 BMSC medium 100,000 683,000 1,200,000 BMSC medium 200,000 855,000 2,200,000

These results indicated that when NSCs and BMSCs were co-cultured in NSC medium, BMSCs did not compete with NSCs and overgrow.

Co-cultivation of BMSCs and NSCs; survival and expansion of NSCs on BMSC-coated plates

Human BMSCs (hBM-03-016, P1) were thawed, washed and counted, and placed in a 12-well plate, 75,000 cells/well. In parallel, another 12-well plate was coated overnight with 15 μg/ml polyornithine followed by 10 μg/ml human fibronectin. The next day, hNSCs grown in medium were collected and resuspended in NSC medium containing EGF and FGF. The NSC used was hHB-007, P7, cultured in NSC medium containing EGF, FGF and LIF. Most of these cells grow as neurospheres. When placing these cells in BMSC or coated wells, the neurospheres were broken down into single cells as much as possible. Three copies of NSC were placed on BMSC or coated plates under the following conditions.

1. 25000NSC+NSC medium without LIF

2.25000NSC+NSC medium+LIF

3.50000NSC+NSC medium without LIF

4.50000NSC+NSC medium+LIF

Cultures were replaced with 50% fresh medium every other day. Phase contrast pictures of cells were taken at different times. After 12-13 days in culture, cells were harvested using trypsin. That is, cells were incubated with 0.05% trypsin for 5 minutes or until all cells detached from the dish. Trypsin was then neutralized using soybean trypsin inhibitor (SBTI). Cells were washed with phosphate buffered saline (PBS) and counted in a hemocytometer using trypan blue staining. Cells of two different sizes, larger BMSCs and smaller NSCs, were observed under the microscope. These two cell populations were counted separately. The first count was at day 12 by mixing two of the three replicate cultures.

NSCs placed on BMSCs adhered to the aggregated areas and spread as monolayers. Changing the medium on these cells is not difficult. On the other hand, NSCs, especially those grown as neurospheres in the presence of exogenous LIF (Figure 2), are not very adherent, so be very careful when changing the medium. Sometimes it is necessary to centrifuge the aerated medium to prevent loss of neurospheres. Meanwhile, NSCs do not survive when the density is low unless they are co-cultured with BMSCs.

Morphologically, NSCs in co-culture formed a network of small circles to ovals on spread larger BMSCs. NSCs formed isolated NSC colonies, sometimes surrounded by fibroblastic BMSCs (Fig. 3). The NSC colony has a greater number of NSCs in each additional colony. NSCs cultured alone survived and proliferated only in the presence of exogenous LIF and at higher seeding densities; however, in co-cultures, NSCs expanded significantly even in the absence of LIF. Thus, BMSCs appear to be able to provide nutritional support to NSCs as a trophoblast. NSCs in co-culture achieved significant expansion compared to NSCs grown on coated (ie polyornithine/fibronectin) dishes supplemented with exogenous LIF. Table 2 describes the number of NSCs and BMSCs collected in culture after 12 days.

Table 2

Expansion of NSCs co-cultured with BMSCs Initial #NSC LIF +BMSC no BMSC 2.5e4 - 8.8e4 1.3e4 2.5e4 + 10e4 7.5e4 5.0e4 - 25e4 11e4 5.0e4 + 24e4 33e4

Characterization of NSCs cultured on BMSCs: expression of nestin

To evaluate nestin expression in NSCs co-cultured with BMSCs by immunostaining, cultures were grown on coverslips. Coverslips (10 mm) placed in 24-well dishes were coated with polyornithine and human fibronectin as described elsewhere herein. One set of coverslips was plated with BMSCs (35,000/coverslip) and cells were allowed to adhere overnight. The next day, NSCs grown in culture (THD-015+LIF, P12) were harvested and counted. 25000/well of NSCs were plated on BMSCs or directly on coated coverslips. Some wells with BMSCs were cultured alone without NSCs. All different cultures were grown in NSC medium or NSC medium + LIF. Cells were replaced with 50% fresh medium every day for 10 days. After 10 days, some cultures were fixed and prepared for nestin staining. Other cultures were allowed to differentiate for 2 weeks.

To fix the culture, the culture was washed once with PBS. Then add 4% paraformaldehyde, incubate at room temperature for 15 minutes, and wash 3 times with PBS. At this point, the fixed cultures were stored in PBS at 4°C or stained immediately.

Nestin staining of subsequent cultures was achieved by staining with anti-nestin + anti-GFAP antibodies. Cultures included BMSC+NSC, BMSC+NSC+LIF, BMSC alone, BMSC+LIF, NSC alone, and NSC+LIF. One replicate sample as a control (no primary antibody)

NSCs cultured alone in NSC medium with or without exogenous LIF expressed nestin. In addition to expressing Nestin, NSCs also co-expressed GFAP when cultured in NSC medium+LIF.

All co-cultures exhibited a large number of nestin positive cells spreading on top of BMSCs (Fig. 4A-4F). BMSCs themselves showed only vague background staining (Fig. 4G-4H). The morphology of nestin-positive cells is heterogeneous. Many nestin-positive cells were also positive for GFAP, while others were nestin-positive and GFAP-negative. Some nestin-positive cells were small round or oval with or without one or both filamentous extensions. Some nestin-GFAP double positive cells are large, flattened astrocyte-like cells. There was no clear difference between the co-cultures with and without the addition of exogenous LIF.

Differentiation of co-cultures

After 10 days of culture in NSC medium, the co-cultures were differentiated for 2 weeks. The differentiation schedule consisted of 2 additions, each of 1) DMEM/F1 supplemented with N2 but no EGF or FGF, 2) DMEM/F12+B27 supplemented, and 3) DMEM/F12+B27+BDNF . Differentiated cultures were fixed in paraformaldehyde and stained using antibody combinations such as Nestin/GFAP, MAP2, GFAP and O4/GFAP. In all conditions, GFAP was detected by red fluorescence (Alexa 594), whereas Nestin, MAP2 or O4 was detected by green fluorescence (Alexa 488). Cultures were counterstained with DAPI for green fluorescent labeling of all nuclei.

NSCs grown on BMSCs maintained their ability to differentiate into neurons and astrocytes (FIGS. 5A-5D). After incubation in differentiation medium, several cells expressed the neural marker MAP-2. These cells are small cells with oval nuclei and bipolar or multistage cell bodies. These protrusions are expansive and form complex neuronal networks on BMSCs or on GFAP-stained cells. GFAP-positive astrocytes were also present throughout the culture. These cells are large, polygonal, and flat compared to neurons. By approximation, the number of neurons is comparable to or greater than that of astrocytes, an exact determination is not possible because the vast network of neurons forms a three-dimensional cellular network. At the dilutions used, O4 antibody staining showed no differentiation of cells into oligodendrocytes. Longer differentiation times may be required to generate oligodendrocytes. However, when the presence of oligodendrocytes was estimated using NG2, a chondroitin sulfate proteoglycan oriented on the surface of oligodendrocyte precursors, as a marker of oligodendrocyte differentiation, few cells are positive for NG2. Nestin staining of differentiated co-cultures revealed that some GFAP-positive astrocytes were weakly positive for Nestin, but these cells appeared morphologically different from the small filamentous cells seen before differentiation (Figure 6). The presence or absence of exogenous LIF during growth brought no difference in the differentiation of NSCs in co-cultures. When NSCs cultured alone were differentiated as described above, they were observed to differentiate into neurons (MAP2 positive) and astrocytes (GFAP positive) ( FIG. 7 ).

When BMSCs were subjected to the same differentiation conditions alone, they showed weak, diffuse staining for Nestin and GFAP, with GFAP staining being somewhat more intense in cells cultured with additional exogenous LIF. The cell morphology remained the same as BMSCs but different from astrocytes. Few cells in culture had a high degree of nestin or MAP2 positivity indicative of differentiation into NSC or neuronal lineages (Figure 8).

Example 2: Removal of BMSCs from BMSC and NSC co-cultures

Cells in BMSC and NSC co-cultures can be isolated by incubating the co-culture with a murine anti-human CD13 antibody (positive on BMSC and negative on NSC). Using magnetic beads linked to pan mouse IgG, BMSCs were able to be isolated from NSCs. Unbound cells representing NSCs were analyzed by FACS or returned to culture medium. For FACS analysis, different BMSC marker antibodies (mouse anti-human CD105) were used to detect infected BMSCs, while mouse anti-human CD133 was used to detect NSCs.

BMSCs were put into 6-well culture dishes, 150,000 cells/well, and allowed to adhere overnight in BMSC medium. NSCs (THD-hWB-015+LIFP17) grown in culture were harvested and resuspended to disrupt neurospheres. The BMSC medium was removed from the BMSC culture and the harvested NSCs were plated on BMSCs (75,000 cells/well) in NSC medium (with EGF and FGE, with or without LIF). Co-cultures were grown in NSC medium for 13 days. Replace the cells with fresh medium every other day or on weekends.

On day 13, co-cultures were harvested using trypsin, followed by soybean trypsin inhibitor. The cell mixture was resuspended in PBS containing 0.1% BSA. Incubate the cell mixture with anti-CD13 for 30 min on ice. Cells were centrifuged and washed with PBS (0.1% BSA). At the same time, the Dynal beads-Pan mouse IgG were washed 3 times with PBS (0.1% BSA), each time they were separated by a magnetic particle concentrator (Dynal-MPC). Washed cells were incubated with washed magnetic beads in PBS (0.1% BSA) for 30 minutes at 4°C on a tilt/rotate device (Dynal sample mixer). The tube containing the cell-bead mixture was placed in the MPC for 2-3 minutes. The magnetic beads, with the cells attached to them, adhere to the side of the tube next to the magnet. Collect the supernatant and place in a separate tube.

A portion of the cells in the supernatant was used for FACS analysis, and a portion of the cells were placed on cell culture slides coated with polyornithine and fibronectin. FACS analysis was performed using anti-CD105 and anti-CD133 (recognizing NSC). Cells on cell culture slides were cultured in NSC medium for 1-2 days and then fixed for immunostaining for nestin/GFAP.

NSCs were expanded on the BMSC layer over a period of 2 weeks. Some large colonies were observed thereon, presumably arising from small neurons. Other NSCs in the form of single cells or a few cells grew into several smaller NSC colonies within 2 weeks.

Following magnetic exclusion of BMSCs, isolated NSCs were analyzed by FACS. More than 90% of the cells were positive for CD133 (Fig. 9B).

Less than 2% were positive for CD105 (Fig. 9A). Cells on cell culture slides adhered to the dish and were morphologically similar to NSC. Nestin immunostaining was used to confirm the presence of NSCs. The BMSC-bead mixture in culture was morphologically similar to BMSCs that incorporated a large number of magnetic beads and attached to tissue culture flasks. The results demonstrate that BMSCs can be isolated from co-cultures by simple incubation with BMSC-binding antibodies followed by magnetic separation, leaving a concentrated population of NSC cells.

Example 3

Co-culture scale-up and enhanced NSC expansion using minimal amount of BMSC as feeder layer

In this experiment, co-cultures were expanded into T75 tissue culture flasks. BMSCs (donors BM-022, P1 and BM-024, P1 ) were placed in BMSC medium at two different seeding densities of approximately 2.5e5 or 5.0e5/bottle. Two days later, cells were washed with NSC medium, and 5.0e5 NSCs (THD-015, P14) were placed on top of BMSCs in 15 ml complete NSC medium. The cells were co-cultured for 15 days, and the cells were replaced with 50% fresh medium every other day or two. After 15 days, cells were harvested by trypsinization. Larger BMSCs and smaller NSCs were counted on a hemacytometer. BMSCs were removed as in Example 2 as described elsewhere herein. Suspend the cells to 5×10e7/ml and incubate with anti-human CD13 antibody for 15 minutes on ice. Cells were washed once and then incubated with washed Pan mouse IgG Dyna-beads for 30 minutes. Bead-bound cells are separated in a magnetic particle concentrator. The supernatant containing NSCs was washed with NSC medium, and the number of recovered NSCs was counted. Some cells were analyzed by FACS, others by differentiation and immunocytochemistry.

The disclosure herein demonstrates that culturing NSCs on a smaller number of BMSC feeder layers results in greater NSC expansion than culturing on a greater number of BMSC feeder layers. By varying the ratio of BMSC to NSC seeding densities, it was found that when BMSCs were at low confluence of approximately 30%, higher NSC doublings were generated relative to confluence of 50-60% or higher. For example, as shown in Table 3, it can be seen that when the BMSC seeding density in T75 flasks was at about 2.5e5 and the NSC seeding density was at 5.0e5, the NSCs were multiplied 82-fold, while the same number of NSCs were placed in double the number of NSCs multiplied 47-fold when BMSCs were on. In comparison, in other experiments when NSCs were cultured in standard NSC medium supplemented with exogenous LIF in the absence of BMSC-coated dishes, the maximum fold increase of NSCs was about 20-25 times. Following NSC expansion in the presence of BMSCs, BMSCs in co-cultures were efficiently removed using anti-BMSC antibodies and immunomagnetic beads with 83-93% recovery (Table 3).

table 3

Expansion of hNSCs on BMSC trophoblasts BMSC donor (seeding density) Starting #NSC #NSC day 15 NSC doubling % recovery after BMSC removal # Recycled NSC (multiplied) BM-03-022 (about 2.5e5) 5.0e5 41.1e6 82.2 times 93% 38.3e6 (76.5 times) BM-04-024 (about 5.0e5) 5.0e5 23.5e6 47 times 83% 19.4e6 (39 times)

Co-culture expanded and isolated NSCs were observed to continue to express markers of progenitor cells, including but not limited to nestin (Figure 10). Isolated NSCs also retained their ability to differentiate into neurons and astrocytes (Figure 11). Those of skill in the art will recognize, based on the present disclosure, that this method for expanding NSCs can be further scaled up and adapted to closed system production processes that have been validated for the production of BMSCs.

Example 4: Co-cultivation of BMSCs and NSCs in Transwell ^™ (contact-independent co-cultivation)

This paper has demonstrated that BMSCs can serve as an excellent feeder/support layer for the propagation and expansion of human neural stem cells (hNSCs). In this example, experiments were designed to address whether physical contact between the two cells is required or whether BMSCs can support NSC propagation in a contact-independent manner by providing soluble trophic factors to support NSC propagation and expansion and proliferate.

BMSCs, designated hBM-03-016-P1 and hBM-012-P2, were thawed, washed with BMSC medium, and placed in Transwell ^™ . Experiments in Transwell ^™ were performed in 6-well Costar dishes. BMSCs were placed in Transwell ^™ , while NSCs were placed at the bottom of the wells. Both cells were cultured in the same NSC medium. The application of Transwell ^TM enables the cultivation of different types of cells, such as BMSCs and NSCs, without physical contact between the two types of cells.

Approximately 30,000 BMSCs were placed on a removable porous filter of Transwell ^(TM) . After allowing the cells to adhere to the Transwell ^™ surface in BMSC medium overnight, the cells were rinsed with plasma-free medium and cultured in complete NSC medium (DMEM/F12+N2 supplement+EGF+FGF). NSCs (designated THD-hWB-015-P13) were thawed, washed and plated at a density of 40,000 cells/well on polyornithine/fibronectin-coated 6-well dishes. The Transwell ^(TM) containing BMSCs were then placed on a 6-well plate containing NSCs. The control was Transwell ^™ including NSCs without BMSCs. Sufficient medium was added to the Transwell ^TM and the bottom of the well. Approximately 50% of the medium was changed every other day. After two weeks in culture, NSCs were collected from each plate well and counted. Cells were subjected to flow cytometry to analyze the expression of NSC markers.

The results indicated that NSCs grown on coated wells co-cultured with BMSCs in Transwell ^™ multiplied better than NSCs cultured on coated wells without BMSCs ( FIG. 12 ). BMSCs were found to support the growth and proliferation of NSCs, even in the absence of direct contact between the two cells. Without wishing to be bound by any particular theory, it is believed that factors secreted from BMSCs can be used to supplement the NSC culture medium to help support NSC propagation and proliferation. FACS analysis of NSCs grown in Transwell ^™ with or without BMSCs showed that the NSCs were phenotypically similar. For example, NSCs grown under both conditions were approximately 100% positive for CD133.

The experiment above was repeated except that the 6-well dishes were not coated with polyornithine/fibronectin. Approximately 50,000 BMSCs were placed in Transwell ^TM . NSCs (designated THD-hWB-015-P12) were thawed and plated in 6-well Falcon or Costar plates. BMSCs grown on Transwell ^TM were placed in 6-well plates and cultured for 2 weeks. As a control, NSCs were cultured alone in NSC medium with or without exogenous LIF. The growth pattern of NSCs co-cultured with BMSCs in Transwell ^TM was similar to that of NSCs cultured alone in NSC culture containing exogenous LIF, while their proliferation was significantly better than that of NSCs cultured alone without exogenous LIF (Figure 13 ). It was also found that Costar dishes provided better results in terms of cell proliferation numbers than Falcon dishes.

Example 5: NSC growth in BMSC conditioned medium

The experimental results in the examples illustrate that BMSCs produce soluble factors that support the growth of NSCs. The next set of experiments used conditioned media derived from cultured BMSCs to further elucidate the role of BMSC-secreted factors on NSC growth. The experiments disclosed here illustrate whether growth and/or other factors secreted by BMSCs can support and enhance NSC growth and proliferation. This experiment was designed to evaluate 1) whether the medium defined by BMSC in culture can replace the NSC medium supplemented with exogenous LIF and bring about the growth-promoting effect on NSC in culture 2) by the BMSC in culture Whether the defined conditioned medium can bring beneficial effects similar to that of culturing NSC on polyornithine/fibronectin-coated medium for NSC culture.

To generate BMSC-CMs, first place BMSCs in T-80 flasks and culture them in BMSC medium. After culturing for a period of time, replace BMSC medium with NSC medium. BMSC-CMs were thus obtained, BMSC-CM1 was obtained by culturing BMSCs with complete NSC medium containing bFGF and EGF, and BMSC-CM2 was obtained by culturing BMSCs with NSC medium without bFGF and EGF. Replace BMSCs with fresh NSC medium every 48 h. At this point, the medium was removed and centrifuged to remove particulate matter and used to culture NSCs or frozen at -80°C for cytokine evaluation. For BMSC-CM experiments, NSCs were cultured using NSC growth medium containing approximately 25-50% BMSC-CMs freshly collected from BMSCs or stored at 4°C for 2 weeks. BMSC-CMs were analyzed for the presence of various cytokines by Cytokine Arrays (RayBiotech Inc.) or ELISA.

To evaluate whether NSCs can proliferate in BMSC-CM, NSCs were placed on polyornithine/fibronectin-coated culture dishes, and 50% of the medium was replaced every other day. The replacement medium used was a) BMSC- CM1, b) BMSC-CM2, c) complete NSC medium with exogenous LIF, or d) complete NSC medium without exogenous LIF. For the first two exchanges of BMSC-CM1 and BMSC-CM2, fresh EGF and FGF were added to each BMSC-CM, and this addition of EGF and FGF was stopped later.

The experimental results showed that the growth of NSC in BMSC-CM or complete NSC medium was equally good. Figure 14 depicts representative regions of NSCs cultured under different conditions. In the presence of BMSC-CM1, it can be seen that NSCs form large sticky neurospheres, and some cells grow out of neurospheres. It can also be seen that some NSCs cultured in the presence of BMSC-CM1 formed monolayers. In the case of culturing NSCs in BMSC-CM2, it was also seen that NSCs formed neurospheres, but compared with NSCs cultured under BMSC-CM1, there were few large neurospheres formed and monolayers rarely appeared. In the presence of complete NSC medium, NSCs grow into several interconnected neurospheres and exhibit cell overgrowth from the neurospheres. In any case, cells in each culture group were harvested and counted after 10 days of culture to compare the effects of different culture conditions on NSC proliferation. The number of cells harvested from BMSC-CM1 (approximately 3.4e6 cells) was comparable to the number of cells harvested from complete NSC medium containing exogenous LIF (approximately 3.3e6 cells). The number of cells obtained in BMSC-CM2 was approximately 2.85e6.

The next set of experiments tested whether NSCs could proliferate in BMSC-CMs, even on uncoated dishes. The 4 groups of NSCs were as follows: 1) NSCs cultured in BMSC-CM1; 2) NSCs cultured in BMSC-CM2; 3) NSCs cultured in complete NSC medium containing exogenous LIF; and 4) cultured in NSCs in complete NSC medium without exogenous LIF. Cells were cultured for 2 weeks, passaged by trypsinization and treated under the same conditions for another period of 2 weeks. It was found that in the first 2 weeks, NSCs had the greatest proliferation in BMSC-CM1, followed by BMSC-CM2, and then NSC medium+LIF (bar graph indicated as P1, Figure 15). After passaging, NSC proliferation of BMSC-CM1 and NSC medium+LIF was similar (indicated as P2, Figure 15). Cells treated with BMSC-CM2 continued to proliferate better than cells treated with LIF-free NSC medium. Therefore, the available data suggest that BMSC-CM is a good source of growth factors for hNSCs and can induce NSC proliferation at a rate comparable to or higher than that of NSC media supplemented with exogenous LIF. In addition, NSCs cultured with BMSC-CMs can be passaged by trypsinization and further proliferated to increase the number of cells.

Cytokine microarray analysis of BMSC-CM

BMSCs were cultured in complete NSC medium. The medium was collected at 48 hours, and the cytokine profile was determined using a cytokine chip. RayBio Human Cytokine Antibody Chip C Series 1000 (combination of Chip VI and VIT) was purchased from RayBiotech (Norcross, GA). Chip membranes were processed using samples as described in the manufacturer's instructions. Final detection of cytokines was performed using the ECL-Plus system (Amersham, Piscataway, NJ). Signals were visualized on X-ray film (Amersham, Piscataway, NJ).

The results of the cytokine curves indicated that various cytokines/other factors were present in BMSC-CMs, and their signal intensity was higher than that of the negative control on the same chip. Cytokines/other factors include (but are not limited to) LIF, brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell-derived Neurotrophic factor (GDNF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, trypsin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, monocyte chemoattractant protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinases (TIMP-1) , TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein (BMP4), IL1 -a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor beta (TGFβ- 1) and TGF beta-3.

Without wishing to be bound by any particular theory, BMSC-CMs contain several factors, including those present in the medium containing EGF and bFGF, in addition to factors secreted by BMSCs or induced by the medium. The factors listed above are those factors whose signal evaluation is higher than the negative control on the chip among the 120 cytokines detected on the chip.

Cytokines detected above background levels include, but are not limited to, EGF and bFGF. Other well-expressed cytokines were HGF (hepatocyte growth factor), M-CSF (monocyte phagocyte colony-stimulating factor), and TIMP-1, TIMP-2 (tissue inhibitors of metalloproteinases 1 and 2, respectively). Neurotrophic factors include, but are not limited to, BDNF, NT3, GDNF, and GNTF. IGFBPs and FGF-6 are shown. Many chemokines, pro-inflammatory cytokines and angiogenic factors, such as VEGF, are also produced. ELISA also confirmed that BMSCs also produced LIF. In the ELISA assay, the actual amount of each of the tested factors secreted by BMSCs was estimated by subtracting the control medium levels from the levels observed in BMSC-CMs.

From what is disclosed here, it can be concluded that BMSCs produce soluble factors that promote the growth of NSCs. Direct contact of NSCs with BMSCs is not necessary, as BMSC conditioned culture can also support the growth and proliferation of NSCs. The present disclosure shows that contact-dependent co-culture produces maximal proliferation of NSCs, indicating a synergistic effect of physical contact of BMSCs or BMSC products and BMSC-generated soluble factors with NSCs on NSC proliferation.

Example 6: NSCs cultured in BMSCs or BMSC-CMs exhibited reduced expression levels of MHC molecules, but maintained the same phenotype as NSCs grown alone under standard conditions.

The NSCs proliferated in Example 3 were analyzed by flow cytometry for the expression of various BMSC and NSC markers. NSCs co-cultured with BMSCs maintained the same phenotypic characteristics (eg, positive for CD56 and CD133, negative for CD105) as those cultured in NSC medium supplemented with exogenous LIF without BMSCs, except that co-cultured with BMSCs Cultured NSCs showed undetectable expression of MHC class II molecules (Figure 16). When exogenous LIF was added to the NSC growth medium, it was observed that MHC class II molecules were induced on NSCs. In contrast, NSCs co-cultured with BMSCs were not observed to express higher than baseline MHC class II molecules that might be beneficial for transplantation. Additionally, NSCs isolated from co-cultures did not express the BMSC marker CD105 above baseline, indicating efficient removal of BMSCs from co-cultures.

Similarly, NSCs cultured with BMSC-CM also maintained the same phenotypic characteristics as NSCs cultured in standard NSC medium+LIF, except that they exhibited baseline levels of MHC class II and reduced MHC class I (Fig. 17). Four groups of NSCs were subjected to FACS analysis of CD133, MHC class II molecules, MHC class I molecules and CD56 expression (Fig. 17). The results confirmed previous observations that NSCs grown in the presence of exogenous LIF expressed MHC class II molecules and exhibited increased mean fluorescence intensity of MHC class I molecules. However, NSCs grown on BMSC-CMs were not observed to express class II MHC molecules and had lower expression levels of class I MHC molecules. The expression of CD133 was similar in all 4 groups of NSCs. All cells tested expressed CD56, although cells cultured in the presence of exogenous LIF exhibited a reduced mean fluorescence intensity of CD56.

Example 7: Regulation of NSC Class I and Class II MHC Molecule Expression by Proliferation on BMSCs

Additional experiments were performed to evaluate the expression of MHC class I and class II molecules of co-cultured NSCs. NSCs were isolated from fetal brains and cultured in NSC medium for passage 13 (THD-WB-015, P13). Then, the NSCs were cultured for 12 days under the following 4 conditions, and the medium was changed every other day.

1.62,500 NSCs were individually cultured in T25 flasks coated with complete NSC medium

2.62,500 NSC were cultured alone in the coated T25 flask of complete NSC medium + LIF

3. 62,500 NSCs were cultured on feeder layers of 250,000 BMSCs (Donor-012) in complete NSC medium

4. NSCs were cultured on feeder layers of 250,000 BMSCs (Donor-016) in complete NSC medium

Cells were harvested after 12 days by trypsinization. Cells were counted and analyzed by flow cytometry for the expression of CD133, CD105, MHC class I and MHC class II molecules. In co-culture, all cells were required and NSCs were gated based on light scatter and CD133 expression. Figure 18 shows the expression of MHC class I and MHC class II molecules by NSCs cultured under various conditions (black = isotype control; gray = class I or II). The results indicated that all NSCs expressed MHC class I. However, when NSCs were cultured in the presence of exogenous LIF, the expression of MHC class I molecules on the cells was significantly increased, as reflected in the increase in Log Median Fluorescence Intensity when compared to NSCs cultured without exogenous LIF. Co-cultured NSCs expressed significantly lower levels of MHC class I molecules compared to NSCs expanded under LIF (Table 4).

Table 4 Culture conditions Log MF Class I MHC Isotype Control cell doubling NSC alone, without LIF 209 8.16X NSC alone + LIF 469 13.5X NSC+BMSC-012 323 16X NSC+BMSC-016 366 26.7X

NSCs do not constitutively express class II MHC molecules. However, LIF induced the expression of MHC class II molecules in 38% of the cells. NSCs cultured with BMSCs were similar to NSCs cultured without LIF in that they failed to express MHC class II molecules above baseline.

The above results illustrate that the advantage of propagating NSCs on BMSCs is to reduce and/or prevent the expression of immunomodulatory MHC class I and II molecules on NSCs, making them more suitable for clinical transplantation.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

It is obvious to those skilled in the art that various adjustments and changes can be made to the methods and compositions of the present invention without departing from the concept or scope of the present invention. Thus, the present invention covers the modifications and changes provided by the claims and their equivalents.

Claims (43)

1. a composition comprises: (a) a kind of isolating marrow stromal cell; (b) the definite developing medium of a kind of chemistry, described developing medium comprises the neural stem cell growth medium and the described marrow stromal cell excretory factor.

2. the described composition of claim 1, wherein said developing medium does not contain exogenous leukaemia inhibitory factor.

3. the described composition of claim 1, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

4. the described composition of claim 1, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

5. the described composition of claim 1 also comprises a kind of isolating neural stem cell.

6. the described composition of claim 5, wherein said neural stem cell is that physics contacts with described marrow stromal cell.

7. the described composition of claim 5, wherein said neural stem cell is not that physics contacts with described marrow stromal cell.

8. the described composition of claim 5, wherein said neural stem cell derives from people's central nervous system.

9. the described composition of claim 1, wherein said bone marrow substrate cell source is in the people.

10. the described composition of claim 5, wherein exogenous genetic material is imported into described neural stem cell.

11. the described composition of claim 1, wherein exogenous genetic material is imported into described marrow stromal cell

12. a marrow stromal cell conditioned medium comprises the developing medium that a kind of chemistry is determined, described developing medium comprises the neural stem cell growth medium and by the isolating marrow stromal cell excretory factor.

13. the described marrow stromal cell conditioned medium of claim 12, wherein said bone marrow matrix conditioned medium does not comprise exogenous leukaemia inhibitory factor.

14. the described marrow stromal cell conditioned medium of claim 12, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

15. the described marrow stromal cell conditioned medium of claim 12, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

16. regulate the method that main histocompatibility complex molecule is expressed for one kind on isolating neural stem cell, described method comprises co-cultured cell, described cell comprises isolating marrow stromal cell and isolating neural stem cell.

17. the described method of claim 16, wherein said cell is lacking cultivation altogether under the exogenous leukaemia inhibitory factor condition.

18. the described method of claim 16, wherein said neural stem cell is that physics contacts with described marrow stromal cell.

19. the described method of claim 16, wherein said neural stem cell is not that physics contacts with described marrow stromal cell.

20. the described method of claim 16, wherein said neural stem cell derive from people's nervous center system.

21. the described method of claim 16, wherein said bone marrow substrate cell source is in the people.

22. the described method of claim 16, wherein exogenous genetic material is imported into described neural stem cell.

23. the described method of claim 16, wherein exogenous genetic material is imported into described marrow stromal cell.

24. regulate the method that main histocompatibility complex molecule is expressed for one kind on isolating neural stem cell, described method comprises with the marrow stromal cell conditioned medium cultivates described neural stem cell, and wherein said marrow stromal cell conditioned medium comprises nerve stem cell culture medium and by the described marrow stromal cell excretory factor.

25. the described method of claim 24, wherein said marrow stromal cell conditioned medium does not comprise exogenous leukaemia inhibitory factor.

26. the described method of claim 24, wherein said marrow stromal cell conditioned medium does not contain marrow stromal cell substantially.

27. the described method of claim 24, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

28. the described method of claim 24, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

29. the described method of claim 24, wherein said neural stem cell derive from people's nervous center system.

30. the described method of claim 24, wherein exogenous genetic material is imported into described neural stem cell.

31. an isolating neural stem cell prepares through the method for the marrow stromal cell of culture of isolated altogether and isolating neural stem cell.

32. the described isolating neural stem cell of claim 31, wherein said neural stem cell show the minimizing of I class MHC molecule and express.

33. the described isolating neural stem cell of claim 31, wherein said neural stem cell shows the baseline values of II class MHC molecule.

34. the described isolating neural stem cell of claim 31, wherein said neural stem cell derives from people's nervous center system.

35. the described isolating neural stem cell of claim 31, wherein exogenous genetic material is imported into described neural stem cell.

36. isolating neural stem cell, through in marrow stromal cell condition developing medium culture of isolated neural stem cell and prepare, wherein said marrow stromal cell condition developing medium comprises the developing medium that a kind of chemistry is determined, comprises the neural stem cell growth medium and the isolating BMSC excretory factor.

37. the described isolating neural stem cell of claim 36, wherein said neural stem cell show the minimizing of I class MHC molecule and express.

38. the described isolating neural stem cell of claim 36, wherein said neural stem cell shows the baseline values of II class MHC molecule.

39. the described isolating neural stem cell of claim 36, wherein said neural stem cell derives from people's nervous center system.

40. the described isolating neural stem cell of claim 36, wherein exogenous genetic material is imported into described neural stem cell.

41. a neuronal cell cultures equipment contains

(a). isolating neural stem cell;

(b). isolating marrow stromal cell;

(c). neural stem cell growth medium, wherein said neural stem cell growth medium comprise from the described isolating marrow stromal cell excretory factor; With

(d). prevent the device of physics contact between described neural stem cell and the described marrow stromal cell.

42. the described equipment of claim 41 also comprises a strainer or film, makes and does not produce the physics contact between described neural stem cell and the described marrow stromal cell.

43. having the hole, the described equipment of claim 42, described strainer or film make the described marrow stromal cell excretory factor by described strainer or film.

Images