Neuro Spheres Holdings Ltd.

Neurospheres, Ltd.

Brent A Reynolds

Samuel Weiss

Joseph P Hammang

E Edward Baetge

Christopher R Bjornson

Angelo L Vescovi

Rod L Rietze

The invention provides in vitro cell culture compositions consisting of neurospheres and culture medium, wherein the neurospheres consist of undifferentiated cells that are nestin, glial fibrillary acid protein (GFAP), neurofilament (NF), and myelin basic protein (MBP)and are not nestin.

Multipotent neural stem cell compositions

Multipotent neural stem cell (MNSC) progeny are induced to generate cells of the hematopoietic system by placing the MNSC progeny in a hematopoietic-inducing environment. The hematopoietic-inducing environment can be either ex vivo or in vivo. A mammal's circulatory system provides an in vivo environment that can induce xenogeneic, allogeneic, or autologous MNSC progeny to generate a full complement of hematopoietic cells. Transplantation of MNSC progeny provides an alternative to bone marrow and hematopoietic stem cell transplantation to treat blood-related disorders.

Generation of hematopoietic cells from multipotent neural stem cells

The invention provides methods for producing myelin forming cells from multipotent self-renewing central nervous system neural stem cells as well as methods of using one or more cells from a multipotent self-renewing central nervous system neural stem cell population to form patches of myelin. Also provided are cell culture systems for forming patches of myelin and methods of treating demyelination diseases in a mammal.

Remyelination of neurons using multipotent neural stem cell progeny

The invention discloses methods of proliferation and differentiation of multipotent neural stem cells. Also provided are methods of making cDNA libraries and methods of screening biological agents which affect proliferation differentiation survival phenotype or function of CNS cells.

Methods of proliferating undifferentiated neural cells

cDNA libraries may be obtained from neural cell cultures produced by using growth factors to induce the proliferation of multipotent neural stem cells. The libraries may be obtained from both cultured normal and dysfunctional neural cells and from neural cell cultures at various stages of development. This information allows for the identification of the sequence of gene expression during neural development and can be used to reveal the effects of biological agents on gene expression in neural cells. Additionally, nucleic acids derived from dysfunctional tissue can be compared with that of normal tissue to identify genetic material which may be the cause of the dysfunction. This information could then be used in the design of therapies to treat the neurological disorder. A further use of the technology would be in the diagnosis of genetic disorders or for use in identifying neural cells at a particular stage in development.

Multipotent neural stem cell cDNA libraries

Methods of screening biological agents

Composition comprising multipotent neural stem cells for generation of hematopoietic cells

Multipotent neural stem cell (MNSC) progeny are transplanted into a recipient wherein they augment host tissue. The stem cells have a universal lineage potential and are capable of producing progeny that, in response to appropriate environmental signals, can differentiate into a variety of differentiated cell types, and not just neural lineages. MNSCs can be proliferated ex vivo to provide an unlimited supply of stem cells and stem cell progeny which give rise to the differentiated cell types of various tissues. The stem cells are readily amenable to genetic modification, if desired. They also have the advantage that they can be obtained from autologous adult human tissue and thus overcome prior art problems of transplant rejections.

Use of multipotent neural stem cell progeny to augment non-neural tissues

The invention provides methods of transplanting multipotent neural stem cell progeny to a host by obtaining a population of cells derived from mammalian neural tissue containing at least one multipotent CNS multipotent neural stem cell; culturing the neural stem cell in a culture medium containing one or more growth factors which induce multipotent neural stem cell proliferation; inducing proliferation of the multipotent neural stem cell to produce neural stem cell progeny which includes multipotent neural stem cell progeny cells; and transplanting the multipotent neural stem cell progeny to the host. Also provided are methods of transplanting neural stem cell progeny to a host by obtaining an in vitro cell culture containing CNS neural stem cells where one or more cells in the culture (i) proliferates in a culture medium supplemented with one or more mitrogens, (ii) retains the capacity for renewed proliferation, and (iii) maintains the multipotential capacity, under suitable culture conditions, to differentiate into neurons, astrocytes, and oligodendrocytes; and transplanting the one or more cells to the hose.

Neural transplantation using proliferated multipotent neural stem cells and their progeny

cDNA libraries may be obtained from neural cell cultures produced by using growth factors to induce the proliferation of multipotent neural stem cells. The libraries may be obtained from both cultured normal and dysfunctional neural cells and from neural cell cultures at various stages of development. This information allows for the identification of the sequence of gene expression during neural development and can be used to reveal the effects of biological agents on gene expression in neural cells. Additionally, nucleic acid derived from dysfunctional tissue can be compared with that of normal tissue to identify genetic material which may be a cause of the dysfunction. This information could then be used in the design of therapies to treat the neurological disorder. A further use of the technology would be in the diagnosis of genetic disorders or for use in identifying neural cells at a particular stage in development.

A culture method for determining the effect of a biological agent on multipotent neural stem cell progeny is provided. In the presence of growth factors, multipotent neural stem cells are induced to proliferate in culture. The multipotent neural stem cells may be obtained from normal neural tissue or from a donor afflicted with a disease such as Alzheimer's Disease, Parkinson's Disease or Down's Syndrome. At various stages in the differentiation process of the multipotent neural stem cell progeny, the effects of a biological agent, such as a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid or a drug or pro-drug on cell activity are determined. Additionally, a method of screening the effects of biological agents on a clonal population of neural cells is provided. The technology provides an efficient method for the generation of large numbers of pre- and post-natal neural cells under controlled, defined conditions. The disclosed cultures provide an optimal source of normal and diseased neural cells at various developmental stages, which can be screened for potential side effects in addition to testing the action and efficacy of different biological agents.

Use of multipotent neural stem cells and their progeny for the screening of drugs and other biological agents

Methods for administering genetic material to dividing neural precursor cell populations in vivo are provided. The genetic material may comprise useful genes for neurotransmitters, growth factors, growth factor receptors, and the like. The genetic material is administered to the brain with one or more growth factors. The growth factors induce proliferation of neural precursor cells, thereby facilitating the incorporation of the genetic material into the cell progeny.

In vivo genetic modification of growth factor-responsive neural

A method is described for inducing in vivo proliferation of precursor cells located in mammalian neural tissue by administering to the mammal a fibroblast growth factor and at least one additional growth factor selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin. The method can be used to replace damaged or missing neurons and/or glia. Another method is described for transplanting multipotent neural stem cell progeny into a mammal. The method comprises the steps of administering growth factors to a mammal to induce in vivo proliferation of neural precursor cells, removing the precursor cell progeny from the mammal, culturing the removed cells in vitro in the presence of one or more growth factors that induces multipotent neural stem cell proliferation, and implanting the multipotent neural stem cell progeny into the mammal.

Growth factor-induced proliferation of neural precursor cells in vivo

A culture method for inducing the expression of tyrosine hydroxylase in neural cells is provided. Mammalian CNS neural cells are cultured in the presence of a fibroblast growth factor and at least one selected from a member of the transforming growth factor beta family, a feeder layer bed of cells, and cell conditioned medium. Cells cultured as provided above may be transplanted to provide dopaminergic cells to a patient. The cells may also be used in methods for drug screening.

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Saltspring, Canada

Brent A Reynolds