The present invention reveals in vitro cultures of region-specific, terminally differentiated, mature neurons derived from cultures of mammalian multipotential CNS stem cells and an in vitro procedure by which the differentiated neurons may be generated. The procedure involves the culturing of multipotential CNS stem cells from a specific region in a chemically defined serum-free culture medium containing a growth factor; replacing the medium with growth factor-free medium; harvesting the stem cells by trypsinization; plating the stem cells at a density of between 100,000 to 250,000 cells per square centimeter; and culturing the stem cells in a glutamic acid-free chemically defined serum-free culture medium.
This application is a continuation-in-part of U.S. patent application Ser. No. 08/719,450, filed Sep. 25, 1996, now U.S. Pat. No. 5,753,506, which claims priority to U.S. provisional patent application Ser. 60/018,206, filed May 23, 1996, the entire contents of each are hereby incorporated by reference and relied upon.
The present invention relates to methods for generating dopaminergic neurons in vitro from embryonic and adult central nervous system cells. Specifically, these cells are isolated, cultured in vitro and stimulated to differentiate into dopaminergic neurons by down-regulating COUP-TFI and/or COUP-TFII expression or increasing NOT1 expression. These newly generated dopaminergic neurons may serve as an excellent source for cell replacement therapy in neurological disorders in which the dopaminergic system is compromised.
A self-renewing restricted stem cell population has been identified in developing (embryonic day 13.5) spinal cords that can differentiate into multiple neuronal phenotypes, but cannot differentiate into glial phenotypes. This neuronal-restricted precursor (NRP) expresses highly polysialated or embryonic neural cell adhesion molecule (E-NCAM) and is morphologically distinct from neuroepithelial stem cells (NEP cells) and spinal glial progenitors derived from embryonic day 10.5 spinal cord. NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin 3 (NT-3) and express a characteristic subset of neuronal epitopes. When cultured in the presence of RA and the absence of FGF, NRP cells differentiate into GABAergic, glutaminergic, and cholinergic immunoreactive neurons. NRP cells can also be generated from multipotent NEP cells cultured from embryonic day 10.5 neural tubes. Clonal analysis shows that E-NCAM immunoreactive NRP cells arise from an NEP progenitor cell that generates other restricted CNS precursors. The NEP-derived E-NCAM immunoreactive cells undergo self renewal in defined medium and differentiate into multiple neuronal phenotypes in mass and clonal culture. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at embryonic day 13.5 in the spinal cord.
The present invention provides methods that utilize compositions containing colostrinin, an constituent peptide thereof, an active analog thereof, and combinations thereof, as an oxidative stress regulator.
The present invention discloses a use of colostrinin, a constituent peptide thereof, and/or an analog thereof as a neural cell regulator in animals including humans.
The present invention relates to a method of producing neurons that express the enzyme tyrosine hydroxylase (TH) by subjecting neural stem cells to FGF-1, a protein kinase A activator, a protein kinase C activator, and dopamine/L-DOPA. Surprisingly, when forskolin is used as a protein kinase A activator, it requires only low levels of FGF-1 and forskolin to efficiently produce TH positive neurons from fetal or adult neural stem cells. Also provided are compositions used to produce TH positive neurons and the resulting neural cell culture, as well as a method of treating disease or conditions which are associated with dopamine neuron loss or dysfunction.
