Lineage-Restricted Neuronal Precursors

a precursor cell and lineage restriction technology, applied in the field of lineage restriction intermediate precursor cells, can solve the problems of insufficient fgf failure to disclose cultured nep cells, and insufficient fgf itself for long-term growth of neurospheres

Inactive Publication Date: 2009-04-02
RAO MAHENDRA S +2
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  • Abstract
  • Description
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  • Application Information

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Benefits of technology

[0040](b) changing the culture conditions of the neuron-restricted precursor cells from proliferating conditions to dif

Problems solved by technology

Further, no method of purifying such precursors from multipotent cells has been described.
FGF itself is not sufficient for long term growth of neurospheres, though FGF may support their growth transiently.
U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L.

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Examples

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example 1

[0099]To determine if a dividing neuron-restricted precursor is normally present in vivo, sections of E13.5 rat spinal cords were analyzed with a panel of early neuronal markers. Sections were cut of embryos fresh frozen at 13.5 days gestation and then were labeled by immunocytochemistry. Staining procedures were carried out according to methods well known in the art. Cells were double-labeled with antibodies against E-NCAM (Developmental Studies Hybridoma Bank, Iowa) and β-III tubulin (Sigma Chemical Co., St. Louis, Mo.) or were stained with E-NCAM and counterstained with DAPI, a nuclear marker for identifying all cells. All secondary monoclonal antibodies were from Southern Biotechnology.

[0100]Polysialated or embryonic N-CAM (E-NCAM) appeared to be a likely marker for neuronal precursors. E-NCAM immunoreactivity was first detected at E13.5. E-NCAM immunoreactive cells could be seen in the margins of the neural tube, but not in the proliferating ventricular zone. Double-labeling wi...

example 2

[0101]To characterize E-NCAM-immunoreactive cells, E13.5 spinal cords were dissociated and E-NCAM-immunoreactive cells were stained with a panel of antibodies (Table 1). Sprague-Dawley rat embryos were removed at embryonic day 13.5 and placed in a petri dish containing Hanks balanced salt solutions (HBSS, Gibco). The trunk segments of the embryos were dissected using tungsten needles, rinsed, and then transferred to fresh HBSS. Spinal cords were mechanically dissected free from the surrounding connective tissue using sharpened No. 5 forceps. Isolated spinal cords were incubated in 0.05% trypsin / EDTA solution for 20 minutes. The trypsin solution was replaced with fresh HBSS containing 10% fetal bovine serum (FBS). The segments were gently triturated with a Pasteur pipette to dissociate cells. Cells dissociated by trituration were plated in PLL / laminin-coated 35 mm dishes (Nunc) at high density and stained after 24 hours.

TABLE 1Antibody / Cell TypeKindSourceAntigen RecognizedRecognizedA...

example 3

[0104]To determine the differentiation potential of E-NCAM-immunoreactive cells, E-NCAM+ cells were purified by immunopanning and plated at clonal density in gridded dishes. E13.5 cells were prepared according to the procedure of Example 2. An E-NCAM+ cell population was purified from these E13.5 cells using a specific antibody-capture technique according to the procedure of L. Wysocki & V. Sato, “Panning” for Lymphocytes: A Method for Cell Selection, 75 Proc. Nat'l Acad. Sci. USA 2844-48 (1978); M. Mayer et al., supra, hereby incorporated by reference. In brief, cells were trypsinized and the resulting cell suspension was plated on an A2B5-antibody-coated dish to allow binding of all A2B5+ cells to the plate. The supernate was removed, and the plate was washed with DMEM supplemented with additives described by J. Bottenstein and G. Sato, Growth of a Rat Neuroblastoma Cell Line in Serum-free Supplemented Medium, 76 Proc. Nat'l Acad. Sci. USA 514-17 (1979), hereby incorporated by ref...

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Abstract

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. Methods for treating neurological diseases are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This patent application is a continuation of U.S. patent application Ser. No. 10 / 911,374, filed Aug. 4, 2004, which is a continuation of U.S. patent application Ser. No. 09 / 109,858, filed Jul. 2, 1998, issued as U.S. Pat. No. 6,787,353, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 909,435, filed Jul. 4, 1997, issued as U.S. Pat. No. 6,734,015, teachings of each of which are herein incorporated by reference in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under a FIRST award and a Multidisciplinary Basic Cancer Research Training Grant Graduate Fellowship from the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]This invention relates to lineage-restricted intermediate precursor cells and methods of making and using thereof. More particularly, the invention relates ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02C12Q1/06A61K35/12A61K48/00C12N5/0797
CPCA61K35/12A61K48/00C12N2501/385C12N2501/115C12N2501/13C12N5/0623A61P25/28
Inventor RAO, MAHENDRA S.MAYER-PROSCHEL, MARGOTKALYANI, ANJALI J.
Owner RAO MAHENDRA S
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