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A human immortalised neural precursor cell line

A human cell line and cell line technology, applied in the field of immortalized human neural precursor cell line NGC-407, can solve ethical and operational problems

Inactive Publication Date: 2008-05-28
NSGENE AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, human fetal donor cells pose ethical and operational challenges, necessitating the development of alternative cell sources for future transplantation

Method used

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  • A human immortalised neural precursor cell line
  • A human immortalised neural precursor cell line
  • A human immortalised neural precursor cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0249] Generation and Characterization of Example 1NGC-407 Cell Line

[0250] This example illustrates the generation and in vitro characterization of the NGC-407 cell line.

[0251] Human mesencephalic cells in primary culture were prepared from first trimester human embryonic brains (obtained from Lund University, Sweden). Get organized in compliance with Swedish laws and regulations. Immediately after dissection of the ventral midbrain, the tissue was placed in a drop of cell dissociation solution (Sigma #C5914) in a Petri dish and sectioned less than 0.5mm 3 of blocks. Move the two blades of the scalpel toward each other to mince the tissue. The tissue was transferred to 2 ml medium consisting of DMEM / F12 (Gibco #31331-028), N2 supplement (Gibco #17502-048) supplemented with 0.5% HSA (Sigma #A1653), 0.6% glucose ( Sigma #G8769), 5mM HEPES (Gibco #15630-056), B27 Supplement (Gibco #17504-044), 40 μg / ml bFGF (R&D Systems #233-FB), 20 μg / ml EGF (R&D Systems #236-EG ), ce...

Embodiment 2

[0267] Transplantation and in vivo transgene expression of embodiment 2NGC-407 cell lines

[0268] This example illustrates lentiviral transduction, stability of transgene expression, and integration of the NGC-407 cell line following experimental transplantation into the rat brain.

[0269]NGC-407 cells were expanded according to the method described in Example 1. Forty-eight hours before transplantation, NGC-407 cells were transduced with a self-inactivating lentiviral vector expressing GFP (LV-GFP-SIN; Zufferey R et al, 1998). The MOI used was 1, resulting in a transduction efficiency of 60-70%. On the day of transplantation, the cells were washed three times, digested with trypsin, centrifuged at 600 rpm for 5 minutes, and the cell pellet was resuspended in Hank's balanced salt solution (HBSS; Gibco, Sweden). Cell numbers were estimated with a hemocytometer and single cell suspensions were made in HBSS at a density of 50,000 cells / μl.

[0270] A total of 40 adult female...

Embodiment 3

[0283] Example 3 Analysis of Gap Junction Communication between NGC-407 and U343MGa-cl 2:6 Cells Using Fluorescent Dye Transfer and Enhancement of the Gap Junction Communication with 4-Phenylbutyrate

[0284] method

[0285] Donor human embryonic neural stem cells (NGC-407) and recipient human glioblastoma cells (U343MGa-cl 2:6) were grown in two independent 35mm culture dishes (1×10 5 / dish). For NGC-407 cells, the culture dish was coated with poly-L-lysine (Sigma), the medium was DMEM / F12 and glutamax I (Invitrogen) 1:1, containing 40ng / ml bFGF2 (R&D Systems), 20ng / ml rhEGF, 1xN2 supplement, 1x non-essential amino acids, 5mM HEPES buffer solution (from Invitrogen), 0.5% human serum albumin and 6g / L D-glucose (from Sigma). Recipient cells were grown in DMEM containing 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin (from Invitrogen). When the cells were approximately 60% confluent, treatment of donor cells with 0 and 0.5 mM 4-phenylbutyrate (PB) was initiated ...

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Abstract

The present invention relates to an immortalised human neural precursor cell line, NGC-407. The cell line has been established from human foetal tissue. The cell line has been immortalised using a retroviral vector containing the v-myc oncogene. The cell line is a neural progenitor cell line capable of differentiating into to astrocytes and neurons including dopaminergic neurons. NGC-407 cells are capable of migrating to glioblastoma tumours implanted into rat brains and form gap junctions with the tumour cells. NGC-407 cells expressing a suicide gene can be be used for delivering activated prodrugs in the form of activated nucleoside analogs to tumours.

Description

[0001] The present invention relates to the immortalized human neural precursor cell line NGC-407. This cell line has been established from human fetal tissue. Background of the invention [0002] The effectiveness of treating neurodegenerative diseases by transplanting human fetal tissue has been shown in animal models [Brundin et al., Behavioral effects of human fetal dopamine neurons grafted in arat model of Parkinson's disease, Exp Brain Res, 65(1986) 235-40 .; Wictorin et al., Reformation of long axon pathways in adult rat central nervous system by humanforebrain neuroblasts, Nature, 347(1990) 556-8.] and shown in patients with Parkinson's disease (PD) and Huntington's disease [Bachoud-Levi et al., Motor and cognitive improvements in patients with Huntington's disease after neural transplantation, Lancet, 356 (2000) 1975-9.; Freed et al., Transplantation of embryonic dopamine neurons for severe Parkinson's disease, N Engl J Med, 344 (2001) 710-9.; Hagell et al., Sequenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/08C12N5/0797C12N5/10
Inventor 本特·朱丽叶森
Owner NSGENE AS
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