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Reversibly immortalised olfactory ensheathing glia and their use to promote neuronal regenaration

An immortalized and reversible technology applied in the field of neuron regeneration

Inactive Publication Date: 2006-08-23
CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the persistence of oncogenes (in this case the gene encoding the SV40 large T antigen) in these cells is of concern because it can increase the risk of malignant transformation after transplantation

Method used

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  • Reversibly immortalised olfactory ensheathing glia and their use to promote neuronal regenaration
  • Reversibly immortalised olfactory ensheathing glia and their use to promote neuronal regenaration
  • Reversibly immortalised olfactory ensheathing glia and their use to promote neuronal regenaration

Examples

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Embodiment 1

[0075] Reverse Immortalization of Human Primary OEG Cells Using the Cre / Lox System

[0076] In the protocol described in this example, human OEG cells were immortalized using a recombinant lentivirus containing the gene encoding SV40Tag flanked by Loxp sites. Cells were characterized before and after treatment with a recombinant adenovirus capable of delivering the gene encoding the Cre recombinase to determine whether this approach would result in a clinically useful OEG cell line for transplantation.

[0077] Human cells are obtained from post-mortem adult donor human tissue. The tissues belonged to male and female donors from bromospheres; the outer layer was dissociated and digested with 0.1% trypsin. The age of the donor appears to have no limit to the success of the culture. Tissues were washed with sterile phosphate buffered saline (PBS) solution containing antibiotics (penicillin / streptomycin) and antimycotics (Primocin). The tissue was then subjected to controlled ...

Embodiment 2

[0085] Analysis of axon regeneration in the adult retina

[0086] Promotion of axonal regeneration from adult rat retinal ganglion neurons (RGN) was used as a standard method to measure regenerative capacity in cell culture. This approach more closely reflects the in vivo repair performance of OEG cells than other neuritogenic assays.

[0087] Briefly, the method used was as follows: retinas were dissected from P60 rat eyes, dissociated with 0.1% trypsin and digested. Digestion was stopped with soybean trypsin inhibitor at 2 mg / ml and a fine suspension of cells was obtained by passing the digested material through a reduced diameter Pasteur pipette. Cells were centrifuged and resuspended in defined medium (Moreno-Flores et al., 2003). RGN were plated on glial cell monolayers: primary OEG or in established cell lines.

[0088]For immunohistochemical analysis, after seven days of culture, neuron-human OEG cell mixed cultures were fixed with 4% paraformaldehyde. Figure 4 show...

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Abstract

The present invention is based on the capacity of the Olfactory Ensheathing Glia (OEG) to foster axonal regeneration in the adult mammalian central nervous system (CNS). This specific capacity is probably due to a combination of several factors, such as the molecular composition of cellular membrane and / or the capacity to secrete some molecules; combined with the capacity to reduce glial scar and accompany new growing axon in the damaged CNS. We have developed immortalised cell lines from primary human OEGs. The cells were cultured from post-mortem human tissue from donors and immortalised using a reversible system. Some of these OEG human clonal cell lines were selected by their ability to promote axonal regeneration from adult rat retinal ganglion neurons in a similar fashion to primary OEGs. These cell lines, alone or in pharmaceutical compositions comprising these cells, may be used to repair neuronal damage in the CNS.

Description

field of invention [0001] The present invention relates to neuronal regeneration. In particular, the present invention provides a specific type of cells, namely, reversibly immortalized and grown in culture olfactory ensheathing glia (OEG), which are functional and safe for transplantation. The invention has particular application in transplantation to neural regions, such as the human brain, spine and / or peripheral nerves, to aid recovery from acute and chronic neurological injuries following surgery or trauma. Background of the invention [0002] Olfactory Ensheathing Glial Cells (OEG) [0003] The ability of adult central nervous system (CNS) neurons to regenerate is known to be extremely limited from studies by Ramón y Cajal. In contrast, neurons from the peripheral nervous system (PNS) have a remarkable regenerative capacity, which may be attributed to factors such as the specific properties of Schwanncell (SC) that sheath PNS axons. This fact stimulated the applicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N15/64C12R1/91A61K48/00A61P25/00A61K38/12C12N5/079C12N5/0793
CPCC12N2506/30C12N2510/04C12N2799/027C12N5/0622C12N2830/00A61K38/00C12N2800/30C12N5/062A61P25/00A61P35/00A61P43/00C12N5/06A61K35/12
Inventor M·T·莫雷诺·弗洛雷斯M·T·马丁·贝尔梅霍J·阿维拉·德·格拉多F·万多谢尔·胡拉多J·迪亚斯·尼多F·利姆E·帕斯特拉纳·伊斯基耶多
Owner CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC)
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