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Transdifferentiation of glial cells

a technology of glial cells and glial cells, which is applied in the direction of nervous system cells, biochemical apparatus and processes, biocide, etc., can solve the problems of unsuitable use of these cells, difficult use of this process for producing cells for human implantation, immunological rejection and potential contamination, etc., and achieves rapid shipping to medical facilities. , the effect of avoiding the need for glial cells

Inactive Publication Date: 2005-12-01
SPINAL CORD SOC
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0015] The invention provides a renewable source of multipotent mammalian cells that will be invaluable for clinical studies and treatments of neural diseases, including neurotrauma and / or neurodegenerative diseases. The invention provides for the transdifferentiation of astrocytes into multipotent cells and for use of the multipotent cells directly or after they have been transdifferentiated into neurons, astrocytes, and / or oligodendrocytes. The cells may be derived from adult tissue and eliminate the need for other types of cells such as human fetal cells. The multipotent cells are derived from astrocytes, which are easily, predictably, and economically obtained compared to current methods of directly isolating stem cells or using fetal tissue. The multipotent cells of the invention are readily obtained in the abundant amounts required for implantation, and are kept on hand locally or may be stored at a central location for rapid shipping to medical facilities.

Problems solved by technology

Fetal neural progenitor, or stem cells are multipotent cells that are useful for various therapeutic applications involving their implantation into humans but significant moral, ethical, and technological issues make the use of these cells undesirable.
Such problems include immunological rejection and potential contamination.
First, nervous system stem cells are isolated from human embryonic or adult brain; the technical and economic challenges of this technique make it difficult to use this process for producing cells for human implantation (Svendsen et al., Experimental Neurology, 137:376-388, 1996; Chalmers-Redman et al., Neuroscience, 76:1121-1128, 1997; Carpenter et al., Experimental Neurology, 158:265-278, 1999; Fricker et al., Journal of Neuroscience, 19:5990-6005, 1999; Kudekov et al., Experimental Neurology, 156:333-334, 1999; Vescovi et al., Experimental Neurology, 156, 71-83, 1999).

Method used

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  • Transdifferentiation of glial cells
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Examples

Experimental program
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Effect test

example 1

6.0 EXAMPLE 1

Analysis of Astrocytes Treated with bFGF

[0043] A series of experiments are grouped together in this example to show conditions for the culture of cells and treatments for transdifferentiation.

6.1 In Vitro Culture of Astrocytes

6.1.1 In Vitro Culture of Astrocytes from Human Neural Progenitor Cell Derived Astrocytes

[0044] Astrocytes derived from human neural stem cells were grown on plastic tissue culture flasks in the presence of 10% FCS in DMEM / F-12 culture medium. The media was changed twice per week. After confluency, the cells were trypsinized (0.25% trypsin for 2 minutes at 37 degrees C.), treated with trypsin neutralization solution (CALBIOCHEM) and the cells were pelleted, resuspended and plated at a ratio of 1:3. In general, it was important to passage the cells at no more than 1:5 ratio; further, at a 1:2 ratio confluency was typically achieved in 7-10 days.

6.1.2 In Vitro Culture of Mammalian Astrocytes from Mammalian Nervous Tissue

[0045] Nervous tissue...

example 2

7.0 EXAMPLE 2

Treatment with bFGF without a Dissociation Step

[0052] The cells were treated as described in Example 1 except as described below, most notably in the omission of the trypsinization step. In brief, monolayers of astrocytes were washed once in DMEM / F-12 and defined medium containing DMEM / F-12 and SUPPLEMENT B27 (GIBCO), 20-ng per ml bFGF (PEPROTECH), and 1 microgram per ml heparin (SIGMA) was added for 2 and 5 days. The cells were then grown in Medium I for 7 days.

[0053] Immunochemistry revealed that there were no MAP2ab or Beta-tubulin III positive cells in either the 2-day or 5-day groups, indicating that no neurons were present. Therefore cell dissociation enhances the production of multipotent cells because cells that underwent the same treatment plus a dissociation step were made multipotent and then differentiated into neurons.

example 3

8.0 EXAMPLE 3

Pretreatment and Treatment of Cells with bFGF

[0054] These experiments were performed as described in Example 1 except for the differences described herein; these results are the results of multiple experiments. Cultured astrocytes were dissociated and plated on tissue culture plastic. The cells were cultured with 20 ng per ml bFGF plus heparin for three to six weeks. The cells were then placed in Medium I, Medium II, or Medium III for 7 and 14 days on laminin-coated substrates.

[0055] After 7 days in Medium I, II, or III, 60%-80% of the cells were GFAP positive astrocytes. Up to 30% of the cells were Beta-tubulin III positive, GFAP negative neurons (FIG. 3A) and 5-10% of the cells were MAP2ab positive. In Media II and III, 0.5%-2.0% cells were positive for the oligodendrocyte markers CNPase and O4. Cells in Medium II marked for CNPase and O4 were more strongly positive for these markers than cells in the other media. In Medium I oligodendrocytes were mainly absent. The...

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Abstract

A process for generating multipotent cells from glial cells using in vitro techniques to dedifferentiate fetal or adult mammalian glial cells into multipotent cells. The multipotent cells may further be differentiated into particular types of nervous system cells, including neurons, astrocytes, and oligodendrocytes. A small sample of astrocytes is used to establish an in vitro culture of cells that is expanded and processed to yield multipotent cells that may be used directly or be differentiated to yield neurons and / or oligodendrocytes and / or astrocytes. The invention includes implanting the generated cells into patients. The invention includes a step of exposing the cells to a growth factor.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 09 / 644,498, filed Aug. 23, 2000, now U.S. Pat. No. 6,897,061, issued May 24, 2005, which claims the benefit of U.S. Provisional Application No. 60 / 212,240, filed Jun. 16, 2000, which is hereby incorporated by reference.1.0 FIELD OF THE INVENTION [0002] The invention relates to the in vitro transdifferentiation of mammalian cells from a glial cell type to neurons, oligodendrocytes, astrocytes, and associated type cells. 2.0 BACKGROUND OF THE INVENTION [0003] Fetal neural progenitor, or stem cells are multipotent cells that are useful for various therapeutic applications involving their implantation into humans but significant moral, ethical, and technological issues make the use of these cells undesirable. Such problems include immunological rejection and potential contamination. [0004] It has been reported that the entire ventricular neuraxis, including the spinal cords of adult mamma...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/02C12N5/0797
CPCA61K35/12C12N5/0623C12N2506/08C12N2501/13C12N2501/115
Inventor SALIN-NORDSTROM, TUIJA HELINA
Owner SPINAL CORD SOC
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