Platelet derived growth factor (PDGF)-derived neurospheres define a novel class of progenitor cells

a neural progenitor cell and platelet-derived growth factor technology, applied in the direction of growth factor/regulator, nervous system cells, animal/human proteins, etc., can solve the problem of inability of these cells or the brain region to carry out the intended function

Inactive Publication Date: 2011-07-28
STEM CELL THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These diseases, which include Alzheimer's Disease, Multiple Sclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease, have been linked to the degeneration of neural vi cells in particular locations of the central nervous system (CNS), leading to inability of these cells or the brain region to carry out their intended function.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

PDGF Induces Production of Primary Neurospheres

[0085]E14 striatum was mechanically dissociated as previously described (for example see U.S. Pat. No. 5,750,376; 5,980,885; or 5,851,832) and plated at 10,000 cells / ml in 6 well plates in defined culture media plus 100 ng / ml of PDGF-AA (Peprotech). The composition of defined culture media is as follows:[0086]DMEM / F12 (1:1)[0087]glucose (0.6%)[0088]glutamine (2 mM)[0089]sodium bicarbonate (3 mM)[0090]HEPES (5 mM)[0091]insulin (25 μg / ml)[0092]transferrin (100 μg / ml)[0093]progesterone (20 nM)[0094]putrescine (60 μM)[0095]selenium chloride (30 nM)

[0096]Neurospheres, which are clusters of neural cells derived from single cells, formed after 7 days of culture in vitro (DIV). Six wells per experiment were counted for neurosphere production and the results are shown below:

Average number of Experiment#neurospheres produced / well114.529.2334.3*48.657.7631.5*average = 17.7 ± 5.0*PDGF appears to be approximately 3 fold more potent when used immedia...

example 2

Tyrphostin AG 1296 Inhibits the Production of Primary Neurospheres by PDGF but not EGF

[0098]In order to determine if the PDGF-induced primary neurosphere formation is mediated by the PDGF receptor kinase, a selective PDGF receptor kinase inhibitor, Tyrphostin AG 1296, was added to neurosphere culture. Primary cells were cultured at 10,000 cells / ml in the presence of either EGF or PDGF, plus 5 μM of Tyrphostin AG 1296 (Sigma) or DMSO in the same volume as Tyrphostin (DMSO being the solvent for Tyrphostin). Results are expressed below as the percentage of neurospheres formed, with the number of PDGF- or EGF-derived neurospheres arbitrarily set at 100%, respectively'.

TABLE 1The Effect of Tyrphostin on neurosphere formation in response to EGF or PDGFPDGF or EGF alonewith DMSOwith TyrphostinPDGF100% 97%10%EGF100%101%55%n = 3 to 9

[0099]Therefore, Tyrphostin AG 1296 almost abolished primary neurosphere formation induced by PDGF, indicating that formation of the PDGF-derived neurospheres is...

example 3

PDGF-Derived Primary Neurospheres can be Subcultured into EGF but not PDGF Containing Media

[0100]Single PDGF primary neurospheres prepared as described in Example 1 were transferred into 96-well plates and mechanically dissociated in either the presence of 20 ng / ml EGF (Peprotech) or 100 ng / ml PDGF-AA. The formation of secondary neurospheres was assayed after 7 or more days in culture in vitro. The results are shown below.

Average # of secondary neurospheres formed / wellPDGF0EGF3.12 ± 1.64 (n = 3)

[0101]Therefore, the cells in PDGF-derived primary neurospheres cannot proliferate in response to PDGF. In contrast, these cells can proliferate and form secondary neurospheres in response to EGF.

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Abstract

The present invention is related to the discovery of a novel class of neural progenitor cells, which proliferate in response to platelet derived growth factor (PDGF) and differentiate into neurons and oligodendrocytes but not astrocytes. Progeny of the progenitor cells can be obtained by culturing brain tissue in PDGF without serum, epidermal growth factor (EGF), fibroblast growth factor 2, or transforming growth factor alpha. Upon subculturing into EGF-containing media, these progeny cells can proliferate and form neurospheres, whereas PDGF has no such effect.

Description

RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 60 / 287,214, filed Apr. 27, 2001, and U.S. Provisional Application Ser. No. 60 / 307,070 filed Jul. 20, 2001, both of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention is related to the discovery of a novel class of neural progenitor cells which can differentiate into neurons and oligodendrocytes but not astrocytes, and methods of proliferating these progenitor cells by using platelet derived growth factor (PDGF).REFERENCES[0003]U.S. Pat. No. 5,750,376.[0004]U.S. Pat. No. 5,980,885.[0005]U.S. Pat. No. 5,851,832.[0006]Dirks R and Bloomers H. 1996. Signals controlling the expression of PDGF. Mol. Biology Reports 22: 1-24,[0007]Ek B. Westermark B, Wasteson A. and Heldin C H. 1982. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature 295(5848):419-420.[0008]Hannink M and Donoghue D J, 1989. S...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/20A61K38/18A61K38/28A61P25/00A61P25/16A61K35/12C12N5/0797
CPCA61K35/12A61K38/1858C12N2503/02C12N2501/11C12N2501/135C12N5/0623A61P25/00A61P25/16
Inventor WEISS, SAMUELCHOJNACKI, ANDREW K.
Owner STEM CELL THERAPEUTICS
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