Methods to prepare and use epidermal stem cells

Patent Number: 6927060

This is a method of isolating stem cells by dye exclusion and applying the principle to epidermal stem cells. It is well known in the art that stem cells are the property to exclude various dyes. This is what causes the existance of the "side population cells" which were known since 1996 (Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996 Apr 1;183(4):1797-806). It will be interesting to determine the actual novelty of this patent. The patent's independent claims are:


A method to prepare isolated mammalian epidermal stem cells, comprising: (a) providing a sample comprising mammalian epidermal stem cells obtained from mammalian epidermal tissue, which sample is contacted with an agent that binds DNA in viable cells and an agent that binds non-viable cells; (b) separating the sample into a first population of cells that are the smallest 30% of the viable cells in the sample and a second population of cells that are larger than the cells in the first population, wherein the first population comprises a substantially pure population of epidermal stem cells; and © isolating a substantially pure population of epidermal stem cells from the first population of epidermal stem cells.

A method to prepare isolated mammalian epidermal stem cells, comprising: (a) providing a sample comprising mammalian epidermal stem cells obtained from mammalian epidermal tissue, which sample is contacted with a dye with low or no cellular toxicity that binds DNA in viable cells and an agent that binds non-viable cells; (b) separating the sample into a first population of cells that are the smallest 30% of the viable cells in the sample and a second population of cells that are larger than the cells in the first population, wherein the first population comprises a substantially pure population of epidermal stem cells; and © isolating a substantially pure population of epidermal stem cells from the smaller cells.

A method to prepare mammalian epidermal stem cells, comprising: (a) contacting a population of mammalian epidermal cells comprising epidermal stem cells with an amount of a first agent under conditions effective for viable cells to retain the first agent, wherein the mammalian epidermal cells are obtained from mammalian epidermal tissue; (b) contacting the population of (a) with an amount of a second agent under conditions effective for non-viable cells to retain the second agent; and © separating the population of(b) into a first population of cells which first population represents the smallest 30% of the viable cells which comprise viable epidermal stem cells and a second population of cells which represents larger cells, which second population does not comprise a substantial portion of epidermal stem cells.

A method to prepare a substantially pure population of mammalian epidermal stem cells, comprising: separating a sample obtained from mammalian epidermal tissue which comprises mammalian epidermal stem cells and epidermal cells which are not epidermal stem cells and contacted with a Hoechst dye and propidium iodine or pyronidine iodine, into a substantially pure population of epidermal stem cells which comprise the hoechst dye but not propidium iodine or pyronidine iodine and into at least one population of cells that does not comprise a substantial portion of epidermal stem cells.

A method to prepare mammalian epidermal stem cells, comprising: separating a sample obtained from mammalian epidermal tissue which comprises mammalian epidermal stem cells and epidermal cells which are not epidermal stem cells and contacted with a Hoechst dye and propidium iodine or pyronidine iodine, into a population which represents the smallest 30% of the viable cells in the sample and into a population which comprises larger cells, wherein the population with the smallest 30% of the viable cells comprises a substantially pure population of epidermal stem cells which comprise the Hoechst dye but not propidium iodine or pyronidine iodine.

A method comprising: (a) providing a sample comprising mammalian epidermal stem cells obtained from mammalian epidermal tissue, which sample is contacted with an agent that binds DNA in viable cells and an agent that binds non-viable cells; and (b) separating the sample into a first population of cells that are the smallest 30% of the viable cells in the sample and a second population of cells that are larger than the cells in the first population, wherein the first population comprises a substantially pure population of epidermal stem cells.

A method comprising: (a) providing a sample comprising mammalian epidermal stem cells obtained from mammalian epidermal tissue, which sample is contacted with a dye with low or no cellular toxicity that binds DNA in viable cells and an agent that binds non-viable cells; and (b) separating the sample into a first population of cells that are the smallest 30% of the viable cells in the sample and a second population of cells that are larger than the cells in the first population, wherein the first population comprises a substantially pure population of epidermal stem cells.

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