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http://www.wipo.int/pctdb/en/wo.jsp?IA= ... LAY=CLAIMS
METHODS FOR EX VIVO PROPAGATION OF SOMATIC HAIR FOLLICLE STEM CELLS
We claim: 1. A method of culturing and expanding somatic hair follicle stem cells in vitro, comprising: a) isolating somatic hair follicle stem cells from a mammal; b) culturing said somatic hair follicle stem cells in a culture medium that permits cell growth under conditions, and for a time sufficient, to permit cell growth, wherein a guanine nucleotide biosynthesis pathway in said somatic hair follicle stem cells is enhanced by an agent present in the culture medium, or by a genetic manipulation to said somatic hair follicle stem cells, resulting in conversion of the stem cells from asymmetric cell kinetics to symmetric cell kinetics resulting in enhanced proliferation of said hair follicle stem cells with a reversibly reduced production of differentiating progeny cells. 2. The method of claim 1 , wherein an agent is present in said media. 3. The method of claim 2, wherein said agent is a guanine nucleotide precursor, an analogue or derivative thereof. 4. The method of claim 3, wherein said guanine nucleotide precursor is xanthosine or hypoxanthine. 5. The method of claim 2, wherein said agent is xanthine. 6. The method of claim 3, wherein said guanine nucleotide precursor is xanthosine. 7. The method of claim 3, wherein said guanine nucleotide precursor is present in an amount of 1 - 5000 ?M. 8. The method of claim 7, wherein said guanine nucleotide precursor is present in an amount of 50 - 1500 ?M.
9. The method of claim 1 , wherein genetic manipulation is used. 10. The method of claim 9, wherein the genetic manipulation results in upregulation of guanine nucleotide biosynthesis. 11. The method of claim 10, wherein the genetic manipulation comprises expressing a gene encode inosine-5 'monophosphate dehydrogenase (IMPDH) or xanthine phosphoribosyltransferase (XPRT) in the cultured somatic hair follicle stem cells. 12. The method of claim 11 , wherein the gene encodes xanthine phosphoribosyltransferase. 13. The method of claim 1 , wherein cells are cultured at a high cell density. 14. A method for administering somatic hair follicle stem cells to a subject, wherein said method comprises: (a) isolating somatic hair follicle stem cells from said individual or a matched individual; (b) culturing said isolated somatic hair follicle stem cells in a medium and under conditions sufficient for culturing; (c) adding a substituent to said medium to enhance guanine nucleotide biosynthesis suppressing asymmetric kinetics; (d) culturing said isolated somatic hair follicle stem cells for at least 10 days after said substituent is added to expand said isolated somatic cells; and, (e) administering said isolated hair follicle stem cells of step (d) to said individual. 15. A method for deriving clonal cells lines of somatic hair follicle stem cells by isolating somatic hair follicle stem cells from a mammal, performing limiting dilution plating or cell sorting of said somatic hair follicle stem cells to isolate single somatic hair follicle stem cells, and culturing and expanding said single hair follicle somatic stem cells using the method of claim 1. 16. A method for identifying molecular probes specific for somatic hair follicle stem cells, comprising culturing and expanding said single somatic hair follicle stem cells using the method of claim 1, and using said population- of expanded somatic hair follicle stem cells for comparison to a second population of non-stem cells to identify differences in gene and/or protein expression between the two said populations. 17. A method of culturing and expanding somatic hair follicle stem cells in vitro, comprising culturing somatic hair follicle stem cells isolated from a mammal in a culture medium which permits cell growth under conditions, and for a time sufficient, to permit cell growth, wherein the expression of a protein downstream of the guanine nucleotide biosynthesis pathway in said somatic hair follicle stem cells is modulated by an agent present in the culture medium or by a genetic manipulation to said somatic hair follicle stem cells such that asymmetric cell kinetics are suppressed. 18. The method of claim 17, wherein the modulation is increased expression of the protein. 19. The method of claim 17, wherein the modulation is decreased expression of the protein. 20. A method for treating baldness comprising administering to a subject a composition that stimulates hair follicle stem cells to replicate by converting from asymmetric cell kinetics to symmetric cell kinetics resulting in enhanced proliferation of said hair follicle stem cells with a reversibly reduced production of differentiating progeny cells. 21. The method of claim 20, wherein the agent that stimulates conversion of the stem cells from asymmetric cell kinetics to symmetric cell kinetics is a guanine nucleotide precursor (rGNPr), a analogue or derivative thereof. 22. The method of claim 21 , wherein said guanine nucleotide precursor is xanthosine or hypoxanthine. 23. The method of claim 20, wherein said composition is administered topically. 24. The method of claim 20, wherein said composition is administered by injection. 25. The method of claim 20, wherein said guanine nucleotide precursor is present in an amount of 1 - 10,000 ?M. 26. The method of claim 20, wherein said guanine nucleotide precursor is present in an amount of 50 - 1,500 ?M. 27. A composition comprising a population of human somatic hair follicle stem cells, wherein the population contains at least 100 human somatic hair follicle stem cells. 28. The composition of claim 27, wherein the composition comprises at least 60% multi-potent somatic hair follicle stem cells. 29. The composition of claim 27, wherein the population contains more than 1 ,000 multi-potent hair follicle stem cells. 30. The composition of claim 29, wherein the composition is at least 60% multi-potent hair follicle stem cells.
http://www.wipo.int/pctdb/en/wo.jsp?IA= ... LAY=CLAIMS
METHODS FOR EX VIVO PROPAGATION OF SOMATIC HAIR FOLLICLE STEM CELLS
We claim: 1. A method of culturing and expanding somatic hair follicle stem cells in vitro, comprising: a) isolating somatic hair follicle stem cells from a mammal; b) culturing said somatic hair follicle stem cells in a culture medium that permits cell growth under conditions, and for a time sufficient, to permit cell growth, wherein a guanine nucleotide biosynthesis pathway in said somatic hair follicle stem cells is enhanced by an agent present in the culture medium, or by a genetic manipulation to said somatic hair follicle stem cells, resulting in conversion of the stem cells from asymmetric cell kinetics to symmetric cell kinetics resulting in enhanced proliferation of said hair follicle stem cells with a reversibly reduced production of differentiating progeny cells. 2. The method of claim 1 , wherein an agent is present in said media. 3. The method of claim 2, wherein said agent is a guanine nucleotide precursor, an analogue or derivative thereof. 4. The method of claim 3, wherein said guanine nucleotide precursor is xanthosine or hypoxanthine. 5. The method of claim 2, wherein said agent is xanthine. 6. The method of claim 3, wherein said guanine nucleotide precursor is xanthosine. 7. The method of claim 3, wherein said guanine nucleotide precursor is present in an amount of 1 - 5000 ?M. 8. The method of claim 7, wherein said guanine nucleotide precursor is present in an amount of 50 - 1500 ?M.
9. The method of claim 1 , wherein genetic manipulation is used. 10. The method of claim 9, wherein the genetic manipulation results in upregulation of guanine nucleotide biosynthesis. 11. The method of claim 10, wherein the genetic manipulation comprises expressing a gene encode inosine-5 'monophosphate dehydrogenase (IMPDH) or xanthine phosphoribosyltransferase (XPRT) in the cultured somatic hair follicle stem cells. 12. The method of claim 11 , wherein the gene encodes xanthine phosphoribosyltransferase. 13. The method of claim 1 , wherein cells are cultured at a high cell density. 14. A method for administering somatic hair follicle stem cells to a subject, wherein said method comprises: (a) isolating somatic hair follicle stem cells from said individual or a matched individual; (b) culturing said isolated somatic hair follicle stem cells in a medium and under conditions sufficient for culturing; (c) adding a substituent to said medium to enhance guanine nucleotide biosynthesis suppressing asymmetric kinetics; (d) culturing said isolated somatic hair follicle stem cells for at least 10 days after said substituent is added to expand said isolated somatic cells; and, (e) administering said isolated hair follicle stem cells of step (d) to said individual. 15. A method for deriving clonal cells lines of somatic hair follicle stem cells by isolating somatic hair follicle stem cells from a mammal, performing limiting dilution plating or cell sorting of said somatic hair follicle stem cells to isolate single somatic hair follicle stem cells, and culturing and expanding said single hair follicle somatic stem cells using the method of claim 1. 16. A method for identifying molecular probes specific for somatic hair follicle stem cells, comprising culturing and expanding said single somatic hair follicle stem cells using the method of claim 1, and using said population- of expanded somatic hair follicle stem cells for comparison to a second population of non-stem cells to identify differences in gene and/or protein expression between the two said populations. 17. A method of culturing and expanding somatic hair follicle stem cells in vitro, comprising culturing somatic hair follicle stem cells isolated from a mammal in a culture medium which permits cell growth under conditions, and for a time sufficient, to permit cell growth, wherein the expression of a protein downstream of the guanine nucleotide biosynthesis pathway in said somatic hair follicle stem cells is modulated by an agent present in the culture medium or by a genetic manipulation to said somatic hair follicle stem cells such that asymmetric cell kinetics are suppressed. 18. The method of claim 17, wherein the modulation is increased expression of the protein. 19. The method of claim 17, wherein the modulation is decreased expression of the protein. 20. A method for treating baldness comprising administering to a subject a composition that stimulates hair follicle stem cells to replicate by converting from asymmetric cell kinetics to symmetric cell kinetics resulting in enhanced proliferation of said hair follicle stem cells with a reversibly reduced production of differentiating progeny cells. 21. The method of claim 20, wherein the agent that stimulates conversion of the stem cells from asymmetric cell kinetics to symmetric cell kinetics is a guanine nucleotide precursor (rGNPr), a analogue or derivative thereof. 22. The method of claim 21 , wherein said guanine nucleotide precursor is xanthosine or hypoxanthine. 23. The method of claim 20, wherein said composition is administered topically. 24. The method of claim 20, wherein said composition is administered by injection. 25. The method of claim 20, wherein said guanine nucleotide precursor is present in an amount of 1 - 10,000 ?M. 26. The method of claim 20, wherein said guanine nucleotide precursor is present in an amount of 50 - 1,500 ?M. 27. A composition comprising a population of human somatic hair follicle stem cells, wherein the population contains at least 100 human somatic hair follicle stem cells. 28. The composition of claim 27, wherein the composition comprises at least 60% multi-potent somatic hair follicle stem cells. 29. The composition of claim 27, wherein the population contains more than 1 ,000 multi-potent hair follicle stem cells. 30. The composition of claim 29, wherein the composition is at least 60% multi-potent hair follicle stem cells.