Differentiation of specialized dermal and epidermal cells into neuronal cells

Abstract

Cells are generated from skin biopsies for use in cell implantation by identifying a source of skin cells that have a surface concentration of at least one cell selected from the group consisting of keritinocytes and/or melanocytes; taking a sample of tissue from the surface area; mechanically disaggregating the tissue samples; collecting the disaggregated cells; washing the disaggregated cells; filtering the washed disaggregated cells; providing a cell suspension with filtered and washed keritinocytes and/or melanocytes; and suspending the cell suspension in a medium. In greater particularity, the method may comprise a) identifying a source of skin cells that have a surface concentration of at least 400 cells per square millimeter of surface area of at least one cell selected from the group consisting of keritinocytes and/or melanocytes; b) taking a sample of those skin cells and culturing them to enrich for cells selected from the group consisting of keritinocytes and melanocytes to form cultured cells for transplantation purposes c) concentrating the cells selected from the group consisting of keritinocytes and/or melanocytes from the cultured mass of cells; and d) differentiating the concentrated cells selected from the group consisting of keritinocytes an/or melanocytes.

What is claimed:  
     
         1 . A method for generating cells for use in cell implantation comprising: 
 a) identifying a source of skin cells that have a surface concentration of at least 400 cells per square millimeter of surface area of at least one cell selected from the group consisting of keritinocytes and melanocytes;    b) taking a sample of those skin cells and culturing them to increase the surface concentration of cells selected from the group consisting of keritinocytes and melanocytes to form a cultured mass of cells;    c) concentrating the cells selected from the group consisting of keritinocytes and melanocytes from the cultured mass of cells; and    d) differentiating the concentrated cells selected from the group consisting of keritinocytes and melanocytes.    
     
     
         2 . The method of  claim 1  wherein the differentiated cells are implanted into a patient.  
     
     
         3 . The method of  claim 1  wherein the identified cells comprise melanocytes.  
     
     
         4 . The method of  claim 1  wherein the identified cells are keritinocytes.  
     
     
         5 . The method of  claim 3  wherein the surface concentration of melanocytes is at least 500 melanocytes per square millimeter of skin.  
     
     
         6 . The method of  claim 3  wherein the surface concentration of melanocytes is at least 700 melanocytes per square millimeter of skin.  
     
     
         7 . The method of  claim 3  wherein the surface concentration of melanocytes is at least 800 melanocytes per square millimeter of skin.  
     
     
         8 . The method of  claim 4  wherein the surface concentration of keritinocytes is at least 500 melanocytes per square millimeter of skin.  
     
     
         9 . The method of  claim 1  further comprising the step of concentrating the cells selected from the group consisting of keritinocytes and melanocytes  
     
     
         10 . The method of  claim 1  wherein said step of differentiating in carried out by a technique selected from the group consisting of genetic manipulation, the use of growth factors or inhibitors, or transplantation into a host.  
     
     
         11 . The method of  claim 1  wherein said step of differentiating in carried out by the technique of genetic manipulation, wherein one or more genes which are inserted are under the control of an inducible promoter system.  
     
     
         12 . The method of  claim 11  wherein said inducible promoter system is selected from the group consisting of tetracycline induced and magnetic resonance imaging induced.  
     
     
         13 . The method of  claim 11  wherein said one or more gene is selected from the group consisting of EGFP and tyrosine hydroxylase.  
     
     
         14 . The method of  claim 1  wherein said source of skin cells is selected from the group consisting of skin biopsy tissue, mole biopsy tissue, and fetal foreskin.  
     
     
         15 . A method for generating cells from skin biopsies for use in cell implantation comprising: 
 identifying a source of skin cells that have a surface concentration of at least one cell type selected from the group consisting of keritinocytes and melanocytes;    taking a sample of tissue from the surface area;    chemically or mechanically disaggregating the tissue sample;    collecting the disaggregated cells;    washing the disaggregated cells;    filtering the washed, disaggregated cells;    providing a cell suspension with filtered and washed keritinocytes or melanocytes;    suspending the cell suspension in a medium.    
     
     
         16 . The method of  claim 15  wherein the source of skin cells has a surface concentration of keritinocytes and melanocytes of at least 400 cells per square millimeter of surface area of skin.  
     
     
         17 . The method of  claim 15  wherein the medium in which the cell suspension is suspended comprises a nutritional medium.  
     
     
         18 . The method of  claim 17  wherein the nutritional medium is selected from the group consisting of DMEM/F12+N2 supplement, bFGF, EGF containing less than 10% fetal calf serum, and Melanocyte basal medium.  
     
     
         19 . The method of  claim 15  wherein said step of providing further includes the step of separating said cell type by the use of specific surface antibodies.  
     
     
         20 . The method of  claim 15  wherein said source of skin cells is selected from the group consisting of skin biopsy tissue, mole biopsy tissue, and fetal foreskin.  
     
     
         21 . A method for the implantation of cells generated from skin biopsies, comprising the steps of a) identifying a source of skin cells that have a surface concentration of at least 400 cells per square millimeter of surface area of at least one cell selected from the group consisting of keritinocytes and melanocytes; 
 b) taking a sample of said skin cells and culturing them to increase the surface concentration of cells selected from the group consisting of keritinocytes and melanocytes to form a cultured mass of cells;    c) concentrating the cells selected from the group consisting of keritinocytes and melanocytes from the cultured mass of cells;    d) differentiating the concentrated cells selected from the group consisting of keritinocytes and melanocytes; and    e) transplanting said differentiated cells into a host.    
     
     
         22 . A method for producing cells for cell implantation or cell transplantation therapies, comprising the steps of: 
 obtaining a sample of skin cells which includes at least one cell selected from the group consisting of keritinocytes and melanocytes at a surface concentration of at least 400 cells per square millimeter;    culturing said sample of skin cells;    concentrating cells selected from the group consisting of keritinocytes and melanocytes from a cultured mass of cells generated in said culturing step; and    isolating whole differentiated implantable or transplantable cells from a concentration of cells generated in said concentrating step.    
     
     
         23 . The method of  claim 22  wherein said obtaining step is performed by skin biopsy.  
     
     
         24 . The method of  claim 22  wherein undifferentiated and differentiated cells remain together during the process until said isolating step.  
     
     
         25 . The method of  claim 22  wherein said obtaining step includes the steps of: 
 disaggregating said sample to produce disaggregated cells;  
 washing and filtering said disaggregated cells; and  
 suspending said disaggregated cells in a suspension medium.  
 
     
     
         26 . The method of  claim 25  wherein said suspension medium includes nutrients for said disaggregated cells.