Use of rna for reprogramming somatic cells

Abstract

The present invention provides methods for de-differentiating somatic cells into stem-like cells without generating embryos or fetuses. More specifically, the present invention provides methods for effecting the de-differentiation of somatic cells to cells having stem cell characteristics, in particular pluripotency, by introducing RNA encoding factors inducing the de-differentiation of somatic cells into the somatic cells and culturing the somatic cells allowing the cells to de-differentiate.

1 . A method for producing cells having stem cell characteristics comprising the steps of (i) providing a cell population comprising somatic cells, (ii) introducing RNA into at least a portion of said somatic cells inducing the development of stem cell characteristics, and (iii) allowing the development of cells having stem cell characteristics. 
     
     
         2 . The method of  claim 1  wherein said RNA is derived from an undifferentiated cell. 
     
     
         3 . The method of  claim 2  wherein said undifferentiated cell is a stem cell. 
     
     
         4 . The method of  claim 3  wherein said stem cell is an embryonic stem cell or an adult stem cell. 
     
     
         5 . The method of  claim 1  wherein said RNA comprises whole-cell RNA. 
     
     
         6 . The method of  claim 1  wherein said RNA essentially comprises RNA specific for said undifferentiated cell. 
     
     
         7 . The method of  claim 1  wherein said RNA has been obtained by in vitro transcription. 
     
     
         8 . A method for producing cells having stem cell characteristics comprising the steps of (i) providing a cell population comprising somatic cells, (ii) introducing RNA capable of expressing OCT4 and RNA capable of expressing SOX2 into at least a portion of said somatic cells and (iii) allowing the development of cells having stem cell characteristics. 
     
     
         9 . The method of  claim 8  wherein the method further comprises introducing RNA capable of expressing NANOG and/or RNA capable of expressing LIN28. 
     
     
         10 . The method of  claim 8  wherein the method further comprises introducing RNA capable of expressing KLF4 and/or RNA capable of expressing c-MYC. 
     
     
         11 . The method of  claim 1  wherein step (iii) comprises culturing the somatic cells under embryonic stem cell culture conditions. 
     
     
         12 . The method of  claim 1  wherein the stem cell characteristics comprise an embryonic stem cell morphology. 
     
     
         13 . The method of  claim 1  wherein the cells having stem cell characteristics have normal karyotypes, express telomerase activity, express cell surface markers that are characteristic for embryonic stem cells and/or express genes that are characteristic for embryonic stem cells. 
     
     
         14 . The method of  claim 1  wherein the cells having stem cell characteristics exhibit a pluripotent state. 
     
     
         15 . The method of  claim 1  wherein the cells having stem cell characteristics have the developmental potential to differentiate into advanced derivatives of all three primary germ layers. 
     
     
         16 . The method of  claim 1  wherein said somatic cells are fibroblasts. 
     
     
         17 . The method of  claim 16  wherein said fibroblasts are lung fibroblasts, foreskin fibroblasts or dermal fibroblasts. 
     
     
         18 . The method of  claim 1  wherein said somatic cells are genetically modified. 
     
     
         19 . The method of  claim 1  wherein said somatic cells are human cells. 
     
     
         20 . The method of  claim 1  wherein said RNA is introduced into said at least a portion of somatic cells by electroporation. 
     
     
         21 . Cells having stem cell characteristics prepared by the method of  claim 1 . 
     
     
         22 . A composition comprising the cells of  claim 21  in a cell culture medium. 
     
     
         23 - 27 . (canceled) 
     
     
         28 . A method of deriving differentiated cell types comprising the step of culturing the cells having stem cell characteristics of  claim 21  under conditions that induce or direct partial or complete differentiation to a particular cell type. 
     
     
         29 . A method for reprogramming an animal differentiated somatic cell to a cell having stem cell properties, comprising the step of introducing RNA capable of expressing one or more factors allowing the reprogramming of said somatic cell to a cell having stem cell characteristics into said somatic cell. 
     
     
         30 . The method of  claim 29  wherein said one or more factors comprise OCT4, SOX2, NANOG and LIN28. 
     
     
         31 . The method of  claim 29  wherein said one or more factors comprise OCT4, SOX2, KLF4 and c-MYC. 
     
     
         32 . An assay to identify one or more factors useful for reprogramming an animal differentiated somatic cell to a cell having stem cell characteristics comprising the steps of introducing RNA capable of expressing one or more factors into said somatic cell and determining whether said somatic cell has developed into a cell having stem cell characteristics. 
     
     
         33 . The assay of  claim 32  wherein said step of determining whether said somatic cell has developed into a cell having stem cell characteristics comprises comparing the gene expression of said cell resulting from said introduction of said RNA with embryonic cells of the same type. 
     
     
         34 . A kit for producing cells having stem cell characteristics comprising RNA capable of expressing OCT4 and RNA capable of expressing SOX2. 
     
     
         35 . The kit of  claim 34  further comprising an embryonic stem cell culture medium. 
     
     
         36 . A composition comprising RNA which when introduced into a somatic cell is capable of inducing the development of stem cell characteristics in said cell.