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 morphology and expressing genes characteristic for pluripotent cells, comprising the steps of
 (i) providing a cell population comprising mammalian somatic cells,   (ii) introducing RNA encoding OCT4, RNA encoding SOX2, RNA encoding KLF4 and RNA encoding c-MYC into at least a portion of said somatic cells to produce RNA transfected cells,   (iii) culturing the RNA transfected cells to develop said stem cell morphology, and   (iv) selecting cells expressing marker genes of pluripotency.   
     
     
         2 . The method of  claim 1  wherein the method further comprises introducing one or both members of the group consisting of RNA encoding NANOG and RNA encoding LIN28. 
     
     
         3 . The method of  claim 1  wherein step (iii) comprises culturing the somatic cells under embryonic stem cell culture conditions. 
     
     
         4 . The method of  claim 1  wherein the stem cell morphology is an embryonic stem cell morphology. 
     
     
         5 . 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. 
     
     
         6 . The method of  claim 1  wherein the cells having stem cell characteristics exhibit a pluripotent state. 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 1  wherein said somatic cells are fibroblasts. 
     
     
         9 . The method of  claim 8  wherein said fibroblasts are lung fibroblasts, foreskin fibroblasts or dermal fibroblasts. 
     
     
         10 . The method of  claim 1  wherein said somatic cells are genetically modified. 
     
     
         11 . The method of  claim 1  wherein said somatic cells are human cells. 
     
     
         12 . The method of  claim 1  wherein said RNA is introduced into said at least a portion of somatic cells by electroporation. 
     
     
         13 . A method for reprogramming mammalian differentiated somatic cells to cells having stem cell morphology and expressing genes characteristic for pluripotent cells, comprising the steps of
 (i) introducing into said somatic cells RNA encoding factors allowing the reprogramming of said mammalian somatic cells to cells having stem cell morphology, said factors including at least OCT4, SOX2, KLF4 and c-MYC;   (ii) culturing the cells containing said RNA encoding factors under embryonic stem cell culture conditions to develop said stem cell morphology; and   (iii) selecting cells expressing marker genes of pluripotency.   
     
     
         14 . The method of  claim 13  wherein said RNA encoding factors further include NANOG or LIN28, or NANOG and LIN28. 
     
     
         15 . The method of  claim 1 , wherein said RNA has been obtained by in vitro transcription. 
     
     
         16 . The method of  claim 1 , wherein said RNA has been obtained by chemical synthesis. 
     
     
         17 . The method of  claim 1 , wherein said cells having stem cell morphology are selected by monitoring expression of a marker gene, said marker gene being selected from the group consisting of endogenous OCT4, endogenous NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and telomerase reverse transcriptase (TERT). 
     
     
         18 . The method of  claim 17  wherein the marker gene is DPPA4. 
     
     
         19 . The method of  claim 13 , wherein said cells having stem cell morphology are selected by monitoring expression of a marker gene, said marker gene being selected from the group consisting of endogenous OCT4, endogenous NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and telomerase reverse transcriptase (TERT). 
     
     
         20 . The method of  claim 19  wherein the marker gene is DPPA4.