Downregulation of sine/alu retrotransposon transcription to induce or restore proliferative capacity and/or pluripotency to a stem cell

Abstract

In certain embodiments methods are provided for inducing and/or restoring and/or maintaining a non-senescent phenotype, or aspects thereof (e.g., proliferative capacity and/or pluripotency) in a mammalian cell. The methods typically involve reducing the level or activity of SINE/Alu retrotransposon transcripts in the cell in an amount sufficient to induce or restore proliferative capacity and/or pluripotency to said mammalian cell.

1 . A method of inducing or restoring or maintaining proliferative capacity and/or pluripotency in a mammalian cell, said method comprising:
 reducing the level or activity of SINE/Alu retrotransposon transcripts in said cell in an amount sufficient to induce or restore proliferative capacity and/or pluripotency to said mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein said method comprises:
 restoring proliferative capacity and/or pluripotency to a mammalian cell that has reduced or lost proliferative capacity and/or reduced or lost pluripotency; and/or   inducing proliferative capacity and/or pluripotency to a mammalian cell that lacks such capacity; and/or   maintaining proliferative capacity and/or pluripotency in a mammalian cell.   
     
     
         3 - 4 . (canceled) 
     
     
         5 . The of  claim 1 , wherein said mammalian cell is selected from the group comprising a stem cell, a non-renewing progenitor cell, a terminally differentiated cell, and a senescent cell. 
     
     
         6 - 8 . (canceled) 
     
     
         9 . The method of  claim 5 , wherein said cell comprises a stem cell selected from the group consisting of an embryonic stem cell, a cord blood stem cell, an adult stem cell, and an IPSC. 
     
     
         10 . The method of  claim 5 , wherein said cell is a stem cell derived from a tissue selected from the group consisting of human adipose tissue, human bone marrow, human neurological tissue, human smooth muscle, human adipose tissue, human cardiomyocytes, human endothelial tissue, human epithelial tissue. 
     
     
         11 . The method of  claim 1 , wherein said cell:
 shows one or more indicators of senescence; and/or   has committed to differentiation to a cell type selected from the group consisting of ectoderm, mesoderm, and endoderm.   
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 11 , wherein said cell has committed to differentiation to a cell type selected from the group consisting of ectoderm, mesoderm, and endoderm. 
     
     
         14 . The method of  claim 11 , wherein said cell has committed to differentiation to a cell type selected from the group consisting of human adipose cells, human blood cells, human nerve cells, human smooth muscle cells, human adipocyte, human chondrocyte, human cardiomyocytes, human endothelial cells, and human epithelial cells. 
     
     
         15 . The method of  claim 1 , wherein said reducing the level or activity of SINE/Alu transcripts comprises introducing into said cell a construct that comprises or encodes a small interfering RNA (siRNA or shRNA) molecule that targets SINE/Alu retrotransposon transcripts. 
     
     
         16 . The method of  claim 15 , wherein said small interfering RNA comprises a molecule selected from the group consisting of a single strand RNA, a paired double strand RNA (dsRNA), a small hairpin RNA (shRNA), and a PIWI RNA (piRNA). 
     
     
         17 . The method of  claim 15 , wherein said construct comprises sh-132Alu. 
     
     
         18 . The method of  claim 15 , wherein said construct produces a stable down regulation of SINE/Alu transcripts. 
     
     
         19 . The method of  claim 15 , wherein said construct comprises a vector. 
     
     
         20 . The method of  claim 19 , wherein the vector is a plasmid vector; or a viral vector. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated vector. 
     
     
         23 . The method of  claim 15 , wherein said introducing is achieved via transformation, transduction, transfection, or infection. 
     
     
         24 . The method of  claim 15 , wherein said introducing is achieved via a lipid or liposome. 
     
     
         25 . The method of  claim 1 , wherein said method does not comprise targeted upregulation of a Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc, or providing one or more heterologous constructs in said cell encoding Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc. 
     
     
         26 . A cell containing:
 a construct that reduces the level or activity of SINE/Alu transcripts in said cell whereby said cell has restored or maintains proliferative capacity and/or pluripotency; and/or   a construct that reduces the level or activity of SINE/Alu transcripts and said cell shows reduced senescence as compared to a stem cell of the same type lacking said construct.   
     
     
         27 . (canceled) 
     
     
         28 . The cell of  claim 26 , wherein said construct comprises or encodes a small interfering RNA (siRNA) molecule that targets SINE/Alu retrotransposon transcripts. 
     
     
         29 . The cell of  claim 28 , wherein said small interfering RNA comprises a molecule selected from the group consisting of a single strand RNA, a paired double strand RNA (dsRNA), a small hairpin RNA (shRNA), and a PIWI RNA (piRNA). 
     
     
         30 . The cell of  claim 26 , wherein said construct produces a stable down regulation of SINE/Alu transcripts. 
     
     
         31 - 34 . (canceled) 
     
     
         35 . The cell of  claim 26 , wherein:
 said cell comprises a stem cell selected from the group consisting of an embryonic stem cell, a cord blood stem cell, an adult stem cell, and an IPSC; and/or   said cell is a stem cell derived from a tissue selected from the group consisting of human adipose tissue, human bone marrow, human neurological tissue, human smooth muscle, human osteoblast and osteoclast, human fetal tissue, human cord tissue, human adipose tissue, human cardiomyocytes, human endothelial tissue, human epithelial tissue; and/or   said cell was formerly or is derived from a cell that has reduced or lost proliferative capacity and/or reduced or lost pluripotency.   
     
     
         36 - 37 . (canceled) 
     
     
         38 . The cell of  claim 35 , wherein:
 said cell was formerly or is derived from a stem shell showing one or more features of senescence; and/or   said cell was formerly or is derived from a stem cell committed to a cell differentiation pathway; and/or   said cell was formerly or is derived from a non-renewing progenitor cell; and/or   said cell was committed to differentiation to a cell type selected from the group consisting of human adipose cells, human blood cells, human nerve cells, human smooth muscle cells, human osteoblast and osteoclast, human pancreatic cells, human adipocytes, human chondrocytes, human cardiomyocytes, human endothelial cells, and human epithelial cells.   
     
     
         39 - 42 . (canceled) 
     
     
         43 . A stem cell line, said cell line comprising stem cells containing a construct that stably down-regulates SINE/Alu transcripts. 
     
     
         44 . A method of providing a differentiated cell, said method comprising:
 providing a cell according to  claim 26 ; and   culturing said cell under conditions that induce or permit differentiation to an embryoid body and/or to a terminally differentiated cell.   
     
     
         45 . (canceled) 
     
     
         46 . A method of transdifferentiating a mammalian cell from a first cell type or lineage into a second cell type or lineage, said method comprising:
 transforming a differentiated cell into a pluripotent cell by the method of  claim 1 ;   culturing said cell under conditions that induce or permit differentiation of said cell;   selecting cells that have differentiated into said second cell type or lineage; and   culturing the cells of said second cell type or lineage.   
     
     
         47 . The method of  claim 46 , wherein said first cell type or lineage is a mesodermal cell type and said second cell type or lineage is a neuroectodermal cell type. 
     
     
         48 . The method of  claim 46 , wherein said first cell type is an adipocyte or a bone marrow cell. 
     
     
         49 - 50 . (canceled)