Immortalized stem cell, Compositions, Preparations and Uses Thereof

Abstract

The purpose of the present invention is to provide immortalized stem cells, which produce a growth factor capable of regenerating various kinds of tissues that have been damaged by a variety of causes, and a method for producing the aforesaid immortalized stem cells. Another purpose of the present invention is to provide a medicinal composition and a medicinal preparation for restoring damaged tissues, and a method for the percutaneous absorption of a culture supernatant. Provided are immortalized stem cells that are obtained by isolating stem cells selected from the group consisting of mammalian mesenchymal cells, an embryo at the early stage of the development and somatic cells, first culturing the cells to give first stage culture cells, transferring four kinds of genes into the first stage culture cells to give transgenic cells, and selecting the desired immortalized stem cells from among the transgenic cells using the expression of STRO-1 as an index. Also provided are a medicinal composition and a medicinal preparation for restoring damaged tissues which comprise a culture supernatant of the immortalized stem cells as the active ingredient.

What is claimed is: 
     
         1 . An isolated immortalized stem cell, wherein said isolated stem cell is selected from the group consisting of a mammalian early-developed embryo stem cell, mesenchymal stem cell and a non-mesenchymal somatic stem cell, wherein said stem cell is obtained by performing a primary culture of said stem cell to obtain a primarily cultured stem cell; transfecting 4 genes into said primarily cultured stem cell to produce a gene transduced stem cell; selecting a gene transduced immortalized stem cell having telomere repairing ability, which divides at least 200 times and which expresses STRO-1 and isolating said selected immortalized stem cell. 
     
     
         2 . The isolated immortalized stem cell according to the  claim 1 , wherein said stem cell is selected from the group consisting of exfoliated dens deciduous, exfoliated dens permanence, removed deciduous teeth, and removed dens permanence. 
     
     
         3 . The isolated immortalized stem cell according to  claim 1 , wherein said early-developed embryo is a blastoderm. 
     
     
         4 . The isolated immortalized stem cell according to  claim 1 , wherein said mammal is selected from the group consisting of human, swine, equine and monkey. 
     
     
         5 . The isolated immortalized stem cell according to  claim 1 , wherein at least 40% of cell number produced from said immortalized stem cell have population doubling times of at least 20 as determined by STRO-1 expression, and said isolated immortalized stem cell has neonatal bone quantity production ability at least equal to those of a primary cultured cell. 
     
     
         6 . The isolated immortalized stem cell according to the  claim 1 , wherein said immortalized stem cell secretes at least IGF-1, VEGF, TGF-β1 and HGF into said culture sup. 
     
     
         7 . A composition comprising said culture sup of said immortalized stem cell according to  claim 1 . 
     
     
         8 . The composition according to  claim 7 , wherein said composition has a dosage forms selected from the group consisting of powder, liquid, gel, spray and percutaneous system. 
     
     
         9 . The composition according to  claim 7 , wherein the content of the culture sup is 50 to 500% (w/v) when the sup produced by the immortalized stem cell is 100%. 
     
     
         10 . A method for restoring damaged tissue in a subject in need thereof comprising administering an amount of the composition of  claim 7  in an amount effective to restore damaged tissue. 
     
     
         11 . The method according to  claim 10 , wherein said damaged tissue is any one of tissue selected from the group consisting of a damaged tissue with ulcer or bedsore, a damaged brain is caused by cell degeneration, a brain tissue partially lost it tissue by a surgical operation. brain tissue damaged by traumatic brain injury, a damaged brain tissue with inflammatory brain disease, a damaged bone tissue, a damaged periodontal tissue, a damaged tissue by a central nervous system disease, and a damaged tissue by refractory skin disease. 
     
     
         12 . The method according to  claim 10 , wherein said cell degeneration is caused by the disease selected from the group consisting of Alzheimer disease, Parkinson's disease, cognitive impairment, schizophrenia, depression disease, cerebral hypoxia, amyotrophic lateral sclerosis, cerebral infarction, cerebellar degeneration, diabetes, and hepatitis. 
     
     
         13 . The method according to  claim 10 , wherein said traumatic brain injury is caused by a traffic accident or fall accident. 
     
     
         14 . The method according to  claim 10 , wherein said inflammatory disease is inflammatory encephalopathy, a disease selected from the group consisting of encephalitis encephalopathy, epileptic, Jakob disease, and polio. 
     
     
         15 . The method according to  claim 10 , said refractory dermatitis is atopic dermatitis. 
     
     
         16 . A method for producing an immortalized stem cell comprising the steps of:
 separating a stem cell from a cell population selected from the group consisting of mammary mesenchymal cell, wherein said mesenchymal stem cell is selected from the group consisting of dental pulp stem cell, bone marrow stem cell, umbilical stem cell and adipose stem cell; early-developed embryo and a non-mesenchymal somatic cell;   culturing said cultured stem cell as a primary culture to obtain a primarily cultured cell;   transfecting 4 genes into said primarily cultured cell to produce a gene-transfected cell; and   selecting a mesenchymal stem cell selected from the group consisting of dental pulp stem cell, bone marrow stem cell, umbilical stem cell and adipose stem cell; early-developed embryo and non-mesenchymal somatic cell having telomere repairing and bone regeneration ability, which divides at least 200 times, and which has a cell doubling time of at least 20 as determined by STRO-1 expression   
     
     
         17 . The method according to the  claim 16 , wherein the dental pulp cell is any one of the teeth selected from the group consisting of exfoliated dens deciduous, exfoliated dens permanence, removed dens deciduous, and removed dens permanence. 
     
     
         18 . The method according to  claim 16 , wherein the primary embryo is an embryo of blastodisc stage. 
     
     
         19 . The method according to  claim 16 , wherein the mammary is any one of the animal selected from the group consisting of human, swine, equine, and monkey. 
     
     
         20 . The method according to  claim 16 , wherein the four genes are selected from the group consisting of hTERT, bmi-1, E6, E7, Oct3/4, Sox2, Klf4, c-Myc, and p16INK4a.