Stem cells from adipose tissue, and differentiated cells from said cells

Abstract

The invention concerns adult multipotent human stem cells, characterized in that they have: i) significant telomerase activity, ii) an HLA Class I negative phenotype, iii) a normal karyotype, iv) a capacity to become quiescent, v) a capacity for self-renewal preserved for at least 130 population doublings.

1 . An adult multipotent human stem cell, characterized in that it has:
 i) significant telomerase activity,   ii) an HLA Class I negative phenotype,   iii) a normal karyotype,   iv) a capacity to become quiescent,   v) a capacity for self-renewal preserved for at least 130 population doublings.   wherein the stem cell is characterized in that it can express at least one transgene.   
     
     
         2 .- 12 . (canceled) 
     
     
         13 . A method for obtaining multipotent human stem cells comprising the following steps:
 (a) culturing cells from a human adipose tissue sample,   (b) selecting two cell sub-populations termed a “CA” population and a “CS” population, the “CA” population having an adhesion rate of less than 12 hours, and the “CS” population having an adhesion rate of more than 12 hours,   (c) enriching the “CA” population until a quiescent cell population is obtained, and   (d) inducing proliferation of stem cells of the “CA” population.   
     
     
         14 . A method according to  claim 13 , comprising the following additional steps prior to step (a):
 (i) enzymatically digesting a sample of adipose tissue; and   (ii) recovering a cell fraction that is free of adipocytes, containing all of the cell types present in the preparation obtained in (i) with the exception of adipocytes;   wherein the culturing in step (a) is carried out using the cell fraction obtained in step (ii) for at least 12 hours.   
     
     
         15 . A method according to  claim 13 , characterized in that the adipose tissue sample derives from a healthy child under 10 years of age. 
     
     
         16 . A method according to  claim 13 , characterized in that the adipose tissue sample is a sample of extramedullary tissue derived from the umbilical region, from the pubic region, from the inguinal region, from the perineal region, from the abdominal region, or from the subcutaneous region. 
     
     
         17 . A method according to  claim 13 , characterized in that proliferation of stem cells in step (d) is an intensive proliferation induced by adding a growth factor. 
     
     
         18 . A method according to  claim 14 , characterized in that the enzymatic digestion in step (i) is carried out by bringing the adipose tissue sample into contact with a collagenase preparation for a maximum period of 10 minutes. 
     
     
         19 . A method according to  claim 14 , characterized in that the recovery of the cell fraction that is free of adipocytes in step (ii) is carried out by centrifugation. 
     
     
         20 . A method according to  claim 14 , characterized in that the cell fraction that is cultured does not undergo any filtration step before culturing. 
     
     
         21 . A method according to  claim 14 , characterized in that the culturing step is carried out in a culture medium supplemented with foetal calf serum without the addition of other growth factors. 
     
     
         22 . A method according to  claim 14 , characterized in that during the culturing step, cell transfer is carried out when the cells reach 80% confluence, transfer being carried out at a seeding density of about 1000 to 3500 cells/cm 2 . 
     
     
         23 . A method according to  claim 13 , characterized in that the “CA” population becomes quiescent after about 60 population doublings. 
     
     
         24 . A method according to  claim 17 , characterized in that the growth factor employed is selected from the group consisting of bFGF, PDGF, EGF, NGF and SCF. 
     
     
         25 .- 28 . (canceled) 
     
     
         29 . A therapeutic product for use in in vivo tissue regeneration which comprises adult multipotent human stem cells having:
 i) significant telomerase activity,   ii) an HLA Class I negative phenotype,   iii) a normal karyotype,   iv) a capacity to become quiescent,   v) a capacity for self-renewal preserved for at least 130 population doublings.   
     
     
         30 .- 32 . (canceled) 
     
     
         33 . A method for producing differentiated cells of the mesodermal lineage, characterized in that adult multipotent human stem cells are cultivated from confluence in the presence of a differentiation medium, wherein the stem cells have:
 i) significant telomerase activity,   ii) an HLA Class I negative phenotype,   iii) a normal karyotype,   iv) a capacity to become quiescent,   v) a capacity for self-renewal preserved for at least 130 population doublings.   
     
     
         34 . A method according to  claim 33 , characterized in that the stem cells are seeded at a density of about 10 000 to 25 000 cells/cm 2 . 
     
     
         35 . A method according to  claim 33 , characterized in that the differentiation medium is a medium allowing differentiation into adipocytes. 
     
     
         36 . A method according to  claim 33 , characterized in that the differentiation medium is a medium allowing differentiation into osteoblasts. 
     
     
         37 . A method according to  claim 33 , characterized in that the differentiation medium is a medium allowing differentiation into myocytes, or an angiogenic medium. 
     
     
         38 . A screening method to identify agents that can modulate the differentiation of cells into cells of the mesodermal lineage, characterized by:
 a) culturing adult multipotent human stem cells under culture conditions that allow their differentiation into cells of the mesodermal lineage, in the presence of a candidate agent, wherein the stem cells have:
 i) significant telomerase activity, 
 ii) an HLA Class I negative phenotype, 
 iii) a normal karyotype, 
 iv) a capacity to become quiescent, 
 v) a capacity for self-renewal preserved for at least 130 population doublings; 
   b) comparing the differentiation of cells in the presence of a candidate agent with differentiation in the absence of the candidate agent.   
     
     
         39 .- 42 . (canceled) 
     
     
         43 . A screening method for identifying agents that may have a lipolytic activity, characterized by:
 a) culturing adult multipotent human stem cells under conditions allowing their differentiation into adipocytes, wherein the stem cells have:
 i) significant telomerase activity, 
 ii) an HLA Class I negative phenotype, 
 iii) a normal karyotype, 
 iv) a capacity to become quiescent, 
 v) a capacity for self-renewal preserved for at least 130 population doublings; 
   b) bringing the adipocytes thus obtained into contact with a candidate agent,   c) evaluating the lipolytic activity of the candidate agent.   
     
     
         44 . A screening method for identifying agents that may have an anti-lipolytic activity, characterized by:
 a) culturing adult multipotent human stem cells under conditions allowing their differentiation into adipocytes, wherein the stem cells have:
 i) significant telomerase activity, 
 ii) an HLA Class I negative phenotype, 
 iii) a normal karyotype, 
 iv) a capacity to become quiescent, 
 v) a capacity for self-renewal preserved for at least 130 population doublings; 
   b) bringing the adipocytes thus obtained into contact with a candidate agent, in the presence of a lipolytic agent,   c) evaluating the anti-lipolytic activity of the candidate agent.   
     
     
         45 . A screening method for identifying agents that may have an insulin-sensitizing activity, characterized by:
 a) culturing adult multipotent human stem cells under conditions allowing their differentiation into adipocytes, wherein the stem cells have:
 i) significant telomerase activity, 
 ii) an HLA Class I negative phenotype, 
 iii) a normal karyotype, 
 iv) a capacity to become quiescent, 
 v) a capacity for self-renewal preserved for at least 130 population doublings; 
   b) bringing the adipocytes obtained into contact with a candidate agent,   c) evaluating the insulins-sensitizing activity of the candidate agent.   
     
     
         46 . (canceled) 
     
     
         47 . A cosmetic composition comprising a plurality of adult multipotent human stem cells having:
 i) significant telomerase activity,   ii) an HLA Class I negative phenotype,   iii) a normal karyotype,   iv) a capacity to become quiescent,   v) a capacity for self-renewal preserved for at least 130 population doublings;   
       in association with an excipient, vehicle, solvent, colorant, fragrance, antibiotic or other additives acceptable in cosmetic products. 
     
     
         48 .- 54 . (canceled) 
     
     
         55 . A pharmaceutical composition comprising a cell population and a physiologically acceptable excipient, wherein the cell population comprises a plurality of adult multipotent human stem cells having:
 i) significant telomerase activity,   ii) an HLA Class I negative phenotype,   iii) a normal karyotype,   iv) a capacity to become quiescent,   v) a capacity for self-renewal preserved for at least 130 population doublings;   
       characterized in that it is free of adipocytes, fibroblasts, preadipocytes, endothelial cells, pericytes, mastocytes, and smooth muscle cells.