Tissue, cell type: Evercyte offers the immortalization / life span extension of different human cell types (e.g. fibroblasts from skin, lung, kidney tissue, keratinocytes, endothelial cells from skin, umbilical vein or mesenchymal stem cells from adipose tissue, placental tissue or bone marrow) Ethical issues:
cells used for life span extension are isolated with ethical approval from the responsible IRB and prior given written informed consent by the donor
Life span extension method:
depending on the cell type, cells are immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT) and/or cell cycle regulators such as cyclin-dependent-kinase 4 (cdk-4) or viral oncogenes (e.g. SV40 early region, SV40 largeT/smallt antigens)
Selection of transfectants:
positively transfected cells are selected using antibiotic resistance genes such as neomycin-phosphotransferase (neo)
selected cell lines are characterized depending on the customers needs; Evercyte offers services to test the presence of cell type specific markers and functions, authentication, freedom from contaminations, tumorigenic potential or RNAseq
Recent example – immortalization of mesenchymal stem cells
Wharton´s Jelly-derived mesenchymal stem cells were isolated from umbilical cord tissue using enzymatic and mechanical disaggregation. After enrichment, the normal cells were transfected with a plasmid carrying the catalytic subunit of human telomerase (hTERT) and transfectants were selected by growing cells in the presence of Geneticin sulfate (G418). The resulting cell line can be grown for a minimum of 50 population doublings with a stable growth rate without showing signs of growth retardation. The cells are characterized by the typical spindle-shaped mesenchymal morphology and express typical markers and functions of mesenchymal stem cells (not shown).
Recent example – reprogramming of urine cells into iPSCs
Evercyte has developed protocols for isolation of different cells (e.g. renal proximal tubular epithelial cells, podocytes, urine-stem cells, mesenchymal stem cells) from human urine sediments. These cells are reprogrammed into induced pluripotent stem cells (iPSCs) that can be differentiated towards cells from the 3 germ layers such as muscle cells, neuronal cells or hepatocyte-like cells.