Genetic stability and cryopreservation of banked neural stem cells

The use of stem cells in medical research is increasing steadily since they, likely, will constitute a renewable source of cells for the treatment and study of human diseases. For this purpose the application of standard quality controls, testing basic behaviour and general sterility of the cell cultures, are not enough to guarantee their safety in clinical settings. Sophisticated testing, as the detection of contaminating pathogens and assessment of genetic stability, represent a fundamental tool to contain the risks and preserve original features of stem cells. For these reasons, we routinely perform on human stem cells (i) viral screening (HSV1-2, EBV, CMV, HBV, HCV, HIV, HPV), (ii) high resolution genetic analysis such as comparative genomic hybridization array (aCGH) and (iii) telomere analysis. Telomere maintenance appears to be essential for the chromosomal integrity and the prolonged persistence of stem cell function. We analyzed hTERT mRNA expression, relative telomerase activity and average telomere length in human Neural Stem (NS) cell lines of different origin: CB660 and CB660SP derived from foetal cortex and foetal spinal cord; Hes4-hNCPC (Neural Crest Progenitor Cells) derived from human embryonic stem cell (hES) and AF22 derived from induced Pluripotent Stem (iPS) cells. Although human NS cells CB660, CB660SP and Hes4-hNCPC preserved a normal karyotype, their telomeric length shortened during expansion due to the absence of telomerase. This implies that human NS cells may undergo replicative ageing. Instead, the AF22 cell line showed a high telomerase activity and a high hTERT expression when compared to hES-derived NS cells. This confirms their great proliferation and differentiation potential even if maintaining high telomerase activity, characteristic of pluripotent cells. Furthermore, if these cells will ever be used in clinical applications, safer cryoprotectants need to be adopted to assure genomic stability after long term storage. We have developed and applied a cryopreservation reagent (trehalose-based) that successfully cryopreserved human fetal- and iPS-derived NS cells. Cell\u2019s viability, stability and differentiation capacity were tested in relation to the standard reagents and method (Cryostor10\uae)