Effects of Temperature on the Change of HSP and the Viability of Mobilized Hematopoietic Stem Cells.

It is well known that HSP is influenced by temperature. Here we searched the effects of temperatures on preservation of hematopoietic stem cells (HSC) and the changes of HSP according to the temperatures and cryopreserving agents. The G-CSF-mobilized peripheral blood HSCs were collected from a healthy donor. DMSO, EG and GLY were used for cryopreserving agents. HSP were measured by real-time quantitative PCR, and cell viability were measured by trypan blue stain and MTS assay, respectively. Testing temperatures were 4°C, room temperature (RT, 20°C) and 37°C. Samples were HSCs stored according to testing temperatures without cryopreserving agents (cycle 0), HSCs thawed after first cryopreservation with various cryopreserving agents (cycle 1). The measure was done daily from D0 to D5 at cycle 0, and in 30 minutes interval (0, 30, 60, 90, 120 min) at cycle 1. Whereas more than 98% of cells were viable till 5 days after the harvest of cycle 0 at 4°C, RT and 37°C showed rapid decrease of viable HSCs. By day 3, less than 10% cells were viable at 37°C. At cycle 1, cell viability was rapidly decreased in both DMSO and GLY after 120 min stay at all temperatures. In case of EG, the same patterns were found at RT and 37°C. However, cell viability remained relatively well at 4°C. The expression of HSP 27 were over-expressed by day 2 and suppressed thereafter at all temperatures of cycle 0. At 4°C of cycle 0, the over-expression of HSP 27 was greater than those at RT and 37°C. The expression of HSP 70 was mostly suppressed along times. The suppression at 37°C was greater than those of other temperatures. In case of HSP 90, the overall expression was suppressed except for day 2 at 4°C and RT. At all cases of cycle 0, the expressions at 37°C were lower than those at 4°C and RT. At 4°C of cycle 1, DMSO showed decreased expression of HSP27 and HSP70. In contrast to DMSO, the expression of HSP27 and HSP70 was increased in case of EG and GLY at 4°C of cycle 1. The over-expression of HSP90 at 4°C of cycle 1 in EG and GLY was very low compared with those in DMSO. Overall expressions of HSPs in GLY were lower than in DMSO or EG at RT of cycle 1 except for HSP27. The expression of HSP27, HSP70, HSP90 were all decreased at 37°C of cycle 1 except for HSP70 in EG and HSP90 in DMSO. In conclusion, we should keep cool when storing HSCs without cryopreserving agent. The rapid apoptosis at 37°C could be caused by changed expression of anti-apoptotic HSPs. The expression of HSPs influenced by temperatures and cryopreserving agents may affect the viability of HSCs through the changes of HSPs.