Embryonic Neural Stem Cell Improved The Survival Of Aging Neurons In Postmortem Human Brain Slices

Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cells can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control subjects with rat embryonic day 14 (E14) neural stem cells. Human Postmortem brain slices from the elderly control and Alzheimer's disease (AD) (n=13) were co-cultured with NSC without direct contact or by cell fusion using separated co-culturing system. Immunohistochemistry and viability staining were used to evaluate the morphological features and survival of the human neurons in neocortical tissue on different days in vitro ranged from 0 to 38.Viability staining, based on the exclusion of ethidium bromide by intact plasma membranes, showed that there were strikingly more viable cells and fewer dead cells in co-cultured slices than in untreated slices. The presence of Alzheimer pathology in the brain slices did not influence this effect, although the slices from Alzheimer patients, in general, contained fewer viable cells. Co-culturing with rat E14 fibroblasts did not improve the viability of neurons in the human brain slices. Since the human slices and neural stem cells were separated by a membrane during co-culturing our data show for the first time that neural stem cells release diffusible factors that may improve the survival of aged and degenerating neurons in human brains.Data analysis revealed that there was no significant changes over time viable (p=0.41), leaky (p=0.72) and dead (p=0.70) by patient. Visual inspection of untreated slices stained with the Live/Dead kit did not show any obvious differences between tissue from control subjects and AD patients. However, cell counts revealed that the number of viable cells in untreated slices of AD patients was reduced by 55 % as compared with untreated slices from control subjects and the reduction was significant (P = 0.02), whereas the differences in leaky, dead and total cell number were not significant. Taking controls and Alzheimer patients together, the effect of treatment with neural stem cells consisted of an average increase of 2600 viable cells per mm³ (P < 0.001). The number of leaky cells roughly remained the same (P = 0.36), while the mean number of dead cells and the mean total number of cells decreased with about 9500 (P < 0.001) and 7800 (P = 0.009) per mm³, respectively. The high variability of the viability in co-cultured slices from AD patients, however, suggests that tissue from some AD patients responded less well to treatment with neural stem cells.To further confirm the neuronal survival in our culture system, Total RNA was isolated from cultured slices of 8 brains (AD n=4, aging control n=4) and real time PCR was performed using SYBR greenI dye to explore the expression of neuronal related genes on transcriptional level, including neuronal enclose 2 (NSE) and JNK-interacting protein1 (JIP1). Our data showed even the input RNA amount was equal at the start of cDNA synthesis, none of so called "reference genes" expressed constantly during culture. In the following study, we, therefore used the method of 2^â–³CT to normalize the target gene externally and determine whether the mRNA amount could be affected by the treatment.â–³Ct'=Ct_(DIV10)- Ct_{(untreated DIV3)}NSE gene is found in mature neurons and cells of neuronal origin. In 4 elderly non-dementia control, mock-treated slices from different elderly control subjects showed similar level of delta Ct', which indicates a relative stable survival system of human postmortem brain slices regardless the day in vitro. Co-cultured human brain slices contained an obvious higher amount of NSE mRNA after 10 days in co-culture than mock-treated slices (p=0.021 by Mann- Whitney), indicating a significant increase of NSE mRNA induced by NSC.Untreated brain slices from AD patients showed too low transcription level of NSE, only 1 AD patient showed reliable NSE template amount (Ct<32) and again, co-cultured slices from this AD patient displayed higher transcription level of NSE after 10 days co-culture. To examine the responsive capacity of AD brains, we further investigated the changes of JIP1, which could be regulated by the environmental stress, as a marker of neuron responsible ability to the environment stress in vitro. JIP1 mRNA was easily detectable in frozen tissue from 1 AD brain (Ct=28.90) and 2 elderly control brains (Ct=28.77 and 29.79). However, under culture conditions, no or only a doubtful amount of mRNA of JIP-1 was detected in all four cultured AD slices, whereas it was detected in larger amounts in all elderly controls subjects (Ct=31.9, 32.7,29.8, 27.8). Electrophoresis of the PCR products showed that even the elderly control patient with a very low total RNA amount (C4) displayed a different pattern of JIP expression as compared with AD (AD5).Our data indicate that human aged brain tissue could make communications with NSC even without direct contact. These findings may have potential for the clinical use of NSC in aging related diseases.