Howard, Linda

Background. A major technical limitation in preclinical cell replacement research is the ability to discriminate between donor and host tissue after transplantation. This problem has been lessened by the availability of transgenic animals that express “reporter” genes, such as green fluorescent protein (GFP). Objective. We determined the usefulness of one such transgenic reporter rat to assess the survival of bone marrow—derived rat mesenchymal stem cells (MSCs) following direct transplantation into the intact adult brain. We also sought to determine if the expression of GFP in the brain affected the survival of the MSCs or the host’s neuroimmune response to the cells. Methods. Rats received intrastriatal injections of sterile transplantation medium, 100 000 normal MSCs, or 100 000 GFP-MSCs and were killed humanely 1, 4, 7, 28, and 42 days posttransplantation for astrocyte and microglial immunohistochemical staining. Results. GFP-MSCs were evident at each examination, although their survival declined over time. Graft volume estimates comparing normal and GFP-MSCs revealed that GFP expression did not adversely affect the survival of the stem cells in the brain. Furthermore, immunostaining for astrocytes and microglia revealed that expression of the reporter protein did not affect the immunogenicity of the stem cells. Conclusions. These data indicate the usefulness of GFP for investigating the survival of MSCs following transplantation to the brain. However, the mechanisms responsible for the poor survival of the stem cells must be elucidated if these cells are to serve cell-based therapies for neurodegenerative disorders.

The aims of this research were to examine the neural expression profile of undifferentiated mesenchymal stem cells (MSCs), to define a serum-free environment that would support the survival of MSCs, and to assess the effects of elevated cyclic adenosine monophosphate (cAMP) levels on MSC morphology and expression of neural markers. The neural profile of MSCs was assessed using immunocytochemistry and real-time polymerase chain reaction (PCR) techniques. These MSCs were then cultured in varying serum-free environments, with flow cytometry analysis of cell viability and apoptosis. The effects of forskolin and 8 bromo-cAMP treatment on MSC morphology and expression of neural markers were assessed using light microscopy, immunocytochemistry and real-time PCR analysis. Expression of the neural markers nestin, vimentin, β III tubulin, glial fibrillary acidic protein, and the tropomyosin-related kinases was demonstrated in normal undifferentiated MSCs. Furthermore, MSCs cultured in serum-free conditions containing ascorbic acid 2-phosphate demonstrated greater long-term survival and reduced apoptosis. In contrast, forskolin increased the percentage of cells undergoing apoptosis. Culture in the presence of forskolin induced a 6-fold increase in β III tubulin expression after a 6-h exposure time, which was accompanied by dramatic changes in morphology. However, this effect was transient, with the cells reverting to their normal morphology by 24 h. Treatment with 8 bromo-cAMP induced similar increases in β III tubulin expression without such dramatic morphological changes. Factors which increase the concentration of cAMP induce transient changes in expression of neural markers that appear to be the result of cellular adaptation to changes in culture conditions rather than a real process of neural differentiation.