for Neurotherapeutics Differentiation Unit: Our laboratories are fully staffed, fully functional and operating in 3 buildings. We have established a number of automated, medium throughput assays in the past year. Weve utilized these assays to screen small compound collections against rat mixed cortical cultures, human neural stem cells and human NSC-derived neurons along with some astrocyte cultures (with M. Rao, CRM). Weve established live imaging based assays with rat hippocampal/cortical neurons utilizing high content imaging endpoints. These assays have been employed to assay compounds and/or blood and tissue samples provided by numerous investigators (Rao-CRM, Pant, Nath-SINS and other investigators within these research groups). Three of these studies have resulted in three publications. Additional high content imaging assays have been initiated with cells treated with human endogenous retroviruses and serum samples from patients with Nodding syndrome. We continue to curate the NINDS SMA collection and have provided about 18 compounds to numerous outside investigators (in collaboration with NIAID) to evaluate the anti-viral activities of these prototypic compounds. At least two of these compounds have shown some significant antiviral activity against SARS and HPV, while 3 additional compounds have moderate activity in these assays. Additional compounds have been sent to investigators, and rescreening in secondary SARS assays is underway. We continue to provide HPLC and LCMS methods development support to quantitate neuroprotective peptides (TFP-5 Pant), antisense oligonucleotides (JCV14L Major/Nath) and other small molecules (Limonin Nath, Atopaxar, NTDU). Pharmacokinetic analyses, metabolite profiling and tissue distribution of these agents will continue in the coming year. Progress Report for Neural Differentiation Unit For the past year, the Neural Differentiation Unit has made continuous progress based on our mission and goals, which are focused on developing novel specific disease-based cell culture models to study the mechanisms of neuroinflammation and facilitate therapeutics development. We also initiated collaborations with other groups within NINDS and NIH. The progress is summarized below under specific aims:
Specific aim 1 : Further characterization of neural cultures from human adult peripheral CD34+ cells. We further characterized the neural cultures derived from adult peripheral CD34+ cells. 1) We compared the neural stem cells (iNS) directly derived from CD34+ cells with primary cultured neural progenitor cells and neural stem cells differentiated from control iPSC cells using microarray as a platform. The results demonstrated these three different cell populations showed similar gene expression profiles of neural stem cell genes, which were very different from original CD34+ cells, indicating that these cells directly derived from CD34 positive cells are neural stem cells. The few differences in gene expression among the three different cell types may due to the differences in culture conditions and cell localizations as the iNS were enriched with neural crest cell properties using GeneSet Enrichment Analysis. Furthermore, three iNS cell lines generated from different donors showed consistent gene expression profiles, indicating the methods used in iNS generation are reliable to generate consistent cell lines, which is a main concern when using directly transformed cells to model diseases. 2) We further determined the neurotransmitter content of neurons generated from the iNS. We found that most of the neurons (more than 50%) generated were glutamatergic neurons, while the others were mainly GABAergic neurons, with 1-2% of dopaminergic neurons. A manuscript has submitted for publication based on these findings, along with the data from an autologous model of neuroinflammation by co-culturing CD34-generated iNSC and T cells.
Specific aim 2 : Study the effect of inflammation on oligodendrocyte progenitor cells. We have developed methods to derive O4+ oligodendrocyte progenitor cells (OPC) from iNS. We also found that OPC media containing human serum enhanced myelin basic protein (MBP) production in OPC. These developments provide us a very useful model to study the effect of T cells activation on OPC proliferation and differentiation. Using supernatants from activated T cells to treat OPC, we found that activated sups from either CD4+ or CD8+ cells increased OPC proliferation by accelerating the number of cells entering cell division. Activated CD4 sups also increased MBP production from OPC. Further study revealed that activated CD4 and CD8 T cells released VEGF into the supernatants. By immunodepleting VEGF from the sups, the effect of activated sups on OPC proliferation was partially attenuated. These findings indicated that activated T cells, especially CD4+ cells, could activate OPC proliferation and maturation by releasing VEGF. These may play an important role in pathogenesis and recovery from relapsing multiple sclerosis attacks. The OPCs generated from disease specific CD34+ cells could be very useful to study diseases involving OPC functions.
Specific aim 3 : study the effect of HERV K on iPS development. We have found that HERV K components, gag, env and pol expression were increased in iPSCs but diminished rapidly after differentiation. We continued to study the expression pattern of HERVK Env and its functions in stem cells. We could not find detectable HERVK virus particles in either sups and cell membrane using PCR and scanning EM. However, immunostaining with anti-HERVK Env shows that increased Env levels in cell membranes, which could be easily removed by detergents such as saponin and Triton X-100. These results indicate Env may be expressed independent of a complete virus. Western blot studies showed that naturally expressed Env was found at different sizes compared to recombinant consensus Env, implying different processing may happen to the natively expressed Env. Using siRNAs to inhibit HERVK expression, we found that inhibition of Env resulted in larger cell volumes and likely due to inhibition of cell-cell contacts. Inhibition of possible Env interactions by using Env specific antibodies showed similar results, and reduced iPS colony formation. These results indicate that HERVK Env may play some role in cell and tissue development. Further study to delineate the possible Env binding partners is ongoing.
Specific aim 4, Study the effect of microRNA (miR) on neurogenesis and neurotoxicity. We continued the study of role of miRs on neuronal toxicity. Using microarray as a platform, we found that inhibition of DAP5 gene expression may account for the miR16 and miR29-induced neurotoxicity. DAP5 is also named EIF4G2, an eIF4G family member and a mediator of cap-independent translation, which plays an important role in cell division and protection against apoptosis under stress. Further, we found that DAP5 expression levels changed among stem cells and differentiated cells, indicating DAP5 may play important roles in stem cell function, thus requiring further study of DAP5 in our other stem cell systems. We are also generating iPS/iNS from cells of ALS (1 cell line) and PLS (3 cell lines). Further characterization of the cells is ongoing. These cells will be used to study the pathogenesis for the corresponding diseases, as well as for therapeutics development.
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