The Neurotherapeutics Development Unit (NTDU) will focus on preclinical development of neuroprotective and neuroregenerative agents. We will work collaboratively with other NIH researchers to facilitate therapeutics development for neurodegenerative disorders. Additionally, this unit will act as liaison between NINDS translational researchers and NCATS. The goals of the NTDU are to: 1) Develop and utilize medium-high throughput assays for identifying novel therapeutic compounds. -Develop and or conduct screening assays for neuroprotection in rat mixed hippocampal neurons and human cortical neurons. -Utilize axo-dendritic degeneration assays in rat and human neuronal cultures to quantitate neurotrophic potential of therapeutics. -Evaluate potential neuroprotective and neuroregenerative compounds in vitro and in vivo. 2) Facilitate preclinical development of potential therapeutic agents, identified internally at NINDS and by external investigators. The core activity of the NTDU is to facilitate preclinical development of compounds collaboratively with NINDS nvestigators. We will utilize medicinal chemistry, neuronal cell biology, molecular biology, pharmacology, biochemistry and biopharmaceutics techniques to achieve these goals. Progress Summary: We have spent the last 10 months establishing the infrastructure of the NTDU. Much of this time was spent relocating the Steiner lab and equipment from JHU to NIH. Additional time was needed to complete the staffing of the NTDU, which will be achieved by Sept.24. Our staff includes one full-time medicinal chemist (MV), one part-time cell culture (JS), one full-time biochemist (MB), one full-time molecular biologist/robotics specialist (KM) and one full-time neuropharmacologist/bio-pharmaceutics/chemist (JPS). Our laboratories have been established in three buildings and are fully functional. We have upgraded our live imaging microscopic and robotic capabilities. We have just completed evaluation of 4 high content imagers for our live imaging axodendritic neurodegeneration assays. Likewise, we are currently evaluating label free systems to quantitate ligand-protein and protein-protein. NTDU has become a repository for the NINDS SMA collection of compounds, and will curate this collection in coordination with extramural NINDS. We have queried and negotiated with numerous chemical companies about obtaining their chemical screening libraries. We have contacted 12 NINDS investigators about potential collaborations with their translational projects. Some of these projects included developing automated screening assays, production and purification of recombinant proteins, chemical library procurement, new compound synthesis, solubility and preformulation work, LCMS methods development, developing PET imaging agents, pharmacokinetic analysis and metabolite profiling. Many of these projects with multiple investigators will continue in the coming year. The goals of Neural Differentiation Unit are 1. To develop models using induced pluripotent stem cells (iPSC) and induced neural stem cells (iNSC) from cells of human patients to identify novel genetic, epigenetic and other molecular targets for novel diagnosis and therapeutic interventions; 2. To study neurogenesis and neurotoxicity in mixed human T cells and neural cell cultures as a model for neuroinflammation; 3, to facilitate the use of our established models in basic and preclinical studies of neuroimmunology in TNC, NINDS and other NIH investigators. During the past year, the neural differentiation unit has made significant progress, which is summarized under the following specific aims:
Specific aim 1 : Derive and characterize neural cells from human adult peripheral CD34+ cells. To develop the disease/patient specific in vitro neural models from other cell types which can be easily accessed, we used Sendai virus vectors containing Sox-2, C-myC, Klf-4 and Oct3/4 to transfect CD34+ cells purified from adult peripheral blood. By culturing the transfected cells in embryonic stem cell media and/or neural stem cell media, we successfully derived iPS and neural stem cells from CD34+ cells. Especially for iNSC, by bypassing the iPSC stage, which can be generated as fast as 2 weeks after blood collection, and hence should result in substantial cost savings. We further characterized the iNSC by immunostaining. We found these cells are positive for neural stem cell markers SOX-2, nestin, PAX-6 but negative for iPSC marker OCT4, confirming their neural stem cell identity. So far, from the iNSC, we have generated action potential firing neurons, GFAP positive astroglial cells, and O4 positive oligodendrocytes.
Specific aim 2 : Create an autologous model of neuroinflammation by co-culturing CD34-generated iNSC and T cells. Since lymphocytes can be simultaneously obtained during the same blood draw, we established an autologous in vitro model of neuroinflammation by co-culturing activated T cells with iNSC generated from CD34+ cells from the same donor. This represents a major advancement in the field since until now the questions of cell to cell contact between lymphocytes and brain cells could not be studied as an allogenic system would have to be used and there would be issues of MHC mismatch. We found that when activated by CD3/CD28 antibodies, autologous T cells formed a tight interaction with iNSC by adhering on them, resulting in inhibited proliferation and neuronal differentiation and possible neurotoxicity. We have further characterized the model using ion abrasion electron microscopy to get a three dimensional image of the cellular interactions, which confirmed the activated T cells formed an active binding with underlying iNSC. Our model will benefit the field of neuroinflammatory studies.
Specific aim 3 : study the effect of Herv K on iPS development. To support a main object of the Center of Translational Neuroscience, Neuropathogenesis of Retroviral Infections, we studied Herv K expression and function in iPSC, iNSC and neurons. Using quantitative PCR, we found that Herv K components, gag, env and pol expressions were increased in only iPSC but diminished rapidly after differentiation. The Herv K env expression was also increased when CD34 cells and fibroblasts were transfected with stem cell transcriptional factors Oct3/4, Sox-2, Klf-4 and C-myc, indicating HervK activation in stem cells. The expression of HervK env protein on iPSC was confirmed by using immunostaining and Western-blot. We are studying the functions of HervK proteins on stem cells by inhibit env and gag expression using siRNA and to determine their effects on the cell proliferation and differentiation.
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