1.Establish neurogenesis models using Induced Pluripotent Stem cells (iPSC) and Induced Neural Stem Cells (iNSC) derived from patients for identifying novel genetic, epigenetic and other molecular targets for novel diagnosis and therapeutic interventions. -Screening assays for neurogenesis and neurotoxicity in human mixed T cells and neural cells co-cultures. -Identify and study the mechanisms of inflammation on neurogenesis and neurotoxicity. 2. Facilitate the utilization of our established iNSC models in basic and preclinical studies of neurological disorders by TNC, NIH and extramural investigators.
Specific aim 1 :Establish in vitro neurogenesis and development models using neural cultures derived from human adult peripheral CD34+ cells. We established two novel in vitro models representing neural development. 1) By using specific motor neural induction medium, we can now derive motor neural progenitor cells directly from human blood. To accomplish this, we first purified CD34 cells from human peripheral blood samples. Then the CD34 cells were infected with Sendai virus containing transcriptional factors Oct4, Sox2, C-Myc and Klf4. The transfection of the factors in CD34 cells activate the cells to an unstable status with the potential to transform to a variety of stem cell types. By using specific medium to facilitate motor neuronal development, we can now get HB9 positive (a marker for motor neural progenitor cells) neurospheres 10 days after the transfection. The motor neural progenitor cells can then be further differentiated to Islet-1 and Chat positive motor neurons in a week. This method provides a rapid process to derive motor neurons from patient samples and could be useful for a rapid genetic/epigenetic test/study in certain motor neuron degenerative disorders. An abstract based on this method has been submitted and will be presented on the neuroscience meeting 2016 in San Diego. 2) Brain-like organoids cultured in suspension. While most of the brain organoids are started with iPSC/ES cells, we established a brain-like organoids cultured in suspension using the induced neural stem cells (iNS) directly derived from human CD34 cells. Using semi-solid collagen as substrate, we seeded iNS and expended them to spheres which were further cultured in neural differentiation medium. The asymmetry neural differentiation with empty spaces within were observed in the organoids. This method provides a novel model to use for the study of neural development. We are using this model to study ZIKA virus-induced microcephaly.
Specific aim 2 : Study the mechanism of inflammation in neural degeneration using T cell and neural co-cultures. We continued to use the iNS model to study the mechanism of inflammation in neurodegeneration. Part of the work was presented as an oral presentation: in vitro modeling of neuroinflammation using neural stem cells derived from CD34 cells from patients with undiagnosed rare diseases in the Stem Cell Models of Neural Regeneration and Disease International Symposium 1-3 February, Dresden, Germany. We are also using the iNS as an in vitro model to study the effect of ZIKA virus. We repeated and confirmed the inhibitory effect of ZIKA virus on iNS. By using Electrical Microscopic scanning, we found welling mitochondrial in the ZIKA virus infected iNS, indicating a possible mechanism for ZIKA neurotoxicity.
Specific aim 3 : Study the effect of HERV K on pluripotent stem cell development. We continued the study of the effect of HERV-K Env on the pluripotent stem cells and neural differentiation. We transfected iPSC with plasmid containing HERV-K Env. The forced HERV-K Env expression during the induced neural differentiation inhibited neural induction process, suggesting an important role HERV-K may have played in stem cell and human neurological system development. Using RNAseq, we also determined that Ch22 be the likely loci for the HERV-K activation in iPSC. A manuscript is ready for submission based on these findings.
Specific aim 4 : Study the epigenetic mechanisms of neurodegenerative disorders. Heterochromatin is the major form of chromatin in higher eukaryotes that impacts various chromosomal processes including genomic stability and global gene expression patterns. Interestingly, it was found that heterochromatin also formed in specific human cell types such as neuronal stem cells and that those genes are associated with various human diseases. We are in collaboration with Dr. Nathan Lee and Dr. Shiv Grewal to provide them with human iNS and neural stem cells derived from embryonic stem cells to study the role of heterochromatin in regulation of genes and diseases in neuronal stem cells. We are also in collaboration with Drs. Dax Hoffman, Tao Wang and Jiahua Hu to study the effect of a familial mutation of Preso1 gene on human neuronal functions. Previous animal studies have found that genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, resulting in sustained, mGluR5-dependent inflammatory pain in mice. We have generated three iPSC lines from patients with a control line from a healthy family member for this project. Neurons will be differentiated from iPSCs to test the effect of Preso1 gene mutation in human.
Showing the most recent 10 out of 15 publications
