Specific aim 1: Use in vitro 3D brain organoids derived from human adult peripheral CD34+ cells to study neural differentiation. The in vitro neurogenesis and development model can also be used to study the mechanism of neurological disorders. In collaboration with Dr. Mary Kay Floeter, we derived iPSC cells from blood samples from patients with primary lateral sclerosis (PLS) and age-matched healthy donors. We further compared the 3D brain organoids derived from these iPSCs. We found that after 2 weeks of the culture, 3D organoids derived from PLS were significantly bigger in size compared with their age matched controls. Furthermore, the 3D organoids from PLS were more symmetric shaped, compared with the controls, which showed asymmetric growth, likely due to cell differentiation. This was confirmed by gene expression study which showed that the expression levels of genes associated with neural differentiation such as neural stem cell marker nestin, neuronal marker MAP-2, glutaminergic neuronal marker GAD and astroglial marker GFAP were all lower in PLS organoids compared with the healthy controls. However, although the absolute level was extremely low, an increase of ChAT expression was observed in the PLS 3D organoids. ChAT is a marker for peripheral motor neurons, which is spared to damage in PLS which only affect primary motor neurons. Our observation that in the brain organoids derived from PLS patients, there was a general delay of neural development in central neural cell types but not in peripheral motors, agreed with PLS pathogenesis which was specific to central nerve system. As we have shown, there was difference in DNA methylation levels between neural stem cells derived from PLS and healthy donors. It is likely that the lower DNA methylation levels in PLS neural stem cells may delay neural differentiation. While there were not enough forces to push the central neural development, as a secondary pathway, motor neuron differentiation may occur, as suggested by other researchers. We have presented the result on the annual international society of stem cell research meeting in Boston.
Specific aim 2 : Study the role of astroglia in the pathogenesis of neural disorders. We used the same 3D model to study the role of astroglia played in the pathogenesis of another motor neuron disorder: autosomal-dominant frontotemporal dementia (FTD), in collaboration with Dr. Michael Ward. It was recently discovered that patients with FTD may be caused by granulin (GRN) mutation. To address the mechanism, Dr. Ward has been generating FTD models with GRN gene modifications in human iPSC-derived neurons. We used the iPSCs for the 3D brain organoids culture and found that GRN mutation resulted in smaller organoids in size and significantly lower gene expression for astroglial markers. Although the number of neurons was higher in the GRN-mutated 3D organoids, due to the lack of astroglia, which may play a protective role to neurons, damage was shown in the neurons as indicated by immunostaining images. This result indicates that GRN mutation may result in defect of astroglial differentiation, thus causing secondary neuronal damage. These results showed that the 3D model could be a very useful tool in modeling neurodegenerative disorders, determining the roles the different cell types may have played. Further study on the pathogenesis based on the model is ongoing.
Specific aim 3 : Study the role of HERV-K on human neural development. We have found that human endogenous retroviruses K (HERV-K) is expressed on human iPSC. Inhibition of HERV-K Env protein enhanced neuronal differentiation, indicating HERV-K plays a role in keep the stem cells from differentiation. We also determined that Ch22 be the likely loci for the HERV-K activation in human iPSCs. Based on the HERV-K gene sequence of Ch22, four specific siRNAs targeting HERV-K Env was designed and synthesized. The siRNAs were used to inhibit the expression of HERV-K Env in four iPSC lines. We found the siRNAs efficiently decreased HERV-K env expression in three of the four iPSC lines. In the affected three lines, Oct-4, a marker for pluripotent stem cells, was also decreased. The result further confirmed our hypothesis that HERV-K activation in iPSCs is important in maintaining the stemness of iPSCs. One of the iPSC line was not affected by siRNA indicates that in this specific human, there may be different gene patterns, thus HERV-K gene polymorphism may play a role in regulating neural differentiation. We collected the total RNA samples from the siRNA treated cells for RNA-seq analysis to study the genes that were affected by HERV-K inhibition. The result confirmed that 27 genes were significantly increased and 25 genes were significantly decreased after siRNA treatment compared to the control cells. These genes were involved in several critical molecular pathways, including DNA methylation, RNA transcriptional regulation and others specifically associated with stem cell function and differentiation. These results provide a clear mechanism by which HERV-K components used in maintaining the stemness of iPSC. By targeting the HERV-K associated pathways, we may develop novel methods to regulate neural differentiation and even treatment for neurodegenerative disorders and brain tumors where HERV-K is involved. We have submitted an abstract based on the finding to the 2017 Retropath Symposium.
Specific aim 4 : Study the effect of aging on central nervous system using iNSCs directly derived from CD34 cells. We found that the iNSCs we generated from cord blood CD34 cells produced much higher level of TIMP2 compared to the iNSCs generated from CD34 cells from adult donors. TIMP2 is a protein that has been reported to decrease during aging and has trophic effect on neurons. Our observation indicated that iNSCs, directly generated from CD34 cells, may maintain the epigenetic information, at least partially from the aging CD34 cells. This is different from CD34-derived iPSC, which have lost the aging information during cell transformation process. We are in collaboration with Dr. Clive Svendsen from Cedars-Sinai Medical Center to further study if the neurons derived from our iNSC from CD34 cells still maintain the epigenetic signature of the aging CD34 cells. If confirmed, our iNSC model will be very useful to study age-related neurodegenerative disorders.
Specific aim 5 : facilitate the research and therapeutic developments for neurological disorders using our models and methods. We are in collaboration with other investigators by providing training of the iNSC/iPSC generation or with cells. We provided iPSC generation training to Dr. Yogita K. Adlakha and Dr. Ashiwani Choudhary from Centre for Neuroscience, Indian Institute of Science. We provide consultancy to Dr. Henry Levi on developing a project studying the retroelements in neurological disorders. In collaboration with Dr. James Pickel, we tested the function of neural generation in the activation of transfected factors. Dr. James has created a marmoset with a CMV-driven transgenic fluorescent maker intend to have a ubiquitous expression on Marmoset tissues. Although fibroblasts derived from the animal skin lost fluorescent expression after passages, it was not known whether neurons express the signal or not. CMV promotor has been reported to have variable regulatory powers in different tissues. We transfected the fibroblasts with pluripotent stem cell factors to generate neural stem cells. We found after transfection, fluorescence was again observed in neuronal like cells derived from the fibroblasts. This indicated that although fibroblasts are not express the marker, once neurons are differentiated, they may still express the maker. This provide another way to answer the question without killing the animals or by doing biopsy.
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