Research Profile
Winter 2012
Principal Investigators:Katerina Semendeferi, Alysson Muotri, Fred Gage
Exploring Human-Chimpanzee Neuronal Differences Using Pluripotent Stem Cells
Accumulating evidence suggests that the evolution of the human brain, after the split from the common ancestor with the chimpanzees, was accompanied by discrete modifications in local circuitry and interconnectivity of selected parts of the brain. These selective changes may have occurred in specific parts of the cortex and/or selected subcortical structures. Our research aims to identify and characterize a neuronal subpopulation that we believe will differ between humans and chimpanzees. We isolate morphologically distinct cortical neurons from humans and chimpanzees from brain tissue using laser-capture procedures. The expression profile of small RNAs from both human and chimpanzee neurons are compared using deep sequencing and bioinformatics analysis. Unique candidates are used to model genetic reporter systems to allow the identification of the putative human specific neuronal population. Modeling neuronal differentiation will be achieved from induced pluripotent stem cells, generated from human and chimpanzee somatic cells. It is our expectation that this work will lead to novel knowledge regarding the developmental and evolutionary of human specific neuronal populations.
Over the past year we have completed a series of experiments to begin to explore the components needed to "reverse engineer" pluripotent stem cells in an evolutionary relevant manner. The goal of this project is to identify, characterize and compare neural subpopulations in humans and chimpanzees in associated brain structures using novel approaches. The main aims are: (I) Golgi staining and characterization of the cells in postmortem tissue; (II) Laser capture and molecular profile of the cells, and (III) Modeling using human and chimp induced pluripotent stem cells (iPSC).
Over the past year, we successfully stained several samples of chimp and human tissue that were determined to be suitable for tracing and quantification. Tissue blocks were selected from Broca's area (BA 44/45) and occipital cortex. We used a small piece of chimp postmortem brain tissue from our great ape brain collection and mouse brain tissue that were stored under equivalent conditions. RNA extraction and isolation were successful and sufficient for deep sequencing. To measure RNA yield and quality, RNA samples were analyzed using the NanoVue and gel electrophoresis. In the chimp brain, most of the RNA was localized in small fragments. However, the amount of RNA was still appropriated for deep-sequencing experiments. Laser Capture Microdissection was performed at the Center for Cytometry and Molecular Imaging (CCMI) facility at the Salk Institute. Approximately 1000 Layer III pyramidal neurons were captured for RNA extraction and isolation.
Further optimization of the technique was postponed until chimpanzee tissue blocks were available for thin section preparations. A new set of samples from chimpanzee tissue totaling 10,000 cells (approximately 2500 cells from four ROIs) have been collected. Thin section preparations (10 µm) appeared to improve the quality of laser capture. However, we are awaiting results of the RNA yield from this experiment before determining the best course of optimization (e.g., further increase in initial cell numbers). We plan to continue to further the aim of the RNA extraction deep sequencing in postmortem brain tissue from human, chimpanzee, gorilla and orangutan.
Overall the Kavli support jumpstarted an exciting long term collaboration that involves cross training of students in the graduate programs of Anthropology and Molecular Biology at UCSD and additional pilot experiments for more extramural grant applications. We are in the process of reapplying to the National Science Foundation. We are grateful to the Kavli Foundation for the support.
