Spatial-temporal epigenetic profiling based on high-throughput single cell Intracellular biopsy
The capability to profile small RNAs responsible for post-transcriptional regulation of genes expression is essential for molecular identification and characterization of cellular phenotypes. However, compared to long RNA, the quantification of small RNA, such as microRNAs (miRNA), in live cells is still challenging. Also, miRNAs are suggested to play important roles in the pathogenesis and progress of human diseases with heterogenous regulation in different types of cells. However, limited technique is available for profiling miRNAs with both expression and spatial dynamics. Here, we describe an intracellular biopsy technique for fast, multiplexed and highly sensitive profiling of miRNAs. The technique employs an array of diamond nanoneedles that are functionalized with size-dependent RNA-binding-proteins, working as the “fishing rods” to directly pull multiple miRNA targets out of cytoplasm while keeping the cells alive. Each nanoneedle works as a separated reaction chamber for parallel in-situ amplification, visualization and quantification of miRNAs as low as femtomolar, which is sufficient to detect miRNAs of a single copy intracellular abundance and is also specific to single-nucleotide variation in closely-related miRNA sequences. The platform was further developed for multiplexed in situ miRNA profiling in acute tissue slices to achieve a quasi-single-cell analysis in a large population of mixed cells of a tissue sample. In addition to a quantitative evaluation of the expression level of particular miRNAs, the technique also provides the relative spatial dynamics of the cellular miRNAs in associated cell populations, which was demonstrated to be useful in analyzing the susceptibility and spatial reorganization of different types of cells in the tissues from normal or diseased animals. Using inCell-Biopsy, we analyze the temporal miRNA transcriptome over the differentiation of embryonic stem cells (ESCs) towards motor neurons. The combinatorial miRNA expression patterns derived by inCell-Biopsy identifies defined cell subpopulations resulted from ESC differentiation, and also reveals the dynamic evolution of cellular heterogeneity. In a MK-801-induced schizophrenia model, we found that astrocytes, instead of neurons, are more heterogeneously affected in the hippocampus of rats underwent repeated injection of MK-801, showing an expression fingerprint related to differentially down-regulated miRNA-135a and miRNA-143; the associated astrocyte subpopulation is also more spatially dispersed in the hippocampus, suggesting an astrocyte dysregulation in the induced schizophrenia animals.
Dr. Peng Shi is a professor in the Department of Biomedical Engineering at City University of Hong Kong. He received his bachelor’s degree in electrical engineering from Wuhan University and a Ph.D. degree in Biomedical Engineering from Columbia University. After his postdoctoral training at MIT in Electrical Engineering and Biological Engineering, he joined CityU Hong Kong and has been a faculty member in the BME department since 2011. Dr. Shi works at the convergence between neuroscience and engineering by taking advantage of an interdisciplinary approach that involves nano-/micro-fabrication, microfluidics, ultra-fast optics, high-resolution microscopy and imaging processing. He focuses on solving important emerging problems in translational neuro-engineering, especially in the development of high-throughput neuro-technology and screening platform for discovery of novel therapeutic targets. His work has led to more than 60 publications in top-tier research journals, including Nature BME, Science Advances, Nature communications etc., and 7 international patents and disclosures, one of which has been the foundation technology of a spin-off biotech company. Dr. Shi received the Simon’s research award in 2010, and was elected to the 1000 China Young Talent program. He also received the President Award for research excellence in 2017, outstanding supervisor award in 2018 at CityU, and a special recognition as Young Scholars by World Cultural Council in 2018. He is an associate editor for the journal Brain Research.