Transient nuclear deformation initiates epigenetic states and facilitates cell reprogramming

At 23:00 Beijing time on August 4, 2022, Professor Li Song’s research group from the Department of Bioengineering at UCLA published a research result entitled “Transient nuclear deformation primes epigenetic state and promotes cell reprogramming” on Nature Materials.

The results report that suspension cells undergo a rapid squeeze of the nucleus as they pass through a narrow microfluidic channel, a reversible nuclear deformation process that can significantly reduce the methylation levels of histone H3K9 and DNA, thereby improving chromatin accessibility and ultimately promoting the reprogramming efficiency of fibroblasts to neuronal cells. The corresponding author of the paper is Professor Li Song, and the first author is Song Yang.

Cell reprogramming techniques can be used to obtain the cell type of interest needed for research or clinical use and have a wide range of applications in regenerative medicine, disease model building, and drug screening. Compared to classical multi-potential stem cell (iPSCs) reprogramming techniques, direct reprogramming can directly transform one adult cell into another desired functional cell type by circumventing the time-consuming differentiation process of stem cells, such as fibroblasts into induced neuronal (iN) cells. However, the inefficiencies of these conversion processes create obstacles for biomedical applications. A key step in direct cell reprogramming is to overcome the epigenetic barrier of heterochromatin and turn on endogenous genes for cell type conversion. Previous studies have focused on the role of transcription factors and biochemical factors in cell phenotypic transformation, but little is known about the effects on biophysical factors. Cells undergo mechanical stimulation on different time scales (from a few seconds to a few days) that may initiate force chemical signaling, cytoskeletal recombination, and chromatin remodeling. To directly determine the effect of nuclear deformation on chromatin remodeling, the authors investigated whether and how to regulate epigenetic status and cell reprogramming by squeezing suspended cells, and explored the feasibility of translating this mechanical stimulation method into industrial application.

In this study, Professor Li Song’s team constructed a microfluidic device with a microchannel array with a width of 7 μm through micromanufacturing technology, and allowed suspension fibroblasts transfected with neuronal-associated transcription factors (Brn2, Ascl1, Mytl1) to pass through the microchannel at a flow rate of 20 μL/min. The study found that fibroblasts whose nuclei were squeezed were 8 times more efficient at direct reprogramming into neuronal cells than in the control group.

Figure 1. After the nucleus is squeezed, the efficiency of direct reprogramming is significantly increased

An exploration of the epigenetic state of cells found that within 24 hours after the cells were squeezed transiently, the methylation level of histone H3K9 and DNA decreased significantly. At the same time, the mechanically induced decline in H3K9me3 and 5 m-C was basically consistent with the decline level after 24 h of combined treatment of cells by inhibitors. In addition, through collaboration with Professor Wang Yingxiao of the University of California, San Diego (UCSD), real-time observation of H3K9me3 levels with the help of live-cell FRET technology found that H3K9me3 levels decreased significantly within 1 minute of fibroblasts entering the microchannel.

Figure 2. Histone H3K9 and DNA methylation decrease after the nucleus is squeezed

In order to further promote the practical application of the above microfluidic devices, Professor Li Song’s team designed a high-throughput microfluidic chip, which can achieve the mechanical stimulation of millions of orders of magnitude cells in a short period of time. In addition, this microfluidic device can facilitate not only fibroblast reprogramming to neuronal cells, but also can not only facilitate the reprogramming of fibroblasts to multipotent stem cells (iPSCs) and macrophages to neuronal cells.

Figure 3. High-throughput chip design and multiple types of reprogramming practices

The study revealed that mechanical deformations of the nucleus affect chromatin tissue form and induce more open chromatin structures, which can be widely used in the field of gene editing. The study was supported by the National Institutes of Health (NIH) and the U.S. Natural Science Foundation. (Source: Science Network)

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