text | "China Science News" reporter Diao Wenhui
How does a fertilized egg develop into a complex individual before a baby is born? Why do normal cells slowly turn into cancer cells?
Cell is the basic unit of life. Understanding its past, present and future will not only help people understand the process of normal development, but is also crucial to understanding the occurrence and development of diseases.
However, there are still significant technical difficulties in "seeing" the "past and present" of cells.
html On August 17, Chen Wanze, a researcher at the Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, published a long article in "natural" as the co-first author, introducing the research team's internationally pioneered live cell transcriptome sequencing technology (Live -seq).
This technology allows single cells to remain viable for the first time after transcription and sequencing, enabling continuous observation of full gene expression in living cells.
"This study realizes the feasibility of using Live-seq technology to perform multiple transcriptome sequencing on multiple isolated parts of the cytoplasm of the same living cell, indicating that this technology is expected to be used in the future to construct the transcriptome of a single living cell series Change dynamics. This study provides a new research strategy for single-cell transcriptome sequencing and provides a powerful means for us to understand the dynamic changes of life processes. It is another major breakthrough in this field. " School of Life Sciences, Peking University. Professor Tang Fuxue commented.
Schematic diagram of live cell sequencing recording cell changes. Image source: Duygu Koldere Vilain
Sequencing without killing cells
Cells in the human body have almost the same genome, but why can they produce a variety of cells? The expression and level of tens of thousands of genes in the genome largely determine the type and function of cells, such as nerve cells, immune cells , various tumor cells, etc.
If we know the changes in gene expression of cells at different times, we can understand the past, present and future of cells.
Currently, single-cell transcriptome sequencing technology is an important means to understand the status of cells.
is like looking at a "high-definition photo". Through single-cell sequencing , you can clearly see the current expression status of all genes in the cell.
However, these techniques face great challenges in understanding the "movie" dynamic changes in cell states.
"The prerequisite for using single-cell transcriptome sequencing technology to observe cell status is to lyse the cells and extract the RNA to measure the expression level of each gene. This will inevitably kill the cells." Chen Wanze said, "In addition, Using single-cell sequencing technology, we can only understand the current state of a cell, but we cannot obtain its past, nor can we know its future function. "Yes."
Through nearly 7 years of hard work, Chen Wanze and his collaborators developed the live cell transcriptome sequencing technology Live-seq. The core is to perform minimally invasive extraction of part of the cytoplasm in living cells and analyze extremely small amounts of cytoplasmic RNA. Amplification is performed to maintain cell survival and function after single-cell transcriptome sequencing, thereby enabling tracking of dynamic changes in cells.
Corresponding author of the paper and Bart Deplancke, a professor at the Ecole Polytechnique Fédérale de Lausanne in Switzerland, said that this technology has both full gene expression resolution and dynamic analysis capabilities. It is currently the only direct and dynamic measurement of single-cell transcriptomes, coupled with the current status of cells and their The only solution for subsequent phenotypes.
hit the wall twice and finally "fished" out RNA
How to see the dynamic changes of cells without killing them?
"The first thing we think of is exosomes, which are small vesicles spit out by cells, which contain protein , RNA and other substances. If we collect all the exosomes from a single cell, and then analyze the RNA in them By measuring, it may be possible to reflect the cell status to a certain extent without killing the cells," said Chen Wanze.
There are only 10 picograms of RNA in a single cell, which is equivalent to one hundred billionth of 1 gram, and the RNA in cellular exosomes is even less.The
research team designed a microfluidic chip to complete processes such as single cell capture and exosome collection. They found that because the amount of RNA in exosomes was too small, it was impossible to observe single cell molecular levels.
Subsequently, Chen Wanze tried to use the atomic force microscope , which is very niche in the field of life sciences-it has a very sharp silicon probe, which is mostly used to detect the surface properties of substances. The
research team carried out surface activation, modification, elution and other modifications to the probe, allowing it to "fish" out RNA in cells.
"This probe is very thin and causes little damage to cells. It is like a 'fishhook'. After modification, it can 'fish' out the RNA in the cells and ensure that the cells continue to survive. We have modified dozens of After using the probe, the result was that the gene was only successfully 'caught' in two cells." Chen Wanze recalled that it cost $800 to purchase an atomic force microscope probe at that time. The research cost was too high and the success rate was too low, which made this research difficult. Blocked again.
The Swiss Federal Institute of Technology in Lausanne has a strong interdisciplinary atmosphere.
In an accidental academic exchange, Chen Wanze and his tutor learned that Julia Vorholt's laboratory at ETH Zurich, Switzerland, had developed a special atomic force microscope that could suck out a portion of the cytoplasm. After some exchanges between
, the two research groups hit it off and started joint research. The joint team optimized a series of experimental processes and solved various problems such as RNA degradation, rapid operation at low temperatures, ultra-micro sample transfer, sampling channel cleaning to avoid cross-contamination, and cell tracking under images to ensure the reliability of experimental results. . The
joint team used the re-engineered Live-seq to sequence a total of 295 cells of 5 types. They found that Live-seq can effectively distinguish different types of cells, and on average each cell can detect the expression of approximately 4112 genes. information.
only sequences a small amount of cytoplasmic , can it represent the status of the cell? "We compared the single-cell sequencing results in parallel and found that the live-cell sequencing results were highly consistent with the ordinary single-cell sequencing results, proving that Live-seq can well reflect the full transcriptome status of cells." Chen Wanze said. How to ensure the survival rate of
cells?
"The tip of the atomic force probe is only a few hundred nanometers in size, and can be sealed with the cell membrane, causing minimal damage to the cells. After absorbing about 5% to 50% of the cytoplasm, the cell volume can quickly return to normal levels, and the survival rate is 85% Between 89% and 89%, cells can divide normally. Through a series of functional analysis and molecular characterization, we found that Live-seq has no significant impact on cell status," said Chen Wanze.
In this regard, the reviewer also wrote in the review comments: "Because the cells still survive after sequencing, Live-seq achieves the continuous measurement of the entire gene expression of the same cell for the first time."
The history of cell sequencing starts from "high-definition pictures"
The leap to " high-definition movie "
In the history of cell observation technology, technologies such as microscopic imaging and gene editing -mediated molecular recording can not only observe processes such as growth, division, and death at the cell level, but also observe cells. single or several gene indicators.
In 2009, single-cell transcriptome sequencing technology provided a revolutionary means to define cell types and states more systematically and comprehensively.
However, people can still only observe the static state of cells, and cannot continuously observe cell dynamics or check the subsequent phenotype of cells.
If observing cells using single-cell transcriptome sequencing technology is likened to looking at a high-definition picture of cells at the molecular level, then using Live-seq to observe cells is like watching a high-definition movie, where you can see the "past and present life" of cells.
"Live-seq can answer how a cell's past determines its present. It not only knows why there are differences in cells, but also where these differences come from." Chen Wanze introduced.
In the verification experiment, the team used Live-seq to directly measure the state changes of in the same macrophage at different times, and found that the expression differences and noise of a few genes (such as Nfkbia, Gsn, etc.) in the initial state of the cell were determined An important reason for differences in subsequent cellular responses.
Relatively speaking, ordinary single-cell transcriptomes cannot find these patterns.
Chen Wanze said that although Live-seq still has many challenges and needs to be further improved, such as low throughput, it cannot be applied in vivo, and it is impossible to achieve whole-cell transcriptome sequencing in cells that are highly polarized and unevenly distributed mRNA. More sampling requires further research, but this technology has achieved continuous observation of living cells for the first time, bringing more possibilities to the development of single-cell sequencing technology. In the future, the team will further improve the practicality of Live-seq technology.
related paper information:
https://doi.org/10.1038/s41586-022-05046-9
