Just as there is no identical leaf in the world, there is no identical cell, either. Science renders it possible for us to “read” every single cell in a living creature.
A research team led by Professor GUO Guoji with ZJU's Center for Stem Cell and Regenerative Medicine published their research findings in the February 22 issue of Cell. The team developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, they analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA)—the world’s first mammalian cell atlas.
Single-cell RNA sequencing (scRNA-seq) technologies examines the sequence information from individual cells with optimized next generation sequencing (NGS) technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. They are poised to reshape the current cell-type classification system and promote the cognition of the secret of life.
“scRNA-seq technologies enable human beings to precisely analyze the separation, regeneration, aging and pathological changes of a single cell,” GUO said. “They are bringing a methodological revolution in cell detection, classification and identification in their wake.”
In October 2017, the United States brought together an international community of scientists with diverse expertise to launch a Human Cell Atlas Project. It aims at creating a comprehensive reference map of all human cells as a basis for understanding human health and diagnosing, monitoring, and treating disease. Very recently, new tools, such as single-cell genomics, have for the first time put this goal within reach.
“By utilizing the microporous matrix, molecular markers and genetic amplification, we are able to make a high-throughput and high-resolution analysis of single cells, thereby resolving such tough problems as insufficient nucleic acids and high costs,” said GUO.
In Microwell-seq, individual cells are trapped in an agarose microarray and mRNAs are captured on magnetic beads. Barcoded beads are synthesized by 3 split-pool rounds. Each oligonucleotide consists of a primer sequence, a cell barcode, a unique molecular identifier (UMI), and a poly T tail. Fabrication of the agarose microarray is simple and inexpensive. The silicon and PDMS chips are reusable, meaning that a single silicon chip can be employed to generate many agarose microarrays. The size of the agarose chip can be readily adjusted by making different-sized PDMS chips for a wide range of input sample sizes and concentrations. Only minutes are required to make an agarose chip for each experiment. An agarose plate with 105 microwells is used to trap 5–10 K individual cells. After cells (50–100 K) are loaded into the wells, the microwell array is inspected under a microscope and rare cell doublets are washed out with a capillary tube. Barcoded magnetic beads are then loaded and trapped into each well by size. Each single bead is conjugated with 107–108 oligonucleotides, which share the same cellular barcode. After incubation of beads and cells in a soft flow of lysis buffer, beads with captured mRNA are retrieved with a magnet. Amplified cDNA is fragmented by a customized transposase that carries two identical insertion sequences. The 3’ ends of the transcripts are then enriched during library generation using PCR and sequenced using the Illumina Hiseq platform.
The Microwell-seq method holds advantages over other related technologies, primarily related to cost and convenience. It is a portable, efficient, and inexpensive high-throughput scRNA-seq platform which will definitely promote the popularity and application of single-cell sequencing technologies in fundamental research and clinical diagnosis. “The cost of sequencing library generation for each cell is estimated to be reduced from roughly 100 yuan to less than 2 yuan,” said GUO.
Using Microwell-seq, researchers profiled more than 50 mouse organ, tissue, and cell lines, constructed a first stage “mouse cell atlas” with more than 400 k single-cell transcriptomic profiles and built the most comprehensive mammalian single-cell data resource to date.
For every organ and tissue, they profiled not only the tissue-specific cell lineages but also the tissue-resident stromal and immune cell types to provide information on tissue microenvironments. Relevant data are available on the MCA website (http://bis.zju.edu.cn/MCA/).
Studies revealed that stromal cells from different tissues are characterized by substantially different genetic expressions and play a crucial role in adjusting tissue-specific microenvironments. This has important implications. In repairing tissues and organs, scientists should repair both tissue cells and stromal cells.
The completion of MCA will be instrumental to the construction of the human cell atlas, thus benefiting varying fields, such as cell biology, developmental biology, neuroscience, hematology and regenerative medicine.