The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells. Single-cell RNA-seq of SFTPC cells and their progenitors demonstrated that their differentiation process and cellular heterogeneity resembled those of developing AT2 cells in vivo. AOs were applicable to drug toxicology studies recapitulating AT2-cell-specific phenotypes. Our methods can help scientists overcome the limitations of current approaches to the modeling of human alveoli and should be useful for disease modeling and regenerative medicine.

Here, we review single-cell sequencing techniques for individual and multiomics profiling in single cells. We mainly describe single-cell genomic, epigenomic, and transcriptomic methods, and examples of their applications. For the integration of multilayered data sets, such as the transcriptome data derived from single-cell RNA sequencing and chromatin accessibility data derived from single-cell ATAC-seq, there are several computational integration methods. We also describe single-cell experimental methods for the simultaneous measurement of two or more omics layers. We can achieve a detailed understanding of the basic molecular profiles and those associated with disease in each cell by utilizing a large number of single-cell sequencing techniques and the accumulated data sets.

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DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis’ extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.