Accumulating evidence has recognized that cancer-associated fibroblasts (CAFs) are major players in the desmoplastic stroma of ovarian cancer, modulating tumor progression and therapeutic response. However, it is unclear regarding the signatures of CAFs could be utilized to predict the clinical outcomes of ovarian cancer patients. To fill in this gap, we explored the intratumoral compartment of ovarian cancer by analyzing the single-cell RNA-sequencing (scRNA-seq) datasets of ovarian carcinoma samples, and identified two distinct CAFs (tumor-promoting CAF_c1 subtype and myofibroblasts-like CAF_c2 subtype). The clinical significance of CAF subtypes was further validated in The Cancer Genomics Atlas (TCGA) database and other independent immunotherapy response datasets, and the results revealed that the patients with a higher expression of CAF_c1 signatures had a worse prognosis and showed a tendency of resistance to immunotherapy. This work uncovered the signatures of the CAF_c1 subtype that could serve as a novel prognostic indicator and predictive marker for immunotherapy.

UNASSIGNED: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.
UNASSIGNED: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.
UNASSIGNED: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.
UNASSIGNED: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

OBJECTIVE: The multiple vital functions of the human liver are performed by highly specialised parenchymal and non-parenchymal cells organised in complex collaborative sinusoidal units. Although crucial for homeostasis, the cellular make-up of the human liver remains to be fully elucidated. Here, single-cell RNA-sequencing was used to unravel the heterogeneity of human liver cells, in particular of hepatocytes (HEPs) and hepatic stellate cells (HSCs).
METHODS: The transcriptome of ~25,000 freshly isolated human liver cells was profiled using droplet-based RNA-sequencing. Recently published data sets and RNA in situ hybridisation were integrated to validate and locate newly identified cell populations.
RESULTS: In total, 22 cell populations were annotated that reflected the heterogeneity of human parenchymal and non-parenchymal liver cells. More than 20,000 HEPs were ordered along the portocentral axis to confirm known, and reveal previously undescribed, zonated liver functions. The existence of 2 subpopulations of human HSCs with unique gene expression signatures and distinct intralobular localisation was revealed (i.e. portal and central vein-concentrated GPC3 + HSCs and perisinusoidally located DBH + HSCs). In particular, these data suggest that, although both subpopulations collaborate in the production and organisation of extracellular matrix, GPC3 + HSCs specifically express genes involved in the metabolism of glycosaminoglycans, whereas DBH + HSCs display a gene signature that is reminiscent of antigen-presenting cells.
CONCLUSIONS: This study highlights metabolic zonation as a key determinant of HEP transcriptomic heterogeneity and, for the first time, outlines the existence of heterogeneous HSC subpopulations in the human liver. These findings call for further research on the functional implications of liver cell heterogeneity in health and disease.
BACKGROUND: This study resolves the cellular landscape of the human liver in an unbiased manner and at high resolution to provide new insights into human liver cell biology. The results highlight the physiological heterogeneity of human hepatic stellate cells.

Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellular heterogeneity emerges from developmental processes by which multipotent progenitor cells make fate decisions and transition to specific cell types through intermediate cell states. Although genetic experimental strategies such as lineage tracing have provided insights into cell lineages, recent developments in single-cell technologies have greatly increased our ability to interrogate distinct cell types, as well as transitional cell states in tissue systems. From single-cell data that describe these intermediate cell states, computational tools have been developed to reconstruct cell-state transition trajectories that model cell developmental processes. These algorithms, although powerful, are still in their infancy, and attention must be paid to their strengths and weaknesses when they are used. Here, we review some of these tools, also referred to as pseudotemporal ordering algorithms, and their associated assumptions and caveats. We hope to provide a rational and generalizable workflow for single-cell trajectory analysis that is intuitive for experimental biologists.