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Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single‐cell RNA‐sequencing (scRNA‐seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets – fibroblast progenitors and stem cells – indicative of an impairment of normal tissue self‐renewal. Tissue‐specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue‐remodeling. In degenerated AF, a novel disease‐associated subset is identified, which expresses disease‐promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFβ/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue‐specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment. Intervertebral (IVD) cells maintain tissue structure and biomechanical function while their depletion and dysfunction are key mechanisms in IVD degeneration and back pain. Using scRNA‐seq, cell type and compartment‐specific are resloved and shared mechanisms and their transcriptional regulators that are dysregulated in disease. These findings define novel genes and gene regulatory networks that enable more precise therapeutic targeting for the prevention and treatment of intervertebral disc degeneration.

Tumor‐infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA‐seq (scRNA‐seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early‐stage non‐small cell lung cancer (NSCLC) patients. Our scRNA‐seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor‐mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte‐to‐M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF‐α signaling via NF‐κB and inflammatory response). Our analysis further identified a co‐regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte‐to‐M2 differentiation, and activated ligand‐receptor interactions (eg, SFTPA1‐TLR2, ICAM1‐ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte‐to‐M2 differentiation. Overall, our study identified the prevalent monocyte‐to‐M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand‐receptor interactions. Single‐cell RNAseq reveals the transcriptional path from monocyte to M2 macrophage. M2 differentiation in NSCLC is driven by a multifaceted co‐regulatory network. Epithelial‐immune cell ligand‐receptor interactions associate with M2 differentiation

Plant anthers are composed of different specialized cell types with distinct roles in plant reproduction. High temperature (HT) stress causes male sterility, resulting in crop yield reduction. However, the spatial expression atlas and regulatory dynamics during anther development and in response to HT remain largely unknown. Here, the first single‐cell transcriptome atlas and chromatin accessibility survey in cotton anther are established, depicting the specific expression and epigenetic landscape of each type of cell in anthers. The reconstruction of meiotic cells, tapetal cells, and middle layer cell developmental trajectories not only identifies novel expressed genes, but also elucidates the precise degradation period of middle layer and reveals a rapid function transition of tapetal cells during the tetrad stage. By applying HT, heterogeneity in HT response is shown among cells of anthers, with tapetal cells responsible for pollen wall synthesis are most sensitive to HT. Specifically, HT shuts down the chromatin accessibility of genes specifically expressed in the tapetal cells responsible for pollen wall synthesis, such as QUARTET 3 (QRT3) and CYTOCHROME P450 703A2 (CYP703A2), resulting in a silent expression of these genes, ultimately leading to abnormal pollen wall and male sterility. Collectively, this study provides substantial information on anthers and provides clues for heat‐tolerant crop creation. Anther development and function have central role in plant reproduction. Here, the first exploration of the spatial expression profiles and regulatory dynamics of different cell types during cotton anther development and in response to high temperature open a new chapter in plant anther research and provides crucial clue for breeding heat‐tolerant crops.

Breast cancer (BRCA) is characterised by complex cellular heterogeneity and differentiation hierarchies, which play a crucial role in bone metastasis and therapeutic resistance. However, existing classification systems remain inadequate in capturing these complexities, limiting their effectiveness in guiding treatment strategies. To address this gap, we integrated single‐cell RNA‐seq profiles, spatial transcriptomes, along with 1097 bulk RNA‐seq profiles of TCGA‐BRCA cohort to dissect the molecular landscape of BRCA. By performing UMAP analysis, we identified nine tumour clusters and three spatially distinct spot types (immune, stromal and malignant spots) and further delineated 11 differentiation states from 2493 malignant spots. Through clustering, monocle 2 pseudo‐time and prognostic analyses, we identified the prognostic BRCA cell fate‐related genes, then constructed a novel BRCA stratification system (four subtypes) with differential prognosis, biological plausibility and clinical significance. Also, least absolute shrinkage and selection operator (LASSO) regression analysis was performed for the BRCA cell fate‐related genes in constructing a prognostic model. The model has modest accuracy and accordance (AUC = 0.708), which could distinguish BRCA patients into high‐risk or low groups. With correlation analysis, regulation networks were constructed for different subtypes based on the key cell fate‐related genes, transcription factors, metastasis‐related pathways, immune components and so on, to investigate the regulatory relationships between primary BRCA and BRCA bone metastasis. Afterwards, we identified the most significant inhibitors (puromycin, MS‐275, megestrol, aesculetin) for bone metastatic BRCA, which might have potential translational significance. In all, we developed a novel molecular stratification system for BRCA based on the cell fate‐related markers of malignant cells, which offered strong translational potential for diagnosis, prognosis and personalised therapeutic interventions.

Human amniotic epithelial cells (hAECs) have shown excellent efficacy in clinical research and have prospective applications in the treatment of many diseases. However, the properties of the hAECs and their proliferative mechanisms remain unclear. Here, single‐cell RNA sequencing (scRNA‐seq) is performed on hAECs obtained from amniotic tissues at different gestational ages and passages during in vitro culture. The results showed that the proliferation of hAECs is associated with epithelial‐mesenchymal plasticity (EMP) during amniogenesis. Freshly isolated, full‐term hAECs are identified as mature epithelial cells. Once cultured in vitro, they are observed to rapidly undergo epithelial‐mesenchymal transition (EMT) and enter a partial epithelial‐mesenchymal transition (pEMT) state to regain their EMP properties and proliferation capacities. With the continuous development of EMT, hAECs eventually enter a senescent state. The addition of SB431542 and microcarrier screening enabled the effective 3D expansion of hAECs by 50 fold while maintaining the EMP status in hAECs for further proliferation. This study not only elucidated the central proliferation mechanism of hAECs during development and expansion but also optimized the in vitro culture system so that it is sufficient to generate hAECs for 50 patients from a single donor amniotic membrane. Freshly isolated human amniotic epithelial cells (hAECs) from full‐term amniotic membranes include mature epithelial cells as well as cells in a senescent state. During in vitro culture, hAECs undergo epithelial‐mesenchymal transition (EMT) and enter a partial epithelial‐mesenchymal transition (pEMT) state (E/M hybrid) resembling early developmental cells, acquiring epithelial‐mesenchymal plasticity (EMP) properties and proliferative capabilities. As the EMT process progresses, cells may enter a senescent state. Inhibiting the EMT process with SB431542 after cells have acquired EMP properties is beneficial for maintaining EMP properties and proliferative capacity in hAECs, thereby enhancing the yield of hAECs.

The inherent heterogeneity of individual cells in cell populations plays significant roles in disease development and progression, which is critical for disease diagnosis and treatment. Substantial evidences show that the majority of traditional gene profiling methods mask the difference of individual cells. Single cell sequencing can provide data to characterize the inherent heterogeneity of individual cells, and reveal complex and rare cell populations. Different microfluidic technologies have emerged for single cell researches and become the frontiers and hot topics over the past decade. In this review article, we introduce the processes of single cell sequencing, and review the principles of microfluidics for single cell analysis. Also, we discuss the common high-throughput single cell sequencing technologies along with their advantages and disadvantages. Lastly, microfluidics applications in single cell sequencing technology for the diagnosis of cancers and immune system diseases are briefly illustrated.

Background The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of individual cells which would provide significant resolution. By integrating single cell RNA-sequencing (scRNA-seq) and population genetics, we apply a framework in which to evaluate cell type-specific effects of genetic variation on gene expression. Results Here, we perform scRNA-seq on 64,018 fibroblasts from 79 donors and map expression quantitative trait loci (eQTLs) at the level of individual cell types. We demonstrate that the majority of eQTLs detected in fibroblasts are specific to an individual cell subtype. To address if the allelic effects on gene expression are maintained following cell reprogramming, we generate scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type-specific eQTLs in iPSCs and show that the eQTLs in fibroblasts almost entirely disappear during reprogramming. Conclusions This work provides an atlas of how genetic variation influences gene expression across cell subtypes and provides evidence for patterns of genetic architecture that lead to cell type-specific eQTL effects.

Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the SPP1 hi and FABP4 hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. KRT5 - /KRT17 + aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.

The bone is an important organ that performs various functions, and the bone marrow inside the skeleton is composed of a complex intermix of hematopoietic, vascular, and skeletal cells. Current single-cell RNA sequencing (scRNA-seq) technology has revealed heterogeneity and sketchy differential hierarchy of skeletal cells. Skeletal stem and progenitor cells (SSPCs) are located upstream of the hierarchy and differentiate into chondrocytes, osteoblasts, osteocytes, and bone marrow adipocytes. In the bone marrow, multiple types of bone marrow stromal cells (BMSCs), which have the potential of SSPCs, are spatiotemporally located in distinct areas, and SSPCs’ potential shift of BMSCs may occur with the advancement of age. These BMSCs contribute to bone regeneration and bone diseases, such as osteoporosis. In vivo lineage-tracing technologies show that various types of skeletal lineage cells concomitantly gather and contribute to bone regeneration. In contrast, these cells differentiate into adipocytes with aging, leading to senile osteoporosis. scRNA-seq analysis has revealed that alteration in the cell-type composition is a major cause of tissue aging. In this review, we discuss the cellular dynamics of skeletal cell populations in bone homeostasis, regeneration, and osteoporosis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271 BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPR CD45 BM-MSCs within the CD271 BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.