Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. METHODS: We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. RESULTS: B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. CONCLUSIONS: We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

Single cell transcriptomic landscape of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Endothelial Reprogramming by Disturbed Flow Revealed by Single-Cell RNA and Chromatin Accessibility Study.

Disturbed flow (d-flow) induces atherosclerosis by regulating gene expression in endothelial cells (ECs). For further mechanistic understanding, we carried out a single-cell RNA sequencing (scRNA-seq) and scATAC-seq study using endothelial-enriched single cells from the left- and right carotid artery exposed to d-flow (LCA) and stable-flow (s-flow in RCA) using the mouse partial carotid ligation (PCL) model. We find eight EC clusters along with immune cells, fibroblasts, and smooth muscle cells. Analyses of marker genes, pathways, and pseudotime reveal that ECs are highly heterogeneous and plastic. D-flow induces a dramatic transition of ECs from atheroprotective phenotypes to pro-inflammatory cells, mesenchymal (EndMT) cells, hematopoietic stem cells, endothelial stem/progenitor cells, and an unexpected immune cell-like (EndICLT) phenotypes. While confirming KLF4/KLF2 as an s-flow-sensitive transcription factor binding site, we also find those sensitive to d-flow (RELA, AP1, STAT1, and TEAD1). D-flow reprograms ECs from atheroprotective to proatherogenic phenotypes, including EndMT and potentially EndICLT.

Photopolymerized microdomains in both lipid leaflets establish diffusive transport pathways across biomimetic membranes.

Controlled transport within a network of aqueous subcompartments provides a foundation for the construction of biologically-inspired materials. These materials are commonly assembled using the droplet interface bilayer (DIB) technique, adhering droplets together into a network of lipid membranes. DIB structures may be functionalized to generate conductive pathways by enhancing the permeability of pre-selected membranes, a strategy inspired by nature. Traditionally these pathways are generated by dissolving pore-forming toxins (PFTs) in the aqueous phase. A downside of this approach when working with larger DIB networks is that transport is enabled in all membranes bordering the droplets containing the PFT, instead of occurring exclusively between selected droplets. To rectify this limitation, photopolymerizable phospholipids (23:2 DiynePC) are incorporated within the aqueous phase of the DIB platform, forming conductive pathways in the lipid membranes post-exposure to UV-C light. Notably these pathways are only formed in the membrane if both adhered droplets contain the photo-responsive lipids. Patterned DIB networks can then be generated by controlling the lipid composition within select droplets which creates conductive routes one droplet thick. We propose that the incorporation of photo-polymerizable phospholipids within the aqueous phase of DIB networks will improve the resolution of the patterned conductive pathways and reduce diffusive loss within the synthetic biological network.