Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage.

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors.

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract.

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.

Nrf2 and SQSTM1/p62 jointly contribute to mesenchymal transition and invasion in glioblastoma.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.