Long-term engrafting multilineage hematopoietic cells differentiated from human induced pluripotent stem cells.

Hematopoietic stem cells (HSCs) derived from human induced pluripotent stem cells (iPS cells) have important biomedical applications. We identified differentiation conditions that generate HSCs defined by robust long-term multilineage engraftment in immune-deficient NOD,B6.Prkdcscid Il2rgtm1Wjl/SzJ KitW41/W41 mice. We guided differentiating iPS cells, as embryoid bodies in a defined culture medium supplemented with retinyl acetate, through HOXA-patterned mesoderm to hemogenic endothelium specified by bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF). Removal of VEGF facilitated an efficient endothelial-to-hematopoietic transition, evidenced by release into the culture medium of CD34+ blood cells, which were cryopreserved. Intravenous transplantation of two million thawed CD34+ cells differentiated from four independent iPS cell lines produced multilineage bone marrow engraftment in 25-50% of immune-deficient recipient mice. These functionally defined, multipotent CD34+ hematopoietic cells, designated iPS cell-derived HSCs (iHSCs), produced levels of engraftment similar to those achieved following umbilical cord blood transplantation. Our study provides a step toward the goal of generating HSCs for clinical translation.

Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber.

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells In Vitro and In Vivo.

Thymic epithelium is critical for the structural integrity of the thymus and for T cell development. Within the fully formed thymus, large numbers of hematopoietic cells shape the thymic epithelium into a scaffold-like structure which bears little similarity to classical epithelial layers, such as those observed in the skin, intestine or pancreas. Here, we show that human thymic epithelial cells (TECs) possess an epithelial identity that also incorporates the expression of mesenchymal cell associated genes, whose expression levels vary between medullary and cortical TECs (m/cTECs). Using pluripotent stem cell (PSC) differentiation systems, we identified a unique population of cells that co-expressed the master TEC transcription factor FOXN1, as well as the epithelial associated marker EPCAM and the mesenchymal associated gene CD90. Using the same serum free culture conditions, we also observed co-expression of EPCAM and CD90 on cultured TECs derived from neonatal human thymus in vitro. Single cell RNA-sequencing revealed these cultured TECs possessed an immature mTEC phenotype and expressed epithelial and mesenchymal associated genes, such as EPCAM, CLDN4, CD90 and COL1A1. Importantly, flow cytometry and single cell RNA-sequencing analysis further confirmed the presence of an EPCAM+CD90+ population in the CD45- fraction of neonatal human thymic stromal cells in vivo. Using the human thymus cell atlas, we found that cTECs displayed more pronounced mesenchymal characteristics than mTECs during embryonic development. Collectively, these results suggest human TECs possess a hybrid gene expression program comprising both epithelial and mesenchymal elements, and provide a basis for the further exploration of thymus development from primary tissues and from the in vitro differentiation of PSCs.

Enduring epigenetic landmarks define the cancer microenvironment.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Combinatorial Ranking of Gene Sets to Predict Disease Relapse: The Retinoic Acid Pathway in Early Prostate Cancer.

BACKGROUND: Quantitative high-throughput data deposited in consortia such as International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) present opportunities and challenges for computational analyses. METHODS: We present a computational strategy to systematically rank and investigate a large number (210-220) of clinically testable gene sets, using combinatorial gene subset generation and disease-free survival (DFS) analyses. This approach integrates protein-protein interaction networks, gene expression, DNA methylation, and copy number data, in association with DFS profiles from patient clinical records. RESULTS: As a case study, we applied this pipeline to systematically analyze the role of ALDH1A2 in prostate cancer (PCa). We have previously found this gene to have multiple roles in disease and homeostasis, and here we investigate the role of the associated ALDH1A2 gene/protein networks in PCa, using our methodology in combination with PCa patient clinical profiles from ICGC and TCGA databases. Relationships between gene signatures and relapse were analyzed using Kaplan-Meier (KM) log-rank analysis and multivariable Cox regression. Relative expression versus pooled mean from diploid population was used for z-statistics calculation. Gene/protein interaction network analyses generated 11 core genes associated with ALDH1A2; combinatorial ranking of the power set of these core genes identified two gene sets (out of 211 - 1 = 2,047 combinations) with significant correlation with disease relapse (KM log rank p < 0.05). For the more significant of these two sets, referred to as the optimal gene set (OGS), patients have median survival 62.7 months with OGS alterations compared to >150 months without OGS alterations (p = 0.0248, hazard ratio = 2.213, 95% confidence interval = 1.1-4.098). Two genes comprising OGS (CYP26A1 and RDH10) are strongly associated with ALDH1A2 in the retinoic acid (RA) pathways, suggesting a major role of RA signaling in early PCa progression. Our pipeline complements human expertise in the search for prognostic biomarkers in large-scale datasets.