Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 107 wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence ‘families’ that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1–3 d.

Stem cell-based methods for myocardial regeneration suffer from considerable cell attrition. Artificial matrices reproducing mechanical and structural properties of the native tissue may facilitate survival, retention and functional integration of adult stem or progenitor cells, by conditioning the cells prior to, and during, transplantation. Here we combined autologous cardiosphere-derived cells (CDCs) with nanotopographically defined hydrogels mimicking the native myocardial matrix, to form in vitro cardiac stem cell niches, and control cell function and fate. These platforms were used to produce cardiac patches that could be transplanted at the site of infarct. In culture, highly anisotropic, but not more randomized nanotopographic, control augmented cell adhesion, migration, and proliferation. It also dramatically enhanced early, and, in the presence of mature cardiomyocytes, late cardiomyogenesis. Nanotopography sensing and transcriptional response was mediated via p190RhoGAP. In a rat infarction model, engraftment of nanofabricated scaffolds with CDCs enhanced retention and growth of transplanted cells, and their integration with the host tissue. The infarcted ventricle wall increased in thickness, with higher cell viability and better collagen organization. These results suggest that nanostructured polymeric materials that closely mimic the extracellular matrix structure on which cardiac cells reside in vivo can be both very effective tools in investigating the mechanisms of cardiac differentiation and the basis for cardiac tissue engineering, thus facilitating stem cell-based therapy in the heart.

Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C2-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C2-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C2-ceramide inhibited growth and caused death of colon cancer cells in time-and concentration-dependent manners, whereas C2-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C2-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C2-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by Inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C2-ceramide increased the number of cells in the A0 peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G2/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis In colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.

Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. The role of the mechanical environment in the coordination of these processes is poorly understood. We show that multipotent cells derived from native cardiac tissue continually monitored cell substratum rigidity and showed enhanced proliferation, endothelial differentiation, and morphogenesis when the cell substratum rigidity closely matched that of myocardium. Mechanoregulation of these diverse processes required p190RhoGAP, a guanosine triphosphatase-activating protein for RhoA, acting through RhoA-dependent and -independent mechanisms. Natural or induced decreases in the abundance of p190RhoGAP triggered a series of developmental events by coupling cell-cell and cell-substratum interactions to genetic circuits controlling differentiation.

Paediatric rhabdomyosarcomas (RMS) are classified into two major subtypes based on histological appearance, embryonal (ERMS) and alveolar (ARMS), but this clinically critical distinction is often difficult on morphological grounds alone. ARMS, the more aggressive subtype, is associated in most cases with unique recurrent translocations fusing the PAX3 or PAX7 transcription factor genes to FKHR. In contrast, ERMS lacks unique genetic alterations. To identify novel diagnostic markers and potential therapeutic targets, we analysed the global gene expression profiles of these two RMS subtypes in 23 ARMS (16 PAX3-FKHR, 7 PAX7-FKHR) and 15 ERMS (all PAX-FKHR-negative) using Affymetrix HG-U133A oligonucleotide arrays. A statistically stringent supervised comparison of the ARMS and ERMS expression profiles revealed 121 genes that were significantly differentially expressed, of which 112 were higher in ARMS, including genes of interest as potential diagnostic markers or therapeutic targets, such as CNR1, PIPOX (sarcosine oxidase), and TFAPbeta. Interestingly, many known or putative downstream targets of PAX3-FKHR were highly overexpressed in ARMS relative to ERMS, including CNR1, DCX, ABAT, ASS, JAKMIP2, DKFZp762M127, and NRCAM. We validated the highly differential expression of five genes, including CNR1, DKFZp762M127, DCX, PIPOX, and FOXF1 in ARMS relative to ERMS by quantitative RT-PCR on an independent set of samples. Finally, we developed a ten-gene microarray-based predictor that distinguished ARMS from ERMS with approximately 95% accuracy both in our data by cross-validation and in an independent validation using a published dataset of 26 samples. The gene expression signature of ARMS provides a source of potential diagnostic markers, therapeutic targets, and PAX-FKHR downstream genes, and can be used to reliably distinguish these sarcomas from ERMS.

Rhabdomyosarcoma is a family of myogenic soft tissue tumors subdivided into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by a frequent 2;13 chromosomal translocation that creates a PAX3-FKHR fusion transcription factor. To identify downstream targets of PAX3-FKHR, we introduced an inducible form of PAX3-FKHR into human RD ERMS cells. Microarray analysis identified 39 genes (29 upregulated and 10 downregulated) that are modulated by PAX3-FKHR in RD cells and differentially expressed between ERMS and PAX3-FKHR-positive ARMS tumors. Functional annotation demonstrated that genes involved in regulation of transcription and development, particularly neurogenesis, are represented in this group. MYCN was one notable neural-related transcription factor-encoding gene identified in this set, and its regulation by PAX3-FKHR was further confirmed at the RNA and protein levels. The findings of cycloheximide inhibition and time-course studies are consistent with the hypothesis that the PAX3-FKHR protein acts directly on the MYCN gene at the transcriptional level. Functional studies established that MYCN cooperates with PAX3-FKHR to enhance oncogenic activity. In conclusion, we identified a selected set of biologically relevant genes modulated by PAX3-FKHR, and demonstrated that PAX3-FKHR contributes to the expression of MYCN and in turn MYCN collaborates with PAX3-FKHR in tumorigenesis.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for use in engineering human tissues for regenerative therapy and drug screening.However, differentiated cardiomyocytes are phenotypically immature, reducing assay reliability when translating in vitro results to clinical studies and precluding hiPSC-derived cardiac tissues from therapeutic use in vivo. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cellular and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces at 14 days of culture and had increased the expression of genes that regulate contractile function. Similar improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial.We also demonstrate that dECM-rGO hydrogels can be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced maturation of these tissues corresponded well with a capability to produce physiologically relevant drug responses.