Generation and molecular characterization of human pluripotent stem cell-derived pharyngeal foregut endoderm.

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.

MiRNA Profiling in Human Induced Pluripotent Stem Cells.

Human iPS cells are capable of differentiation towards all three germ layer lineages. As well as being of use for the study of developmental biology, these cells also provide an excellent resource for disease modeling and cell replacement therapies. iPS cells derived from an individual with a disease type of interest can be differentiated towards the cell type afflicted in that particular disease. Such differentiated cell types can be used to test patient-specific drug responses in vitro, for generation of replacement cells for transplantation, or as a starting material for gene editing to correct disease-causing mutations/deletions. With such a vast array of potential applications, there is a great need to understand the exact mechanisms controlling the maintenance of pluripotency, and the distinct cues signaling these cells to differentiate towards lineages of interest. This chapter focuses on microRNA profiling using TaqMan Human MicroRNA Arrays to examine expression of 754 miRNAs in human iPS cells in the pluripotent state, and iPS derived definitive endoderm.

Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells.

CRISPR-Cas9 effector systems have wide applications for the stem cell and regenerative medicine field. The ability to dissect the functional gene regulatory networks in pluripotency and potentially in differentiation intermediates of all three germ layers makes this a valuable tool for the stem cell community. Catalytically inactive Cas9 fused to transcriptional/chromatin effector domains allows for silencing or activation of a genomic region of interest. Here, we describe the application of an inducible, RNA-guided, nuclease-deficient (d) Cas9-KRAB system (adapted from Streptococcus pyogenes) to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region. This chapter outlines a detailed protocol for generation of a stable human embryonic stem cell line containing both Sp-dCas9-KRAB and sgRNA, followed by inducible expression of Sp-dCas9-KRAB to analyze functional effects of dCas9-KRAB at target loci in human embryonic stem cells.

MicroRNA expression analysis: techniques suitable for studies of intercellular and extracellular microRNAs.

MicroRNAs, the class of small ribo-regulators, have been implicated in the regulation of a range of different biological processes, including development and differentiation, proliferation, and cell death. Only for a small fraction of identified microRNAs has a function been elucidated; therefore, a great deal of research remains to be performed to fully understand the role and implications of microRNAs.This chapter discusses protocols for the isolation of microRNAs, reverse transcription, PCR, and large scale profiling using TaqMan low density miRNA arrays for analysis of microRNA expression levels.

Identification of microRNAs with a role in glucose stimulated insulin secretion by expression profiling of MIN6 cells.

MicroRNAs (miRNAs) are a family of endogenous small non-coding RNAs which regulate mRNAs at the post-transcriptional level. MiRNAs have been identified in both normal physiological and pathological conditions. To date, a limited number of miRNAs have been shown to be involved in the regulation of insulin secretion. We have identified a panel of 10 miRNAs down-regulated in glucose non-responsive MIN6 cells compared to glucose responsive cells using TaqMan Low Density miRNA Arrays. Of these 10 miRNA targets, subsequent functional investigations involving knockdown of mir-200a, mir-130a and mir-410 levels suggested that they may decrease the capability of MIN6 cells to secrete insulin in response to stimulatory levels of glucose. Conversely, experiment with over-expression of mir-410 suggest that it may enhance levels of glucose stimulated insulin secretion. In this study, we have also identified 21 miRNAs not previously known to have a potential murine homologue.

Molecular medicine of microRNAs: structure, function and implications for diabetes.

MicroRNAs (miRNAs) are a family of endogenous small noncoding RNA molecules, of 19-28 nucleotides in length. In humans, up to 3% of all genes are estimated to encode these evolutionarily conserved sequences. miRNAs are thought to control expression of thousands of target mRNAs. Mammalian miRNAs generally negatively regulate gene expression by repressing translation, possibly through effects on mRNA stability and compartmentalisation, and/or the translation process itself. An extensive range of in silico and experimental techniques have been applied to our understanding of the occurrence and functional relevance of such sequences, and antisense technologies have been successfully used to control miRNA expression in vitro and in vivo. Interestingly, miRNAs have been identified in both normal and pathological conditions, including differentiation and development, metabolism, proliferation, cell death, viral infection and cancer. Of specific relevance and excitement to the area of diabetes research, miRNA regulation has been implicated in insulin secretion from pancreatic beta-cells, diabetic heart conditions and nephropathy. Further analyses of miRNAs in vitro and in vivo will, undoubtedly, enable us determine their potential to be exploited as therapeutic targets in diabetes.