Directed differentiation into insulin-producing cells using microRNA manipulation

Our commentary is focused on three studies that used microRNA overexpression methods for directed differentiation of stem cells into insulin-producing cells. Islet transplantation is the only cell-based therapy used to treat type 1 diabetes mellitus. However, due to the scarcity of cadaveric donors and limited availability of good quality and quantity of islets for transplant, alternate sources of insulin-producing cells are being studied and used by researchers. This commentary provides an overview of distinct studies focused on manipulating microRNA expression to optimize differentiation of embryonic stem cells or induced pluripotent stem cells into insulin-producing cells. These studies have used different approaches to overexpress micro-RNAs that are highly abundant in human islets (such as miR-375 and miR-7) in their differentiation protocol to achieve better differentiation into functional islet beta (β)-cells

MicroRNAs as prognostic markers in acute coronary syndrome patients : a systematic review

Background: The potential utility of microRNAs (miRNAs) in the diagnosis, prognosis, and treatment of multiple disease states has been an area of great interest since their discovery. In patients with cardiovascular disease, there is a large pool of literature amassed from the last decade assessing their diagnostic and prognostic potential. This systematic review sought to determine whether existing literature supports the use of miRNAs as prognostic markers after an Acute Coronary Syndrome (ACS) presentation. Methods: A systematic review of published articles from 2005-2019 using MEDLINE and EMBASE databases was undertaken independently by two reviewers. Studies addressing prognosis in an ACS population yielded 32 studies and 2 systematic reviews. Results/conclusion: 23 prospective studies reported significant differences in miRNA levels and 16 compared the predictive power of miRNAs. The most common miRNAs assessed included miR-133a, -208b, -21, -1, -34a, -150, and -423, shown to be involved in cell differentiation, apoptosis, and angiogenesis. Barriers to the use of miRNAs as prognostic markers include bias in miRNA selection, small sample size, variable normalization of data, and adjustment for confounders. Therefore, findings from this systematic review do not support the use of miRNAs for prognostication post-ACS beyond traditional cardiovascular risk factors, existing risk scores, and stratifications tools

An optimised step-by-step protocol for measuring relative telomere length

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease

Postpartum circulating cell-free insulin DNA levels are higher in women with previous gestational diabetes mellitus who develop type 2 diabetes in later life

Background. Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β-cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin (INS) DNA correlate with dying β-cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM. Methods. We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM. Results. In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM (P=0.02). There was no difference in cell-free circulating unmethylated and methylated INS DNA levels between NGT women and women who developed T2DM (P=0.09 and P=0.07, respectively). Conclusions. In women with a previous index GDM pregnancy, postpartum levels of cell-free circulating INS DNA are significantly higher in those women who later developed T2DM

Postpartum circulating cell-free insulin DNA levels are higher in women with previous gestational diabetes mellitus who develop type 2 diabetes in later life

Background. Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β-cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin (INS) DNA correlate with dying β-cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM. Methods. We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM. Results. In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM (P=0.02). There was no difference in cell-free circulating unmethylated and methylated INS DNA levels between NGT women and women who developed T2DM (P=0.09 and P=0.07, respectively). Conclusions. In women with a previous index GDM pregnancy, postpartum levels of cell-free circulating INS DNA are significantly higher in those women who later developed T2DM

Manipulating cellular microRNAs and analyzing high-dimensional gene expression data using machine learning workflows

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a)

Continuous subcutaneous insulin infusion alters microRNA expression and glycaemic variability in children with type 1 diabetes

To determine whether continuous subcutaneous insulin infusion (CSII) vs. multiple daily injections (MDI) therapy from near-diagnosis of type 1 diabetes is associated with reduced glycaemic variability (GV) and altered microRNA (miRNAs) expression. Adolescents (74% male) within 3-months of diabetes diagnosis (n = 27) were randomized to CSII (n = 12) or MDI. HbA1c, 1-5-Anhydroglucitol (1,5-AG), high sensitivity C-peptide and a custom TaqMan qPCR panel of 52 miRNAs were measured at baseline and follow-up (median (LQ-UQ); 535 (519-563) days). There were no significant differences between groups in baseline or follow-up HbA1c or C-peptide, nor baseline miRNAs. Mean +/- SD 1,5-AG improved with CSII vs. MDI (3.1 +/- 4.1 vs. - 2.2 +/- - 7.0 mg/ml respectively, P = 0.029). On follow-up 11 miRNAs associated with diabetes vascular complications had altered expression in CSII-users. Early CSII vs. MDI use is associated with lower GV and less adverse vascular-related miRNAs. Relationships with future complications are of interest

Epigenetic and transcriptome profiling identifies a population of visceral adipose-derived progenitor cells with potential to differentiate into an endocrine pancreatic lineage

Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of pancreas donors. To develop an alternative cell source for transplantation therapy, we carried out the epigenetic characterization in nine different adult mouse tissues and identified visceral adipose-derived progenitors as a candidate cell population. Chromatin conformation, assessed using chromatin immunoprecipitation (ChIP) sequencing and validated by ChIP-polymerase chain reaction (PCR) at key endocrine pancreatic gene promoters, revealed similarities between visceral fat and endocrine pancreas. Multiple techniques involving quantitative PCR, in-situ PCR, confocal microscopy, and flow cytometry confirmed the presence of measurable (2–1000-fold over detectable limits) pancreatic gene transcripts and mesenchymal progenitor cell markers (CD73, CD90 and CD105; >98%) in visceral adipose tissue-derived mesenchymal cells (AMCs). The differentiation potential of AMCs was explored in transgenic reporter mice expressing green fluorescent protein (GFP) under the regulation of the Pdx1 (pancreatic and duodenal homeobox-1) gene promoter. GFP expression was measured as an index of Pdx1 promoter activity to optimize culture conditions for endocrine pancreatic differentiation. Differentiated AMCs demonstrated their capacity to induce pancreatic endocrine genes as evidenced by increased GFP expression and validated using TaqMan real-time PCR (at least 2–200-fold relative to undifferentiated AMCs). Human AMCs differentiated using optimized protocols continued to produce insulin following transplantation in NOD/SCID mice. Our studies provide a systematic analysis of potential islet progenitor populations using genome-wide profiling studies and characterize visceral adipose-derived cells for replacement therapy in diabetes

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultrahigh content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest

Decrease in plasma miR-27a and miR-221 after concussion in Australian football players

Introduction: Sports-related concussion (SRC) is a common form of brain injury that lacks reliable methods to guide clinical decisions. MicroRNAs (miRNAs) can influence biological processes involved in SRC, and measurement of miRNAs in biological fluids may provide objective diagnostic and return to play/recovery biomarkers. Therefore, this prospective study investigated the temporal profile of circulating miRNA levels in concussed male and female athletes. Methods: Pre-season baseline blood samples were collected from amateur Australian rules football players (82 males, 45 females). Of these, 20 males and 8 females sustained an SRC during the subsequent season and underwent blood sampling at 2-, 6- and 13-days post-injury. A miRNA discovery Open Array was conducted on plasma to assess the expression of 754 known/validated miRNAs. miRNA target identified were further investigated with quantitative real-time PCR (qRT-PCR) in a validation study. Data pertaining to SRC symptoms, demographics, sporting history, education history and concussion history were also collected. Results: Discovery analysis identified 18 candidate miRNA. The consequent validation study found that plasma miR-221-3p levels were decreased at 6d and 13d, and that miR-27a-3p levels were decreased at 6d, when compared to baseline. Moreover, miR-27a and miR-221-3p levels were inversely correlated with SRC symptom severity. Conclusion: Circulating levels of miR-27a-3p and miR-221-3p were decreased in the sub-acute stages after SRC, and were inversely correlated with SRC symptom severity. Although further studies are required, these analyses have identified miRNA biomarker candidates of SRC severity and recovery that may one day assist in its clinical management