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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm [scRNA-Seq]

ABSTRACT: Differentiation of human pluripotent stem cells toward definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA-sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. We further show that differentiation-arrested phenotype is inversely correlated with zinc concentration in the differentiation media. This study improves our understanding of in-vitro DE differentiation and provides actionable options to improve DE differentiation efficiency.

ORGANISM(S): Homo sapiens

PROVIDER: GSE109979 | GEO | 2018/02/15

REPOSITORIES: GEO

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Project description:Steroid-refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection. Eighty-three kidney transplant recipients (1995-2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (P<0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT-1 as an independent covariate (AUC=0.88, P<0.0000001). In the validation set, MT-1 expression was also significantly associated with steroid resistance (P=0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that non-response to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc-requiring anti-inflammatory effect of the glucocorticoid receptor. Transcriptional profiles of 40 renal allograft biopsy samples were analyzed using Illumina HumanRef-8 v3.0 BeadChips (Illumina, San Diego, CA). Expression profiles were investigated in total RNA isolated from biopsy samples of 18 patients with steroid responsive acute rejection and 18 patients with steroid resistant acute rejection. Four biopsies, taken during acute decrease of graft function but with no histomorphologic indication of rejection, were included as controls.

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Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm [scRNA-Seq]

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