Stem cell research
Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells (Avior et al. 2017, Stem Cell Research)
I love reading good papers reporting modelling of early-onset human diseases in human stem cells. There is one in Stem Cell Research that caught my attention this week, by Avior et al., modelling ablation of retinoblastoma protein (Rb) in human embryonic stem cells (hESCs). This is a well-known tumour suppressor and an inherited loss of Rb protein leads to retinoblastoma development, the most common primary intraocular pediatric cancer (95 % of cases diagnosed before the age of 5). In 6 % of cases, patients develop trilateral retinoblastoma characterised by neuroectodermal tumour occurence in additional to retinal tumours in the eye. Combined with results from conditional knockout mice, the Rb protein has been suggested to play a role in neural development. In the current study, following CRISPR/Cas9 gene editing to knockout the Rb protein, modified hESCs are subjected to an array of assays to study the resulting phenotype. After observing normal neural stem cell generation in a 2D directed differentiation protocol, despite expectations of a phenotype compared to wildtype hESCs, the authors conclude that a more complex differentiation paradigm is needed. As a result, they move on to generate teratomas in immunocomprised mice and observe a substantial expansion of neural structures in Rb-null teratomas. This highlights the importance of using multiple methods to assess a phenotype reliable. I have come to the same conclusion in my studies as 2D differentiation protocols often rely on non-physiological concentrations of various drugs and peptides, whereby a phenotype may remain masked. This study also reveals mitochondrial dysfunction in Rb-null hESCs and establishes a framework for high-throughput testing of hundreds of FDA-approved chemotherapies in this human cell models.
High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells (D’Antonio et al. 2017, Stem Cell Research)
This paper made me aware of fluorescent cell barcoding as an efficient way to pool multiple stem cell samples together and save on antibodies during initial characterisation. To make pluripotency and multilineage analysis across multiple stem cell lines available, the authors provide an Excel file which users can use to generate their own scores and heatmaps using Ct values of a reference gene of interest and any chosen marker genes.
Another paper from the same lab: Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics (Panopoulus et al. 2017 Cell Stem Cell)
A lot of effort is invested into understanding how iPSCs differ from hESCs and whether somatic memory may limit their utility in disease modelling and as potential treatment strategies. It is not well understood whether aberrant methylation of CpG sites in iPSCs is mainly caused by genetic variability or are artefacts of the reprogramming factors. The current study reports the following: “Here we generated 22 iPSC clonal lines from six individuals (three pairs of older monozygotic twins). We profiled the 22 iPSC lines, at early (passages 5 [p5] and 9 [p9]) and late (passage 20 [p20]) passages, as well as fibroblasts (tissue of origin) using genome-wide methylation arrays and RNA sequencing (RNA- seq) data.We estimate aberrantmethylation of the iPSCs relative to ESCs, and we show that aberrant methylation affects gene expression and is enriched for CpGs associated with MYC and MYC-related protein motifs. We then identify genome-wide associations between CpG methylation variation and genetic background, clone, and passage, andwe showthat these associ- ationslikely result fromrelevant biological processes.Weexamine whether aberrant CpGs are enriched for CpGs associated with genetic and non-genetic effects, and we show that aberrant methylation preferentially occurs at CpGs showing clone-associ- ated effects and is less enriched at sites associated with genetic background. Our study shows that non-genetic regulatory mechanisms associated with clone-specific effects most strongly underlie iPSC aberrancy.”
My immediate reservations without having read the study in detail is the limited number of twin pairs and therefore cells examined as well as the fact that they are all female.
iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types (Panopoulus et al. 2017 Stem Cell Research)
An iPSC resource from the same lab (very productive!). Will be useful for examining phenotypic diversity across individual induced pluripotent stem cell lines. Pertinent to the same topic, a study in Cell Stem Cell examines the influence of genetic variation on gene expression in human iPSCs: Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells (DeBoever et al. 2017 Cell Stem Cell). Actually, this seems to be a theme in the current Cell Stem Cell issues and multiple other papers examine the same topic from different perspectives. For instance, Warren et al. provide proof-of-concept that large cohorts of human iPSCs can be used to performed GWAS studies in a dish, here in the context of metabolic disease.
An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells (Guo et al. 2017 Protein & Cell)
A variation on a system that has been introduced in the past – the ability to activate genes from their endogenous locus via a Dox-inducible expression of dCas9 fused to a transcriptional activation domain.
Other bits and pieces that I have not read in detail, but that caught my interest – mostly metabolism-related
Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling (Bertholet et al. 2017 Cell Metabolism) – up until now all beige adipocytes were assumes to function like brown adipocytes, utilising UCP1 dissipate energy as heat; this paper suggests that there is a subset of beige adipocytes in mice, in epididymal fat in particular, that are UCP-negative yet perform thermogenesis by engaging in futile creatine cycling. Quite biophysical, by the way! If of interest, there is a Preview commentary on this article as well: Now UCP(rotein), Now You Don’t: UCP1 Is Not Mandatory for Thermogenesis (Szabo and Zoratti 2017, Cell Metabolism).
Another paradigm-shaking paper in Cell Metabolism modifies our understanding of the incretin effect mediated by GLP1. I have actually only read the Preview by Habener and Stanojevic (Pancreas and Not Gut Mediates the GLP-1-Induced Glucoincretin Effect) and it is always best to have a look at the actual paper and its data instead, so acknowledging the absence of appropriate assessment on my own, it might still be worth a read for those interested in this field!
Surprising results are the theme in Cell Metabolism this week; another one: FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms by BonDurant et al., providing evidence that adiponectin is dispensable for the metabolic effects of FGF21.
A paper in PNAS by Steptoe and Wardle (Life skills, wealth, health, and wellbeing in later life) looks at the importance of 5 key life skills – conscientiousness, emotional stability, determination, control, and optimism – in later life (cross-sectionally and longitudinally), concluding that (after adjustment of multiple confounding factors) these life skills are associated with wealth, income, subjective wellbeing, less depression, low social isolation and loneliness, more close relationships, better self-rated health, fewer chronic diseases and impaired activities of daily living, faster walking speed, and favorable objective biomarkers (concentration of high-density lipoprotein cholesterol, vitamin D and C-reactive protein, and less central obesity). They were also associated with greater psychological well-being and less loneliness, and a lower incidence of new chronic disease and physical impairment over a 4-y period. I like this study – a good reason to practice mindfulness throughout life!