The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential.

BACKGROUND: Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear. RESULTS: Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells. CONCLUSIONS: Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.

[Pathological conditions of the ocular surface -- a clinical and confocal laser-scanning microscopy study]. / Oberflächenerkrankungen des Auges unterschiedlicher Genese - klinische und konfokalmikroskopische Untersuchungen.

Confocal in vivo laser scanning microscopy is an established technique to visualise morphology of the cornea and conjunctiva, whereby the image interpretation needs experience. We report about changes of the ocular surface in the pathological conditions of infectious, metabolic and traumatic genesis and discuss their relevance. The micromorphology of the corneal epithelium and stroma in respect to pathogens (bacterial, fungal) is discussed. Metabolic disease induces multifaceted corneal alterations which can be visualised and used for assessment of the disease progression. Follow-up microscopic investigations allow for an assessment of the wound healing dynamics and enable a prognosis to be made for corneal recurrence. Taken together, confocal in vivo microscopy allows a non-invasive microscopy on the cellular level and thus complements clinical diagnostics.

[In vivo imaging of limbal epithelium and palisades of Vogt]. / In-vivo-bildgebung des limbusepithels und der Vogt-palisaden.

Limbal disorders can lead to loss of stem cell population and deficiency in corneal integrity. The aim of this study is to describe the morphological features of this region by using confocal laser scanning microscopy (CLSM) and spectral domain optical coherence tomography (SD-OCT) in healthy subjects, contact lens wearers and in patients with surface disorders. On examination hyper- and hyporeflective finger-like structures were found with numerous basal epithelial cells within and in between crypts. In contrast, detailed imaging of limbal palisades of Vogt was not possible on SD-OCT, the corresponding hyperreflective line was identified. These typical crypts were less pronounced or absent in cases with corneal alterations as has been demonstrated. Additionally morphological changes at the level of intermediate and basal cell layers were discussed. A better understanding of the corneal limbus can provide valuable clues on understanding cell integrity and wound-healing processes. In future, quantitative analyses of limbal epithelium in different corneal disorders will be preferable.

Quantitative assessment of central and limbal epithelium after long-term wear of soft contact lenses and in patients with dry eyes: a pilot study.

PurposeAnalysis of microstructural alterations of corneal and limbal epithelial cells in healthy human corneas and in other ocular conditions.Patients and methodsUnilateral eyes of three groups of subjects include healthy volunteers (G1, n=5), contact lens wearers (G2, n=5), and patients with dry eyes (G3, n=5) were studied. Imaging of basal (BC) and intermediate (IC) epithelial cells from central cornea (CC), corneal limbus (CL) and scleral limbus (SL) was obtained by in vivo confocal microscopy (IVCM). An appropriate image analysis algorithm was used to quantify morphometric parameters including mean cell area, compactness, solidity, major and minor diameter, and maximum boundary distance.ResultsThe morphometric parameters of BC and IC demonstrated no significant differences (P>0.05) between groups. Comparison between three corneal locations (CC, CL, and SL) within the groups showed significant differences (P<0.05) with mean values of cell area, compactness, solidity, and major and minor diameter of BC that increase from CC to limbus. The BC were round and regular in the central cornea (P<0.05) compared with CL and SL.ConclusionsIVCM enables high-quality confocal images from central corneal and limbal epithelium. This quantitative study demonstrated morphological differences in the basal and intermediate epithelium between limbus and central cornea, and found no differences between contact lens wearers, dry eyes, and normal subjects.