A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome

Human brain development is a complex process where multiple cellular and developmental events are co-ordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS), is a X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1 that encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated, but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a “disease-in-a-dish” model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development.

Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing

ABSTRACT Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

Loss of p19Arf Promotes Fibroblast Survival During Leucine Deprivation

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here we show that prolonged leucine deprivation resulted in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation.

The matrix in focus: new directions in extracellular matrix research from the 2021 ASMB hybrid meeting

ABSTRACT The extracellular matrix (ECM) is a complex assembly of macromolecules that provides both architectural support and molecular signals to cells and modulate their behaviors. Originally considered a passive mechanical structure, decades of research have since demonstrated how the ECM dynamically regulates a diverse set of cellular processes in development, homeostasis, and disease progression. In September 2021, the American Society for Matrix Biology (ASMB) organized a hybrid scientific meeting, integrating in-person and virtual formats, to discuss the latest developments in ECM research. Here, we highlight exciting scientific advances that emerged from the meeting including (1) the use of model systems for fundamental and translation ECM research, (2) ECM-targeting approaches as therapeutic modalities, (3) cell-ECM interactions, and (4) the ECM as a critical component of tissue engineering strategies. In addition, we discuss how the ASMB incorporated mentoring, career development, and diversity, equity, and inclusion initiatives in both virtual and in-person events. Finally, we reflect on the hybrid scientific conference format and how it will help the ASMB accomplish its mission moving forward.

Potentiating Vγ9Vδ2 T cell proliferation and assessing their cytotoxicity towards adherent cancer cells at the single cell level

Vγ9Vδ2 T cells is the dominant γδ T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for Vγ9Vδ2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise Vγ9Vδ2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly-thawed human serum potentiated Vγ9Vδ2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and consistently enabled Vγ9Vδ2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallyl monophosphate stimulated Vγ9Vδ2 T cell proliferation. We developed a method where the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the Vγ9Vδ2 T cell cytotoxicity towards colon cancer cells. In summary we have developed an easily interpretable method to assess the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells, found that Vγ9Vδ2 T cell proliferation can be potentiated media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of Vγ9Vδ2 T cell immunotherapy.

Novel protocol to observe the intestinal tuft cell using transmission electron microscopy

Tuft cell is a chemosensory cell, a specific cell type sharing the taste transduction system with a taste cell on the tongue, of which the existence has been known in various tissues such as gastrointestinal tract, gall bladder, trachea, pancreatic duct, etc. To date, electron microscopic approaches have shown various morphological features of the tuft cell such as long and thick microvilli, tubulovesicular network at the apical side, prominent skeleton structures, etc. Recently, it has been reported that the small intestinal tuft cell functions to initiate type2 immunity in response to helminth infection. However, the mechanisms by which such distinguished structures are involved with the physiological functions are poorly understood. To address this question, the combination of physiological study regarding the tuft cells using genetic models and its morphological study using electron microscopy will be required. However, it is a challenge to observe tuft cells by electron microscopy due to their extremely low frequency on the epithelium. Therefore, in this paper, we suggest the advanced protocol to observe the small intestinal tuft cell efficiently by transmission electron microscopy using serial semi-thin sections on the Aclar film.

First person – Natalí Delorme and Leonardo Zamora

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Natalí Delorme and Leonardo Zamora are co-first authors on ‘ A new method to localise and quantify oxidative stress in live juvenile mussels’, published in BiO. They are both researchers in the laboratory of Serean Adams at the Cawthron Institute, Nelson, New Zealand. Natalí's research interests centre around ecophysiology of marine invertebrates, particularly on the organisms' stress response. Leonardo is investigating the biology of commercially, ecologically and culturally relevant marine invertebrates throughout their life cycle.

First person – Yixing Wu and Ying Bai

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Yixing Wu and Ying Bai are co-first authors on ‘ Palmitoylated small GTPase ARL15 is translocated within Golgi network during adipogenesis’, published in BiO. Yixing is a research fellow in the lab of Frances Wiseman at UCL Queen Square Institute of Neurology, London, UK, investigating Down's syndrome and Alzheimer's disease-related endo-lysosomal pathways and cathepsin deficits. Ying is a postdoc in the lab of Roger D. Cox at MRC Harwell Institute, Didcot, UK, investigating how fat cells are formed, and genes that are involved in regulating body fat distribution.

Dynamic shapes of the zygote and two-cell mouse and human

ABSTRACT Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder–distorted–rounder shapes were revealed, as were changing patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis. During cell division, two vortices developed in each nascent cell and they rotated in opposite directions at each end of the cell, a pattern that sometimes persisted for up to 10 h. Exchange with the environment may have been promoted by these shape and area cycles and persisting circulation in the cytoplasm may have a similar function between a cell's interior and periphery. Some of these movements were sporadically also seen in human zygotes with abnormal numbers of pronuclei and the two-cell stages that developed from these compromised human zygotes.

Redundant functions of the SLC5A transporters Rumpel, Bumpel, and Kumpel in ensheathing glial cells

Neuronal processing is energy demanding, and relies on sugar metabolism. To nurture the Drosophila nervous system, the blood-brain barrier forming glial cells take up trehalose from the hemolymph and then distribute the metabolic products further to all neurons. This function is provided by glucose and lactate transporters of the solute carrier (SLC) 5A family. Here we identified three SLC5A genes that are specifically expressed in overlapping sets of CNS glial cells, rumpel, bumpel and kumpel. We generated mutants in all genes and all mutants are viable and fertile, lacking discernible phenotypes. Loss of rumpel causes subtle locomotor phenotypes and flies display increased daytime sleep. In addition, in bumpel kumpel double mutants, and to an even greater extent in rumpel bumpel kumpel triple mutants, oogenesis is disrupted at the onset of the vitollegenic phase. This indicates a partially redundant functions between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporters, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, our results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.