Derivation of endothelial cells from human embryonic stem cells in fully defined medium enables identification of lysophosphatidic acid and platelet activating factor as regulators of eNOS localization

The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34⁺KDR⁺ endothelial progenitor cells after 6 days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.     

The Australian Multiple Sclerosis (MS) Immunotherapy Study: A Prospective,Multicentre Study of Drug Utilisation Using the MSBase Platform

Objective

To prospectively characterise treatment persistence and predictors of treatment discontinuation in an Australian relapsing-remitting multiple sclerosis (RRMS) population.

Methods

Tertiary MS treatment centres participating in the MSBase registry prospectively assessed treatment utilisation, persistence, predictors of treatment discontinuation and switch rates. Multivariable survival analyses were used to compare treatment persistence between drugs and to identify predictors of treatment discontinuation.

Results

1113 RRMS patients were studied. Patients persisted on their first disease-modifying therapy (DMT) for a median of 2.5 years. Treatment persistence on GA was shorter than on all IFNβ products (p<0.03). Younger age at treatment initiation and higher EDSS were predictive of DMT discontinuation. Patients persisted on subsequent DMTs, for 2.3 years. Patients receiving natalizumab (NAT) as a subsequent DMT persisted longer on treatment than those on IFNβ or GA (p<0.000). The primary reason for treatment discontinuation for any drug class was poor tolerability. Annualised switch or cessation rates were 9.5–12.5% for individual IFNβ products, 11.6% for GA and 4.4% for NAT.

Conclusion

This multicentre MS cohort study is the first to directly compare treatment persistence on IFNβ and GA to NAT. We report that treatment persistence in our Australian RRMS population is short, although patients receiving IFNβ as a first DMT persisted longer on treatment than those on GA. Additionally, patients receiving NAT as a subsequent DMT were more likely to persist on treatment than those switched to IFNβ or GA. EDSS and age at DMT initiation were predictive of DMT discontinuation. Treatment intolerance was the principal reason for treatment cessation.     

BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells

WNT and bone morphogenetic protein (BMP) signaling are known to stimulate hemogenesis from pluripotent embryonic stem (ES) cells. However, osteochondrogenic mesoderm was generated effectively when BMP signaling is kept to a low level, while WNT signaling was strongly activated. When mesoderm specification from ES cells was exogenous factor dependent, WNT3a addition supported the generation of cardiomyogenic cells expressing lateral plate/extraembryonic mesoderm genes, and this process involved endogenous BMP activities. Exogenous BMP4 showed a similar effect that depended on endogenous WNT activities. However, neither factor induced robust chondrogenic activity. In support, ES cell differentiation in the presence of either WNT3a or BMP4 was associated with elevated levels of both Bmp and Wnt mRNAs, which appeared to provide sufficient levels of active BMPs and WNTs to promote the nonchondrogenic mesoderm specification. The osteochondrogenic mesoderm expressed PDGFRα, which also expressed genes that mark somite and rostral presomitic mesoderm. A strong WNT signaling was required for generating the mesodermal progeny, while approximately 50- to 100-fold lower concentration of WNT3a was sufficient for specifying axial mes(end)oderm. Thus, depending on the dose and cofactor (BMP), WNT signaling stimulates the generation of different biological activities and specification of different types of mesodermal progeny from ES cells.     

Leukemia Inhibitory Factor Protects Axons in Experimental Autoimmune Encephalomyelitis via an Oligodendrocyte-Independent Mechanism

Leukemia inhibitory factor (LIF) and Ciliary Neurotrophic factor (CNTF) are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE) model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG_35–55 EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1±0.14 vs 2.6±0.19; P<0.05). These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540±207 µm²−/s vs 1310±175 µm²−/s; P<0.05), and optic nerve (−12.5%) and spinal cord (−16%) axon densities; and increased serum neurofilament-H levels (2.5 fold increase). No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.     

Assessing the contribution of bacteria to the heat tolerance of experimentally evolved coral photosymbionts

Coral reefs are extremely vulnerable to ocean warming, which triggers coral bleaching—the loss of endosymbiotic microalgae (Symbiodiniaceae) from coral tissues, often leading to death. To enhance coral climate resilience, the symbiont, Cladocopium proliferum was experimentally evolved for >10 years under elevated temperatures resulting in increased heat tolerance. Bacterial 16S rRNA gene metabarcoding showed the composition of intra- and extracellular bacterial communities of heat-evolved strains was significantly different from that of wild-type strains, suggesting bacteria responded to elevated temperatures, and may even play a role in C. proliferum thermal tolerance. To assess whether microbiome transplantation could enhance heat tolerance of the sensitive wild-type C. proliferum, we transplanted bacterial communities from heat-evolved to the wild-type strain and subjected it to acute heat stress. Microbiome transplantation resulted in the incorporation of only 30 low-abundance strains into the microbiome of wild-type cultures, while the relative abundance of 14 pre-existing strains doubled in inoculated versus uninoculated samples. Inoculation with either wild-type or heat-evolved bacterial communities boosted C. proliferum growth, although no difference in heat tolerance was observed between the two inoculation treatments. This study provides evidence that Symbiodiniaceae-associated bacterial communities respond to heat selection and may contribute to coral adaptation to climate change.