Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

Understanding the relationship between the inbreeding coefficient and multilocus heterozygosity: Theoretical expectations and empirical data

Geneticists have been interested in inbreeding and inbreeding depression since the time of Darwin. Two alternative approaches that can be used to measure how inbred an individual is involve the use of pedigree records to estimate inbreeding coefficients or molecular markers to measure multilocus heterozygosity. However, the relationship between inbreeding coefficient and heterozygosity has only rarely been investigated. In this paper, a framework to predict the relationship between the two variables is presented. In addition, microsatellite genotypes at 138 loci spanning all 26 autosomes of the sheep genome were used to investigate the relationship between inbreeding coefficient and multilocus heterozygosity. Multilocus heterozygosity was only weakly correlated with inbreeding coefficient, and heterozygosity was not positively correlated between markers more often than expected by chance. Inbreeding coefficient, but not multilocus heterozygosity, detected evidence of inbreeding depression for morphological traits. The relevance of these findings to the causes of heterozygosity-fitness correlations is discussed and predictions for other wild and captive populations are presented

Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis.

Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution

Evidence for effect of l-serine, a novel therapy for GRIN2B-related neurodevelopmental disorder.

RATIONALE: To date, causal therapy is potentially available for GRIN2B-related neurodevelopmental disorder (NDD) due to loss-of-function (LoF) variants in GRIN2B, resulting in dysfunction of the GluN2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). Recently, in vitro experiments showed that high doses of NMDAR co-agonist d-serine has the potential to boost the activity in GluN2B LoF variant-containing NMDARs. Initial reports of GRIN2B-NDD patients LoF variants, treated with l-serine using different regimens, showed varying effects on motor and cognitive performance, communication, behavior and EEG. Here, this novel treatment using a standardized protocol with an innovative developmental outcome measure is explored further in an open-label observational GRIN2B-NDD study. METHODS: Initially, in vitro studies were conducted in order to functionally stratify two de novo GRIN2B variants present in two female patients (18 months and 4 years old). Functional studies showed that both variants are LoF, and thus the patients were treated experimentally according to an approved protocol with oral l-serine (500 mg/kg/day in 4 doses) for a period of 12 months. Both patients showed a heterogeneous clinical phenotype, however overlapping symptoms were present: intellectual developmental disability (IDD), behavioral abnormalities and hypotonia. Outcome measures included laboratory tests, quality of life, sleep, irritability, stool, and performance skills, measured by, among others, the Perceive-Recall-Plan-Perform System of Task Analysis (PRPP-Assessment). RESULTS: Both patients tolerated l-serine without adverse effects. In one patient, improvement in psychomotor development and cognitive functioning was observed after 12 months (PRPP mastery score 10% at baseline, 78% at twelve months). In the most severe clinically affected patient no significant objective improvement in validated outcomes was observed. Caregivers of both patients reported subjective increase of alertness and improved communication skills. CONCLUSION: Our observational study confirms that l-serine supplementation is safe in patients with GRIN2B-NDD associated with LoF variants, and may accelerate psychomotor development and ameliorate cognitive performance in some but not all patients. The PRPP-Assessment, a promising instrument to evaluate everyday activities and enhance personalized and value-based care, was not performed in the severely affected patient, meaning that possible positive results may have been missed. To generate stronger evidence for effect of l-serine in GRIN2B-NDD, we will perform placebo-controlled n-of-1 trials

Evidence for effect of l-serine, a novel therapy for GRIN2B-related neurodevelopmental disorder.

RATIONALE: To date, causal therapy is potentially available for GRIN2B-related neurodevelopmental disorder (NDD) due to loss-of-function (LoF) variants in GRIN2B, resulting in dysfunction of the GluN2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). Recently, in vitro experiments showed that high doses of NMDAR co-agonist d-serine has the potential to boost the activity in GluN2B LoF variant-containing NMDARs. Initial reports of GRIN2B-NDD patients LoF variants, treated with l-serine using different regimens, showed varying effects on motor and cognitive performance, communication, behavior and EEG. Here, this novel treatment using a standardized protocol with an innovative developmental outcome measure is explored further in an open-label observational GRIN2B-NDD study. METHODS: Initially, in vitro studies were conducted in order to functionally stratify two de novo GRIN2B variants present in two female patients (18 months and 4 years old). Functional studies showed that both variants are LoF, and thus the patients were treated experimentally according to an approved protocol with oral l-serine (500 mg/kg/day in 4 doses) for a period of 12 months. Both patients showed a heterogeneous clinical phenotype, however overlapping symptoms were present: intellectual developmental disability (IDD), behavioral abnormalities and hypotonia. Outcome measures included laboratory tests, quality of life, sleep, irritability, stool, and performance skills, measured by, among others, the Perceive-Recall-Plan-Perform System of Task Analysis (PRPP-Assessment). RESULTS: Both patients tolerated l-serine without adverse effects. In one patient, improvement in psychomotor development and cognitive functioning was observed after 12 months (PRPP mastery score 10% at baseline, 78% at twelve months). In the most severe clinically affected patient no significant objective improvement in validated outcomes was observed. Caregivers of both patients reported subjective increase of alertness and improved communication skills. CONCLUSION: Our observational study confirms that l-serine supplementation is safe in patients with GRIN2B-NDD associated with LoF variants, and may accelerate psychomotor development and ameliorate cognitive performance in some but not all patients. The PRPP-Assessment, a promising instrument to evaluate everyday activities and enhance personalized and value-based care, was not performed in the severely affected patient, meaning that possible positive results may have been missed. To generate stronger evidence for effect of l-serine in GRIN2B-NDD, we will perform placebo-controlled n-of-1 trials

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized “trunk-like structures” (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish