Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids

Abstract

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.

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

“…We performed screening with H9 cells that were cultivated on Matrigel in 5% O 2 as hypoxia is favourable for NMPs. 35 The screening of Wnt/β-catenin, FGF2, TGF-β and BMP modulators resulted in the formation of cell lines in the CHIR99021/FGF2/SB-431542 (abbreviated CFS), and without FGF2 (abbreviated CS) conditions. The observed down-regulation of OCT4 and NANOG in three independently derived lines indicated the non-pluripotent state of CFS and CS cell lines.…”

“…The importance of cultivation of AxSCs under hypoxia might be connected with the enhanced stability of NMPs in vitro when maintained in hypoxic conditions. 35 Thereby, the capture of AxSCs lays an essential groundwork for exploring the epigenetic mechanisms and other pivotal factors that drive the differentiation of NMPs and neural tube progenitors. Such insights hold the potential to guide the direct reprogramming of NM-AxSCs from somatic cells.…”

Integrated Molecular-Phenotypic Profiling Reveals Metabolic Control of Morphological Variation in Stembryos

“…49 We recently reported that in hypoxic gastruloids the relative fraction of NMPs and their somitic progeny is increased. 50 Hence, a hypoxia-induced shift from OxPhos to glycolysis could be a physiological means to balance neural versus mesodermal decision-making of NMPs. We speculate that small variations in size could set the stage for such divergent metabolic activity.…”

“…The multiplexed single-cell experiment was performed as previously described. 23,50 Briefly, structures were generated as described above. Following wide-field imaging, 72 structures at 48h, 48 at 72h, 24 at 96h and 24 at 120h were picked with a p200 cut tip and transferred in a new well of a 96-well plate.…”

“…Following MULTI-seq barcode labeling, single-cell RNA-seq (scRNA-seq) experiments were performed as previously described 23,50 . Briefly, single-cell suspensions were subjected to GEMs encapsulation and libraries generated according to the manual to recover the cDNA fraction.…”