PhD Candidate University of California, Los Angeles Santa Monica, California, United States
Introduction:: Human cerebral organoids enable the discovery of cellular mechanisms underlying neurodevelopment and neurological disorders. Unfortunately, such valuable models lack in maturity compared to primary tissue in terms of recapitulation of developmental trajectories, cell type specifications, and structural morphology. One major limitation of these in vitro cultures is the restricted oxygen and solute diffusion within the organoids which leads to increased cellular stress and apoptosis. To address these challenges, previous studies have utilized fluidic devices and hyper-oxygenation (hyperoxia, 40% O2) to improve the brain organoid phenotype and sustain growth. However, little is known about how these stimuli facilitate key aspects of brain organoid development such as cortical layer differentiation and expression of neuronal transcriptional signatures. Here, we uncover the individual and additive effects of laminar fluidic flow (flow) and hyperoxia. We demonstrate that both flow and hyperoxia function to reduce oxidative stress and enhance cortical phenotype. In addition, through transcriptomics and metabolomics we show that hyperoxia reduces metabolic stress genes and facilitates a shift from glycolysis towards oxidative metabolism. Understanding how the extracellular environment impact neural development will greatly advance our cerebral organoid models.
Materials and Methods:: We generated forebrain organoids from a human induced pluripotent stem-cell line following the Watanabe protocol [1]. Laminar flow was applied using the Microwell Flow Device, as established in our previous work [2], and hyperoxia was controlled using an oxygen regulator. After 18 days of static normoxia culture (20% O2), we split the organoids into four conditions: static + normoxia (SN), static + hyperoxia (SH), flow + normoxia (FN), and flow + hyperoxia (FH). The organoid growth and tissue architecture was characterized using brightfield and immunofluorescent (IF) microscopy, respectively at Days 35 (D35) and 56 (D56). Preparation for IF imaging was performed by fixing, cryopreserving, and cryosectioning samples onto slides. We performed transcriptomic analysis using both bulk RNA-sequencing and single-cell RNA-sequencing (scRNAseq) at D35 and D56 to understand the transcriptional signatures between conditions and within cellular subtypes. Organoid metabolism was analyzed via carbon isotope tracing in conditioned media and Seahorse assay.
Results, Conclusions, and Discussions:: Brightfield analysis showed accelerated growth in all experimental conditions (FN, SH, FH), with maximal growth observed in FH samples, indicating an additive advantage of flow and hyperoxia (Fig. 1A). On D35, we immunostained for forebrain markers FOXG1, LHX2, and neural progenitor markers N-Cadherin and NESTIN to visualize neuroepithelial rosette-structures; as a result, we found both flow and hyperoxia significantly enhance early development of neural rosettes (Figs. 1B-C).
By D56, flow samples displayed an increased cortical plate region compared to static samples, suggesting flow-induced mechanotransduction stimulates cortical plate expansion. By analyzing the angle of division of mitotic radial glia progenitors and basal radial glia (bRGs) via phospho-histone H3 and phospho-vimentin staining, we observed that hyperoxia conditions featured more vertically dividing cells and a greater presence of bRGs. From scRNAseq, we identified major neuronal subtypes (Fig. 1D) and found that hyperoxia increased the populations of progenitor and mature neuron subtypes. Furthermore, FH samples featured a reduced inhibitory neuron population by almost 10-fold compared to all other conditions. Importantly, genes associated with glycolytic stress were significantly reduced under hyperoxia (Fig. 1E), suggesting reduction of metabolic stress markers correlates with improved neural development. We further investigated impacts of oxygenation on metabolic phenotype via Seahorse and metabolomics on early-stage (~D21 & D35) organoids, revealing that even short exposure to hyper oxygenated environments enhances metabolic phenotype and TCA metabolite excretion.
By systematically testing two canonical modes of applying stimulation (i.e., flow and hyper-oxygenation), we deduced their individual and combined contributions on advancing organoid development. Collectively, our findings suggest that hyperoxia impacts organoid growth via upregulation of neural progenitor transcriptional behavior and improvements in early-stage metabolic efficiency, whereas the fluidic flow generally improves the intra-tissue solute transport and imposes mechanotransductive cues on the organoid periphery. Optimizing how environmental factors influence cellular stress in cerebral organoids will revolutionize our models of neurodevelopment and disease.
Acknowledgements (Optional): :
This material is also based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE- 2034835.
References (Optional): : [1] Watanabe, Momoko, et al. "Self-organized cerebral organoids with human-specific features predict effective drugs to combat Zika virus infection." Cell reports 21.2 (2017): 517-532. [2] Payne, Marie C., et al. "Microwell‐based flow culture increases viability and restores drug response in prostate cancer spheroids." Biotechnology Journal (2023): 2200434.