Inter-gastruloid heterogeneity revealed by single cell transcriptomics time course: implications for organoid based perturbation studies

Recent advances in organoid and genome editing technologies are allowing for perturbation experiments at an unprecedented scale. However, before doing such experiments it is important to understand the gene expression profile in each of the organoid’s cells, as well as how much heterogeneity there is between individual organoids. Here we characterise an organoid model of mouse gastrulation called gastruloids using single cell RNA-sequencing of individual organoids at half-day intervals between day 3 and day 5 of differentiation (roughly corresponding to E6.5-E8.75 in vivo). Our study reveals multiple differentiation trajectories that have hitherto not been characterised in gastruloids. Intriguingly, we observe that individual gastruloids displayed a strong bias towards producing either mesodermal (largely somitic) or ectodermal (specifically neural) cell types. This bifurcation is already seen at the earliest sampled time point, and is characterised by increased activity of WNT-associated pathways in mesodermally-biased gastruloids as compared to neurally-biased gastruloids. Notably, at day 5, mesodermal gastruloids show an increase in the proportion of neural cells, while neural gastruloids do not produce notably more mesodermal cells. This is in line with previous studies on how the balance between these cell types is regulated. We demonstrate using in silico simulations that without proper understanding of the inter-organoid heterogeneity, perturbation experiments have either very high false positive or negative rates, depending on the statistical model used. Thus in future studies, modelling of inter-organoid heterogeneity will be crucial when designing organoid-based perturbation studies. MULTI-seq (10X + individual gastruloid barcoding) for gastruloids between day 3 and day 5 of differentiation at half day intervals. A total of 4 experiments were performed. In the first experiment, day 3 and day 4 gastruloids were sequenced using 10X without MULTI-seq. The first MULTI-seq experiment involved 24 day 5 gastruloids spread over 3 10X lanes. The second MULTI-seq experiment involved 3 pools of day 3 and day 3.5 gastruloids (“exp2A_d3_d3.5”, “exp2C_d3_d3.5”, “exp2D_d3_d3.5”; note pools B and D were not sent for 10X sequencing), as well as 3 pools of day 4 and day 4.5 gastruloids (“exp3A_d4_d4.5”, “exp3B_d4_d4.5”, “exp3C_d4_d4.5”). Unfortunately, the day 3 and day 3.5 gastruloid data quality was too poor and these data were excluded in downstream processinng. For the day 4 and day 4.5 gastruloids, out of the 3 pools (pool A, B, and C), only pools B and C were sequenced, and from these only lanes (samples) 2 and 3 from pool B, and lanes 1 and 2 from pool C. The third experiment involved 1 pool of 24 day 3 gastruloids, spread across 2 10X lanes (“exp4_d3”), as well as one pool of 16 day 3.5 and 16 day 4 gastruloids, spread across 4 10X lanes (“exp5_d3.5_d4”), and finally 16 day 4.5 and 16 day 5 gastruloids, spread across 3 10X lanes (“exp6_d4.5_d5”). The MULTI-seq barcodes for this third experiment were additionally resequenced at higher depth and using 16 PCR amplification cycles during library preparation due to low yield.

类器官（organoid）与基因组编辑技术的最新进展，使得扰动实验的规模达到了前所未有的水平。然而，在开展此类实验前，有必要先明确每一类器官细胞的基因表达谱，以及不同类器官个体间的异质性程度。本研究针对名为类原肠胚（gastruloids）的小鼠原肠胚形成（gastrulation）类器官模型展开表征，在分化第3天至第5天期间以半日为间隔，对单个类器官进行单细胞RNA测序（single cell RNA-sequencing），该时间窗大致对应体内发育阶段E6.5-E8.75。本研究揭示了此前在类原肠胚中尚未被报道的多条分化轨迹（differentiation trajectories）。值得注意的是，本研究观察到单个类原肠胚呈现出显著的分化偏向性：要么倾向于生成中胚层（以体节细胞为主），要么倾向于生成外胚层（特指神经细胞）。这种分化分支在最早的采样时间点即可观测到，且与偏向神经分化的类原肠胚相比，偏向中胚层分化的类原肠胚中WNT相关通路的活性显著上调。值得注意的是，在分化第5天时，偏向中胚层的类原肠胚中神经细胞的占比有所上升，而偏向神经分化的类原肠胚则未显著生成更多中胚层细胞。该结果与此前关于这两类细胞类型间平衡调控机制的研究结论相符。本研究通过计算机模拟（in silico simulations）证实，若未能充分理解类器官间的异质性，扰动实验的假阳性（false positive）或假阴性（false negative）率会极高，具体结果取决于所采用的统计模型。因此，在未来的研究中，在设计基于类器官的扰动实验时，对类器官间异质性的建模将至关重要。

本研究针对分化第3天至第5天、以半日为间隔采样的类原肠胚，采用MULTI-seq技术（结合10X平台与单个类器官条形码标记）进行测序，共开展4组实验。
第1组实验中，研究人员未使用MULTI-seq技术，仅通过10X平台对分化第3天和第4天的类原肠胚进行测序。首次MULTI-seq实验共纳入24个分化第5天的类原肠胚，分布于3个10X测序泳道中。第二次MULTI-seq实验包含3组分化第3天和第3.5天的类原肠胚混合样本（分别命名为"exp2A_d3_d3.5"、"exp2C_d3_d3.5"、"exp2D_d3_d3.5"；需注意样本池B与D未送至10X平台进行测序），以及3组分化第4天和第4.5天的类原肠胚混合样本（分别命名为"exp3A_d4_d4.5"、"exp3B_d4_d4.5"、"exp3C_d4_d4.5"）。遗憾的是，分化第3天和第3.5天的类原肠胚测序数据质量极差，因此在后续分析中被剔除。针对分化第4天和第4.5天的类原肠胚样本，在3组混合样本（A、B、C池）中，仅对B池与C池进行了测序；其中B池仅使用泳道2与3，C池仅使用泳道1与2。第3组实验包含1组24个分化第3天的类原肠胚混合样本，分布于2个10X测序泳道（命名为"exp4_d3"）；以及1组包含16个分化第3.5天和16个分化第4天的类原肠胚混合样本，分布于4个10X测序泳道（命名为"exp5_d3.5_d4"）；此外还有1组包含16个分化第4.5天和16个分化第5天的类原肠胚混合样本，分布于3个10X测序泳道（命名为"exp6_d4.5_d5"）。由于该组实验的测序文库产量较低，第三次实验的MULTI-seq条形码序列在文库制备阶段额外进行了更高深度的重测序，并采用了16个PCR扩增循环。