In Vitro model验证 | Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types

Transcriptional benchmarking of in vitro cells to in vivo with single-cell rna-seq - 简介

Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types - 原文

目前发育生物学的实验基础有两类，

一是活体小鼠，作为模式动物；为了把小鼠上的研究造福于人类（诊断、筛查、药物开发、治疗），就必须在人体上得以确认，但根据伦理，所有的破坏性实验都是不能用人的，所以一大批的曲线救国策略出现了：诱导干细胞、类器官。

二就是诱导干细胞和类器官等模拟策略，虽说和活体有很大的区别，但已经是妥协下的最优策略了。模拟的细胞与真人的细胞“同宗同源”，是最高大上的实验材料。

（斑马鱼、线虫什么的就不说了，太偏理论了）

一个显然的问题：

诱导干细胞和类器官是人为制造的，虽说遗传物质是跟真人一样，但重编程已经导致基因表达发生了很大的变化，我们的目的就是让诱导干细胞和类器官尽可能的接近in Vivo的状态。

单细胞测序技术的出现，使得细致的评估in Vitro和In Vivo的model成为可能，也就是这篇文章在做的事。

难点：

• 基因表达是表型，由很多因素驱动，直接比较基因表达得出的结论不一定可靠；最好还有表观的数据
• 基因表达差异太大，30k的基因，模式不计其数，简单的对应关系是不存在的；
• confounder太多，比较极难
• 跨平台数据的整合

我们看看这篇文章做了什么。

Using the PC to benchmark cell type representation of conventional organoids against their in vivo counterparts

碎碎念：这里就是要找对应关系，其实很难，维度太高，粗暴的无监督比对不是很可靠，当然肯定是能给出一个结果的。

To relate the organoid-derived PC state to in vivo PCs, we first generated an unbiased reference in vivo scRNA-seq data set. 确实，需要构建一个好的reference，比对的方向要明确。

We assessed quality metrics for the number of genes, unique molecular identifiers (UMIs), mitochondrial genes, and ribosomal genes, all of which fell within expectations (all cells average: 1043 genes, 2168 UMIs, 5.4% ribosomal genes, 10.4% mitochondrial genes). 这里很良心，核糖体和线粒体基因都考察了。

Within the spectrum of cell types, we identified two clusters (2 and 11) enriched for Lyz1 expression (Fig. 1b, c), of which we determined cluster 11 to be fully mature PCs (n = 189 cells) based on uniform expression of a set of associated antimicrobial peptide marker genes such as Defa22, Defa21, and Ang4 (receiver operating characteristic (ROC) test, area under the curve (AUC) > 0.99 for markers listed; cluster 11 average: 866 genes, 3357 UMI, 3.5% ribosomal genes, 4.8% mitochondrial genes) (Additional file 1: Table S1).

We further utilized these genes (genes with AUC > 0.65 for in vivo PC) throughout our study to relate organoid-derived cell states to in vivo PCs. They are fully inclusive of the 14 high confidence markers described for Paneth cells from the terminal ileum in the recently published mouse small intestinal atlas [3]. Of note, we extended our gene list beyond truly specific marker genes that are not expressed in other cell types as we were interested in a more comprehensive set of PC-enriched genes for further comparison.

还是离不开聚类，特定marker的标定（antimicrobial peptide），用AUC来评估marker，再与已知的数据库对比一下。

We next performed scRNA-seq using Seq-Well on conventional organoids derived from a single donor ISC-enriched state (Fig. 1a).

Following scRNA-seq, we computationally identified six clusters (amongst 2513 cells × 16,198 genes meeting quality standards, see Methods) in ENR organoids, which we label as ENR1-4, and EEC-1 and -2 for two EEC types (Fig. 1d).

然后就是测序in Vitro的细胞了，这里居然还用了effect size，显然在炫技。

Having identified ENR-4 as the cell state of interest in organoids, we directly compared the top 200 most PC-like cells in ENR-4 to in vivo PCs by performing differential expression analysis (Fig. 1g).

纯化In Vitro，然后拿出来去和In Vivo比对。

Beyond these selected genes, we note a global reduction in the fraction of the transcriptome of ENR-4 cells producing the total cadre of in vivo PC marker genes (effect size 1.25, InVivo vs. ENR, *t test p < 2.2 × 10−16), suggesting that the current in vitro organoid-derived PCs are suboptimal for physiological studies (Fig. 1i).

Specifically, modulating Wnt and Notch signaling has been suggested in the literature to increase the frequency and magnitude of Lyz1 expression and protein in ISC-derived cells [29, 32,33,34].

As a result, we sought to comprehensively test if driving Wnt and inhibiting Notch truly results in a more physiologically representative PC versus the organoid-derived PC, beyond bulk measures of increased Lyz1 expression.

Rationally guided chemical induction of Wnt and inhibition of Notch drives PC marker enrichment

Chemically induced PC proteome is enriched for components of secretory lineages

Single-cell RNA sequencing profiles heterogeneity of chemically induced PCs, revealing subsets with improved transcriptional similarity to in vivo

Chemically induced PCs mimic in vivo stimulant-induced secretion and demonstrate selective modulation of bacteria in co-culture

Chemically induced PCs provide niche support and enhance conventional organoid survival

Mapping of in vivo PC-associated transcription factors to in vitro proteome and transcriptome reveals Nupr1 as important in epithelial survival