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

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

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

 

目前發育生物學的實驗基礎有兩類，

一是活體小鼠，做爲模式動物；爲了把小鼠上的研究造福於人類（診斷、篩查、藥物開發、治療），就必須在人體上得以確認，但根據倫理，全部的破壞性實驗都是不能用人的，因此一大批的曲線救國策略出現了：誘導幹細胞、類器官。

二就是誘導幹細胞和類器官等模擬策略，雖然說和活體有很大的區別，但已是妥協下的最優策略了。模擬的細胞與真人的細胞「同宗同源」，是最高大上的實驗材料。

（斑馬魚、線蟲什麼的就不說了，太偏理論了）

 

一個顯然的問題：

誘導幹細胞和類器官是人爲製造的，雖然說遺傳物質是跟真人同樣，但重編程已經致使基因表達發生了很大的變化，咱們的目的就是讓誘導幹細胞和類器官儘量的接近in Vivo的狀態。

單細胞測序技術的出現，使得細緻的評估in Vitro和In Vivo的model成爲可能，也就是這篇文章在作的事。

 

難點：

• 基因表達是表型，由不少因素驅動，直接比較基因表達得出的結論不必定可靠；最好還有表觀的數據
• 基因表達差別太大，30k的基因，模式不可勝數，簡單的對應關係是不存在的；
• confounder太多，比較極難
• 跨平臺數據的整合

 

咱們看看這篇文章作了什麼。

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

碎碎念：這裏就是要找對應關係，其實很難，維度過高，粗暴的無監督比對不是很可靠，固然確定是能給出一個結果的。

To relate the organoid-derived PC state to in vivo PCs, we first generated an unbiased reference in vivo scRNA-seq data set. 確實，須要構建一個好的reference，比對的方向要明確。

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). 這裏很良心，核糖體和線粒體基因都考察了。

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.

仍是離不開聚類，特定marker的標定（antimicrobial peptide），用AUC來評估marker，再與已知的數據庫對比一下。

 

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). 

而後就是測序in Vitro的細胞了，這裏竟然還用了effect size，顯然在炫技。

 

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).

純化In Vitro，而後拿出來去和In Vivo比對。

 

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