Nature:如何利用單細胞mRNA測序發現罕見細胞類型?

                                          

2015年8月24日訊 /生物谷BIOON/ --近日，來自荷蘭的科學家在著名國際學術期刊nature上發表了一項最新研究進展，他們利用一種新的計算方法結合轉錄組測序發現了小腸中一些罕見的細胞類型，這對於深入了解器官的細胞組成，探究健康和疾病狀態下的組織生物學具有重要意義。

 

理解一個器官的發育和功能需要對組成該器官的所有細胞類型的特性有一個清晰的認識。傳統發現和分離細胞亞群的方法是基於幾個已知的標記基因表達出來的信使RNA或蛋白質實現的。但是對於一些罕見的細胞類型來說，鑑定出它們特定的標記基因目前仍存在很大挑戰。而發現一些罕見的細胞類型，如幹細胞，短暫存在的前體細胞，癌症幹細胞或循環腫瘤細胞，對於深入理解正常和疾病狀態下的組織生物學具有非常重要的意義。

 

為解決這一問題，研究人員首先從培養的小腸類器官中隨機挑選了幾百種不同的細胞類型進行轉錄組測序，這種培養的小腸類器官包含了哺乳動物小腸所有的細胞譜系。由於目前可用的計算方法只能對一些豐度較高的細胞類型進行確定，因此研究人員開發了一種叫做RaceID的算法，用於在複雜的單細胞群體中發現一些罕見的細胞類型。

 

研究人員利用這種計算方法發現Reg4是一種罕見的腸內分泌細胞的新標記基因，隨後他們利用Reg4富集這種罕見細胞，檢測這一細胞群體的異質性，結果表明這群細胞中存在已知的腸內分泌細胞譜系，同時還發現了一些新的細胞亞型。在對RaceID這一算法進行了驗證之後，研究人員又利用這一算法分離了Lgr5陽性的幹細胞以及由這些細胞形成的後代細胞，結果表明Lgr5陽性細胞代表了一群同種的豐度較高的幹細胞群體，同時混雜了一種罕見的Lgr5陽性的分泌細胞群。

 

這項研究通過開發一種算法分析轉錄組測序結果，能夠在複雜的單細胞群體中發現一些罕見的細胞類型及其特定標記基因，對於了解正常的和疾病狀態下的組織生物學具有重要意義。（生物谷Bioon.com）

 

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相關會議：2015第三屆非編碼RNA學術研討會

 

 

Single-cell messenger RNA sequencing reveals rare intestinal cell types

 

Dominic Grün,Anna Lyubimova,Lennart Kester,Kay Wiebrands,Onur Basak,Nobuo Sasaki,Hans Clevers & Alexander van Oudenaarden

 

Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids1, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances2. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells3. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5-positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.