10X空间转录组数据分析之细胞单元

hello，大家好，今天我们来分享一个新的、很有意思的内容，细胞单元，在我之前分享的文章里面10X空间转录组的位点注释和临近通讯，提到过这个内容，但是不够系统，今天我们就来详细分析一下什么是细胞单元。

先来看一下概念，细胞单元：by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP（造血干细胞和多能祖细胞） ‘pocket-like’ units（细胞单元） (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors.也就是说，一个细胞单元的组成包括目标细胞类型和围绕在其周围的细胞类型，这几种细胞类型关系密切，相互合作行使一种重要的生物学功能，这为我们空间转录组的发展提供了很好的机会和借鉴，毕竟，研究组织并不能割裂的研究，而是要放在一个联合的背景下进行深入分析。

当然，细胞单元的部分有些占据了很大的部分，有的占有的部分比较小，Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS.这些在细胞单元显著富集的细胞类型，行使更加重要的生物学功能，我们来详细看看整个的分析过程。

首先是单细胞的部分，当然，做了很好的个性化分析，包括细胞注释和通讯分析，这部分内容是基础，需要很大的精力，基础的问题也是最根本的问题。

其中单细胞分析的部分这里有一个部分是对HSC/MPP亚群的一个分析，很值得注意，需要重点分析一下：

CD93-enriched HSCs/MPPs exhibit enhanced stem cell properties

To further dissect the transcriptome features of HSCs/MPPs, we focused on HSC/MPP 1–3 clusters and explored the underlying differences among them.GO and DEG analyses indicated enrichment of hematopoietic regulation and differentiation, active translation processes, and cell cycle regulation in HSC/MPP 1–3, respectively。（亚群的富集分析）

In HSC/MPP1, the expression of Mycn, Mecom, and Hlf was enriched; in HSC/MPP2, metabolic and lineage-specific genes were highly expressed; and in HSC/MPP3, cell cycle-related genes showed highly enriched expression（三个群体现了不同的基因表达模式）。To further evaluate the stem cell properties of heterogeneous HSCs/MPPs, we calculated hscScore(这个地方我们随后分享) as previously reported,and found that the highest score was seen in HSC/MPP1; meanwhile, trajectory analysis among HSC/MPP subsets and downstream myeloid and lymphoid progenitors revealed that HSC/MPP1 occupied the top of hematopoietic hierarchy. Overall, these results indicate that HSC/MPP1 exhibits the most robust stem cell transcriptome feature, compared to HSC/MPP2–3.（这里大家注意对一种细胞类型干性的研究，首先是hscScore，分数越高，干性越高，轨迹分析发现这种细胞类型位于发育的起点，这些手段都是来辅助证明这种细胞类型的干性很强，值得大家借鉴）。

我们来科普一下，什么是hscScore，有机会我们详细分享一下这个算法，简单来讲，就是根据现有的数据进行建模，建立一个细胞干性和表达谱的一个数学模型，以此来预测细胞类型的干性分数，这个大家先了解即可。

当然了，其中的第一个群高表达CD93，Given that Cd93 (also known as AA4.1) has been shown to be expressed in hematopoietic progenitors。we asked whether the combination of CD93 and other HSC/MPP markers, such as Lineage–Sca-1+c-Kit+ (LSK), Flt3–LSK, and CD150+CD48–LSK (SLAM-LSK),29,30 can further purify HSCs/MPPs with robust stem cell properties.（当然，研究也证明，CD93确实可以提高该细胞类型的干性）。当然之后作者也做了一些其他的验证，Taken together, CD93 is not only a surface marker for characterizing HSC/MPP heterogeneity, but also functionally required for FL HSC/MPP development.（这个地方是大家最应该关注的地方）。

接下来用单细胞数据进行细胞通讯分析（CellPhoneDB）,当然，单细胞的通讯分析确实识别了很多重要的细胞交流。

单细胞分析识别的信号交流在空间上是如何分布的？接下来就是单细胞空间的联合分析，这部分之前就分享过了，这里回顾一下。

接下来我们的重点，Identification of expansion units of HSCs/MPPs（识别细胞单元）

确定细胞间相互作用的架构基础，三种spot的定义，点内，点间和其他。

然后对目标细胞类型的三种spot进行细胞类型的富集分析，发现点内得分最高的 EC 接近 HSC/MPP，hepatoblast and stromal cell with the highest scores respectively for inter-spots and other distant spots were less close to HSCs/MPPs;（方法确实很赞，很有创新性），unexpectedly, macrophage with higher scores for intra- and inter-spots than that for other distant spots was considered as a novel niche component spatially close to HSCs/MPPs（巨噬细胞在点内和点间都显示出富集效果），

To quantitatively compare the interaction between HSCs/MPPs and different niche cells, we defined an enrichment fold based on the ratio of the enrichment score median for each spot type to the enrichment score median for all spots（富集倍数的定义），Consequently, we found that macrophage showed a 11.52-fold enrichment in the intra-spots and a 1.31-fold in the inter-spots, EC showed a 1.62-fold enrichment in the intra-spots, while hepatoblast and stromal cell showed less enrichment in the intra-spots（结果确实显现了富集效果，不是随机出现的）。

Furthermore, to validate the spatial relationship at nearly single-cell resolution, we analyzed the mouse E13.5 FL ST data based on Stereo-seq（这个技术大家可以关注一下，能实现空间的亚细胞精度）, which is a sequencing-based spatially resolved transcriptomic technology with subcellular resolution.As a result, we found that macrophages showed a high enrichment both in intra-spotsStereo-seq and interspotsStereo- seq(多种技术证明了点内和点间的细胞类型富集效果).Finally, the consistent result was validated by CSOmap(这个是张泽民团队开发的软件，依据单细胞数据推断细胞之间的空间位置，感兴趣大家可以看一看), an analytic method to reconstruct the cell spatial organization de novo based on scRNA-seq data。Taken together, the results from three analytic methods of spatial information support that macrophage serve as an important niche cell with the closest relationship with HSCs/MPPs.（说白了，几种细胞类型在空间上处于显著的临近状态）。

At molecular level, we examined the spatial expression of interactive signals predicted by CellPhoneDB analysis, and found that genes encoding ligands, such as MDK and PTN, were highly expressed in niche cells of intra-spots and inter-spots（单细胞识别的显著配受体在空间上也体现出了很强的共定位现象）。

and genes related to receptors, such as LRP1 and PTPRS, were enriched in HSCs/MPPs-localized spots.这些结果表明空间邻近性促进了功能上支持 HSC/MPP 扩展的信号交互。 鉴于intra-spots和inter-spots的特点是细胞之间的空间接近性和丰富的交互信号，我们将它们定义为扩展单元，其中HSCs/MPPs位于斑点的核心并与周围的小生境细胞斑点相互作用。 ，我们证明 FL HSCs/MPPs 在许多单位中扩展，其中巨噬细胞和几种生长因子，包括 MDK 和 PTN，高度富集。

怎么样，这样的细胞单元分析，是不是打开了我们的思路呢？？后续作者还做了荧光验证，证实这个结果。

生活很好，有你更好