10X单细胞空间联合分析表征Ureter的形态和相互作用

hello，又到周五，一周的收官之战，我们要继续我们的单细胞空间联合分析的大业，今天的参考文献在Ureter single-cell and spatial mapping reveal cell types, architecture, and signaling networks，看看这一次单细胞空间的联合分析又给我们带来什么样的分析内容。

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搜集一下marker

免疫

• markerast cells (KIT, TPSAB1),
• NK cells (KLRD1, CD247)
• B cells (CD79A, JCHAIN)
• dendritic cells (HLA-DRA, HLA-DRB1)
• macrophages (CD14, C1QA)
• classical (S100A8, S100A9) and non-classical (MS4A7, LILRB2, SELPLG) monocytes.
• regulatory T (Treg) cells (FOXP3, CTLA4),
• central memory T (Tcm) cells (CCR7)
• resident memory T (Trm) cells (ZNF683, CD69, ITGA1).
• Expression of cytokines INFG and TNF in cluster 14 Trm cells suggests that this cluster may have been recently activated
• two clusters of CD8 effector T cells, the GZMK+ cluster 5 and the GZMH+ cluster 9.
• mucosal-associated invariant T (MAIT) cells (KLRB1, IL23R, IL18R1)

间质

• two PECAM1+ endothelial cell clusters, which could be separated into venous endothelial cells (NRP1, NRP2), and arterial endothelial cells (GJA5, BMX).
• smooth muscle cell cluster (ACTA2, MYH11)
• （4个成纤维细胞群）Cluster 5 fibroblasts expressed high levels of COL1A1 and was the most distinct from the other fibroblasts in the ureter based on its position on the UMAP. The others, while clustered closely together, could still be clearly distinguished by unique markers and were labeled HAS1^hi, APOE^hi, and GAS1^hi 。

Summary

Tissue engineering offers a promising treatment strategy for ureteral strictures, but its success requires an in-depth understanding of the architecture, cellular heterogeneity, and signaling pathways underlying tissue regeneration（Tissue engineering为ureteral strictures提供了一种很有前景的治疗策略，但其成功需要深入了解组织再生的结构、细胞异质性和信号通路 ）. Here we define and spatially map cell populations within the human ureter using single-cell RNA sequencing, spatial gene expression, and immunofluorescence approaches（单细胞、空间 + 免疫荧光）. We focused on the stromal and urothelial cell populations（基质和尿路上皮细胞群） to enumerate distinct cell types composing the human ureter and inferred potential cell-cell communication networks（推断细胞通讯网络）* underpinning the bi-directional crosstalk between these compartments（细胞类型之间的串扰）. Furthermore, we analyzed and experimentally validated the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells supported organoid generation in vitro and accurately predicted the differentiation trajectory from basal stem cells to terminally differentiated umbrella cells（看来也研究了发育）. Our results highlight essential processes involved in adult ureter tissue homeostasis and provide a blueprint for guiding ureter tissue engineering（构建图谱，进行指导）.

Introduction

The ureter is a tube that carries urine from the kidney to the bladder; it is often overshadowed by the two organs that it connects(两个连接的器官) and neglected in molecular studies. However, congenital defects（先天缺陷） and injuries of this structure can lead to significant medical issues, which severely compromise patients’ quality of life. Ureteral stricture is a common sequelae of iatrogenic injury from surgery or radiation but can also be caused by benign conditions such as impacted ureteral calculus, retroperitoneal fibrosis, or abdominal aortic aneurysm（Ureteral stricture是手术或放疗引起的医源性损伤的常见后遗症，但也可由良性病症引起，例如阻生性输尿管结石、腹膜后纤维化或腹主动脉瘤。）. If left untreated, ureteral stricture can lead to pain, repeat infections, kidney stones, and permanent loss of renal function（后遗症还挺严重）. The surgical repair approach depends on stricture location, length, density, and other factors such as previous treatments and/or radiation. Long ureteral strictures can be particularly challenging to repair and can involve the use of non-native tissue such as buccal mucosal graft, appendiceal flap, or ileal ureteral interposition. Long-term complications of bowel use in the urinary tract include recurrent urinary tract infection (UTI), metabolic derangements, and potential renal function deterioration(还真挺严重，跟临床很相关)。

Tissue engineering（组织工程） of human ureters is an emerging field and could be an alternative treatment approach in these situations. However, for successful ureter tissue engineering, several problems need to be considered, including physiological characteristics（生理特征） and local environment of the tissue（组织微环境）, the type of scaffold used（支架的使用）, and, most importantly, a detailed understanding of the individual cell types in the adult human ureter（最重要的就是对组织结构和细胞类型的表征）. Advancements in single-cell RNA sequencing technologies have revolutionized our understanding of the cellular complexity for a myriad of different tissue types, both in normal and diseased states. Single cell transcriptomics has been used to study the mouse and human kidney; however, these analyses have focused on interrogating the cellular composition of the kidney with a focus on the kidney interstitium and nephrons, but have not described the urothelial compartment（其实就是单细胞无法表征组织区室）. In fact, with respect to the urothelium, single cell transcriptomic studies have been reported only on the human bladder, but not the human ureter. Historically, the urothelium of the ureter has been overlooked and/or considered continuous with the urothelium of the bladder. While they share many characteristics（看来这里组织存在连续性，必需更多的方法来表征空间分布）, the ureter and bladder are fundamentally different tissues with the ureter arising from the intermediate mesoderm and the bladder arising from the endoderm. Therefore, an in-depth analysis of the human ureter is critical to uncover and understand novel developmental networks and physiological aspects of this tissue（发育和分析网络）。

In this study, we defined the single-cell landscape of human ureter tissues comprised of 30,942 cells from ten different patients. Analysis of these data revealed a previously underappreciated complexity in the immune, stromal and urothelial compartment（免疫、间质和尿路上皮区室的复杂性）. We identified a proliferative stem-like basal urothelial cell population, with potential functional roles in response to tissue injury/inflammation, as well as the ability to support the in vitro generation of organoids. We also investigated the detailed spatial relationships among four types of fibroblasts by complementing single-cell transcriptomic data with spatial transcriptomics and immunofluorescence（空间转录组和免疫荧光）. Furthermore, we interrogated potential cellular communications（同时也要分析细胞之间的相互交流） between fibroblasts and urothelial cells, enhancing our understanding of the putative pathways involved in homeostasis and regeneration. These results promote a detailed understanding of the cellular heterogeneity and signaling networks within the human ureter and serve as the foundation for future tissue engineering endeavors（这些结果促进了对人类输尿管内细胞异质性和信号网络的详细理解，并为未来组织工程的努力奠定了基础）。

Results

Single-cell profiling and unbiased clustering of human ureter cells（单细胞图谱）

To uncover the cellular complexity of the human ureter, we performed single-cell RNA sequencing (scRNAseq) on human ureter tissues（首先运用单细胞技术）. Normal human ureter tissue was procured from patients undergoing radical cystectomy, immediately dissociated into single cell suspensions, and processed for scRNAseq. After quality control and filtering, a total of 30,942 single cells from ten different human ureter samples were segregated into 20 different clusters using Seurat’s unsupervised clustering algorithm（现在的单细胞文章都已多样本、大细胞量著称）. These clusters were visualized by Uniform Manifold Approximation and Projection (UMAP), where each color represents a different cell population ordered from the largest (4,200 cells in cluster 0) to the smallest (94 cells in cluster 19). Patient samples and sexes were uniformly distributed throughout the UMAP with the exception of cluster 5（性别差异也考虑了进来）, which was dominated by female samples, with the highest proportion of cells coming from sample U7. We then performed differential gene expression analysis to aid in the classification of each cell cluster（差异表达基因）. Leveraging established markers, we could separate the clusters into three major compartments, urothelial (eight clusters expressing KRT20, KRT8, KRT17, MIK67, KRT5, KRT13, UPK3A), stromal (four clusters expressing ACTA2, FLT1, COL1A2, DCN), and immune (eight clusters expressing TRAC, CD8A, NKG7, TPSB2, CD163, FCN1, LST1)（还是依据marker来定义细胞类型）. We noted an absence of neuronal cell types in our dataset, but this is consistent with other reports describing difficulties in isolating intact neurons during tissue dissociation. Pearson correlation coefficient based on the expression of the top 2,000 variable genes between all possible cluster pairs confirmed that clusters within each compartment share more similar expression profiles when compared to clusters in other compartments, corroborating our classification（clusters之间的相关性分析）. We thus proceeded to examine each subset separately to improve the resolution of cell types.

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Classification of the immune cells（免疫细胞的划分）

Reanalysis of the immune subset resulted in 16 clusters, with minimal patient-specific and sex-specific effects（去除了批次效应）. Following differential gene expression analysis, we identified one cluster each for mast cells (cluster 13: KIT, TPSAB1), NK cells (cluster 3: KLRD1, CD247), and B cells (cluster 6: CD79A, JCHAIN). We also observed several clusters within the monocyte lineage (clusters 0, 7, 8, 10, and 12) grouped together on the UMAP, with varying expression of the monocyte marker CD14. Higher levels of CD14 were observed in dendritic cells (cluster 7: HLA-DRA, HLA-DRB1) and macrophages (cluster 8: CD14, C1QA). Three CD14/CD68-expressing monocyte clusters with high expression of ficolin 1 (FCN1) were also identified, and they were further divided into classical (clusters 0 & 10: S100A8, S100A9) and non-classical (cluster 12: MS4A7, LILRB2, SELPLG) monocytes. Interestingly, cluster 0 classical monocytes appeared to co-express intermediate markers, including BCL6 and STAT3（细胞定义的marker我们最还是搜集一下）。

We also identified eight different clusters of T cells (clusters 1, 2, 4, 5, 9, 11, 14 & 15)（T细胞的详细划分）. The largest T cell cluster was negative for CD4 and CD8 expression and was referred to as double negative T cells (cluster 1). Also amongst the T cell clusters were regulatory T (Treg) cells (cluster 11: FOXP3, CTLA4), central memory T (Tcm) cells (cluster 4: CCR7), and two clusters of tissue resident memory T (Trm) cells (clusters 2 & 14: ZNF683, CD69, ITGA1). Expression of cytokines INFG and TNF in cluster 14 Trm cells suggests that this cluster may have been recently activated. We also identified two clusters of CD8 effector T cells, the GZMK+ cluster 5 and the GZMH+ cluster 9. Finally, we resolved a cluster of mucosal-associated invariant T (MAIT) cells (cluster 15: KLRB1, IL23R, IL18R1), which are innate-like T cells that play an important role in the antibacterial respons（全是一些细胞定义的分析结果，虽然是基础，但最为重要）。

It is important to note that the ten ureter tissues used for the single cell study were free from any existing injury or infection, based on medical record and pathologist examination of the tissues, with the potential exception of patient U7, who had a history of chronic urinary tract infections (UTIs). Furthermore, in the urinary tract, it has been reported that the adaptive immune response is limited（获得性的免疫反应很有限）, requiring an efficiently primed innate immune component. Therefore, the monocyte cell types (clusters 0, 10, and 12) likely play important homeostatic roles. Nonetheless, the heterogeneity of immune cell types in normal human ureters was strikingly high（免疫细胞类型的异质性非常高 ）。

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Classification of the stromal cells（间质细胞的划分）

The analysis of the stromal cell subset resulted in seven clusters, with even distribution across patient samples and sexes. Each stromal cluster expressed a unique gene set, allowing for unambiguous annotation of each cell type. As expected, we identified two PECAM1+ endothelial cell clusters, which could be separated into venous endothelial cells (cluster 1: NRP1, NRP2), and arterial endothelial cells (cluster 6: GJA5, BMX). We also identified one smooth muscle cell cluster (cluster 2: ACTA2, MYH11) and four distinct fibroblast clusters (clusters 0, 3, 4, and 5). Cluster 5 fibroblasts expressed high levels of COL1A1 and was the most distinct from the other fibroblasts in the ureter based on its position on the UMAP. The others, while clustered closely together, could still be clearly distinguished by unique markers and were labeled HAS1^hi (cluster 0), APOE^hi (cluster 3), and GAS1^hi (cluster 4)（还是一些细胞定义的结果，需要好好搜集一下）。

To gain insight into the functional differences between the four fibroblast clusters（成纤维群之间的功能差异）, we performed gene set enrichment analysis on differentially expressed genes across the fibroblasts（基因富集分析）. Each fibroblast cluster had unique Gene Ontology (GO) term enrichments, suggesting that they were functionally distinct. Cluster 5 fibroblasts (COL1A1^hi) likely serve a prominent structural role and were significantly enriched for GO terms related to extracellular matrix binding and collagen binding. Cluster 3 fibroblasts (APOE^hi) were enriched for terms related to chemokine activity, and in the skin, apolipoprotein E (APOE) expressing fibroblasts were thought to have pro-inflammatory properties. Cluster 4 fibroblasts (GAS1^hi) were enriched for terms related to unfolded protein and heat shock protein binding. In fact, growth arrest specific-1 (GAS1) is an anti-mitogenic factor that mediates growth arrest of fibroblasts. The Saccharomyces cerevisiae homologue of GAS1 plays an important role in the endoplasmic reticulum stress response. Thus, this population of fibroblasts might play a role in upregulating pathways involved in protein homeostasis. The largest cluster of fibroblasts, cluster 0 (HAS1^hi), was enriched for the term “fibronectin binding”. Hyaluronan synthase 1 (HAS1) is responsible for the production of hyaluronan, which is a key constituent of the extracellular matrix and is involved in cell migration, wound healing, and tissue repair. Hyaluronan has also been implicated in regulating the deposition of fibronectin and collagen, which together provide the framework for the ingrowth of blood vessels and fibroblasts during tissue healing and regeneration(这部分探索群之间的功能差异，还是GO富集).

In a different attempt to better understand the four fibroblast populations, we leveraged the Visium Spatial Gene Expression platform to generate spatial transcriptomic profiles of human ureter cross sections（为了更好地了解四种成纤维细胞群体，利用 Visium 空间基因表达平台来生成人类输尿管横截面的空间转录组谱）. While this analysis did not reach single-cell resolution, it allowed us to clearly distinguish the urothelial layer (KRT5⁺ basal cells and UPK1A⁺ umbrella cells) from the outer stromal layer (ACTA2⁺ smooth muscle cells and DCN⁺ fibroblasts). Therefore, by querying the markers used to specify each fibroblast cluster in the scRNAseq analysis(单细胞空间联合), we could visualize how each fibroblast subtype was distributed in the stromal layer. The markers chosen were SEMA3C, GPC3, GAS1, and LAMC3 for clusters 0, 3, 4, and 5, respectively. Clusters 0 (HAS1hi) and 3 (APOEhi) were diffusely spread throughout the interstitial layer. Cluster 4 (GAS1^hi) was localized towards the periphery of the interstitial layer. Finally, Cluster 5 (COL1A1^hi) was localized closest to the urothelium in contrast to the other fibroblast clusters（这部分用单细胞空间联合的方法表征成纤维不同细胞群的空间位置）.

Finally, we used commercially available antibodies that specifically distinguished, either alone or in combination, the fibroblast subtypes（免疫荧光验证）. Cluster 0 was identified by being LIF1⁺/COL15A1^- and Cluster 5 by LIF1^-/COL15A1⁺. Cluster 5 could also be uniquely visualized as FOXF1⁺ cells, which are localized just below the urothelium and is consistent with the spatial gene expression data. Moreover, cluster 3 fibroblasts clearly stained for APOE and could be seen interspersed between the ACTA2⁺ smooth muscle bundles. The immunofluorescence staining not only validated the RNA expression-based classification of the fibroblasts but also confirmed their spatial distributions within the tissue. Together, these data demonstrate a so far unappreciated heterogeneity of the ureter interstitium that, like in the kidney, could be involved in providing critical inputs for ureter homeostasis（总之，这些数据证明了输尿管间质的迄今为止未被重视的异质性，就像在肾脏中一样，可能参与为输尿管稳态提供关键 input）。

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Classification of the urothelial cells（上皮细胞的划分）

The urothelial compartment could be resolved into eight clusters, where samples and sexes were evenly distributed across clusters（没有明显的批次）. We identified two umbrella cell clusters (4 & 7), three intermediate cell clusters (0, 1, & 5), and three basal cell clusters (2, 3, & 6) Umbrella, intermediate, and basal cells are the known major cell types within the urothelium, which span the differentiation spectrum from the least differentiated basal cells to the terminally differentiated umbrella cells（伞状细胞、中间细胞和基底细胞是尿路上皮内已知的主要细胞类型，其分化范围从分化程度最低的基底细胞到终末分化的伞状细胞）. Uroplakin (UPK) genes are routinely used as markers for umbrella cells, but studies have shown that the umbrella cells of the ureter are more heterogeneous with respect to UPK expression than the bladder. Cluster 4 umbrella cells were defined by high expression of UPK genes (UPK1A, UPK2, UPK3A) and labeled as UPK^hi. Conversely, cluster 7 cells expressed low levels of UPK genes but showed KRT20, which is often used as a terminal differentiation marker. Interestingly, cluster 7 umbrella cells expressed high levels of ferritin light (FTL) and heavy (FTH1) chain genes. This was independently confirmed with immunofluorescence staining, which revealed FTL⁺ or KRT20⁺ umbrella cells as scattered in the expected abundance throughout the lumen-facing umbrella layer(上皮细胞的划分可能更多依赖形态学和空间区域了)。

We next examined the genes differentially expressed by the three intermediate cell clusters（又是功能富集）. Cluster 0, which is the largest and most diffuse cluster, had very few distinguishing genes with the exception of cadherin-related family member 5 (CDHR5). Cluster 1 was defined by high expression of cellular retinoic acid-binding protein 2 (CRABP2) and LY6/PLAUR domain containing 3 (LYPD3). Cluster 5 showed high expression of FOXA1 transcription factor, which is involved in bladder differentiation, and grainyhead like transcription factor 3 (GRHL3), which drives terminal differentiation and barrier formation in the urothelium. Interestingly, in contrast to clusters 0 and 5, cluster 1 intermediate cells showed expression of UPK genes, suggesting that these cells might be differentiating into the umbrella cells. Indeed, immunofluorescence analysis demonstrated that intermediate cells with the highest levels of LYPD3 were just below the UPK3A⁺ umbrella cells. Cluster 5 intermediate cells (FOXA1^hi) were located deeper in the urothelial cell layer as shown by low GATA3/LYPD3 staining pattern.（功能验证的同时对群进行进一步的注释）。

Finally, there were three KRT5⁺ basal cell subtypes. Cluster 2 (SHH^hi) had the highest proportion of sonic hedgehog signaling molecule (SHH) expressing cells. Cluster 3 (JUN/FOS^hi) also contained a small number of SHH-expressing cells but was saliently marked by robust expression of JUN and FOS transcription factors. Cluster 6, the smallest of the KRT5⁺ basal cells, was unique in expressing high levels of proliferative genes including MKI67, PCLAF, TYMS, TK1, BIRC5, and RRM2 and was referred to as MKI67^hi. The basal cell clusters could be validated by immunofluorescence staining. A gradient of KRT5 and KRT17 expression marked cluster 2 with strong double staining and cluster 3 with weaker double staining while cluster 6 (MKI67^hi) was distinctly positive for MKI67 within the KRT5⁺ cell layer. Previous studies have indicated KRT14⁺/KRT5⁺ basal urothelial cells as the proliferating pool of basal cells responsible for replenishing the bladder urothelium. However, we did not detect significant levels of KRT14 in any of the KRT5⁺ basal cell clusters. This is in agreement with studies showing that, unlike the bladder urothelium, KRT14 is found in very low levels in the adult ureter urothelium, indicating basal cell heterogeneity between the two organs（异质性无处不在）.

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Reconstructing the differentiation trajectory of the human ureter urothelial cells（轨迹分析）

It is well-accepted that cells of the basal cell layer can differentiate into intermediate and umbrella cells during development as well as during adult tissue regeneration（细胞类型之间有分化关系）. In the developing ureter, a SHH-FOXF1-BMP4 signaling axis has been shown to regulate cellular pathways underlying ureter elongation and differentiation. In mouse bladder tissues, a SHH-expressing basal stem cell population can regenerate all the urothelial cell types following tissue injury or infection. Sonic hedgehog signaling molecule (SHH) is a secreted protein and a potent inducer of WNT stromal expression, stimulating proliferation of both the stromal and urothelial cells. This signaling cascade and associated proliferation are also necessary for the restoration of the bladder urothelium following injury or infection. Our scRNAseq data clearly identified a population of SHH-expressing basal cells in the adult ureter tissue, although the role of SHH in adult ureter homeostasis has not been investigated（利用单细胞的数据来推断轨迹发育）。

We began exploring the relationship among the ureter urothelial cell types with respect to SHH signaling by performing a cell trajectory analysis using Monocle3(Monocle3来推断细胞轨迹) and designating the cells with the highest SHH expression (in cluster 3) as the starting point for the trajectory. Remarkably, this analysis recapitulated urothelial differentiation from SHH^hi basal cells, through the intermediate cells, to the terminally differentiated UPK-expressing umbrella cells. Interestingly, this trajectory did not exist as a single path, but instead had four paths for basal-to-intermediate cell differentiation and two paths for intermediate-to-umbrella cell differentiation. This suggested a higher degree of plasticity than previously anticipated（发育大致方向都是有先验知识的）。

To better understand how signaling pathways are changing along the differentiation paths, we performed KEGG pathway enrichment analysis of the top 200 differentially expressed genes from each differentiation trajectory（用轨迹发育的差异基因来进行富集分析）. Surprisingly, only one of the basal-to-intermediate paths (#2) traversed through the MKI67^hi proliferative cluster and was unique for enrichment of the KEGG pathway terms “cell cycle” and “DNA replication”. The overarching theme for the four basal-to-intermediate trajectories was enrichment in “PPAR signaling” as well as “fat digestion and absorption”. Of note, peroxisome proliferator-activated receptor (PPAR) signaling has been shown to be an important upstream regulator of urothelial differentiation in mouse bladders. The two intermediate-to-umbrella trajectories were not enriched for “PPAR signaling” or “fat digestion and absorption”, suggesting that these signaling networks are only critical for the early stages of differentiation. Consistent with this notion, key genes in the PPAR signaling network were highly expressed in the basal and intermediate cells but downregulated in umbrella cells. Finally, the intermediate-to-umbrella trajectory 1 was enriched for “cell adhesion molecules”, signifying barrier formation. Taken together, our cell trajectory analysis showed that the manual selection of SHH-expressing basal cells as the starting point can accurately recapitulate the differentiation trajectory of basal cells to umbrella cells. Pathway enrichment analysis of the genes that were dynamic across the differentiation trajectories highlighted a potential role for PPAR signaling in the early stages of differentiation and a switch towards the acquisition of barrier function during terminal differentiation(对跨分化轨迹动态基因的通路富集分析突出了 PPAR 信号在分化早期的潜在作用，以及在终末分化过程中获得屏障功能的转变).

Determining the cell-cell communication networks in the adult human ureter(细胞通讯)

The bidirectional crosstalk（细胞串扰） between the urothelium and the stroma is essential for the proper development of the urothelium and smooth muscle in both bladder and ureter. To examine such communication networks between the urothelial and non-urothelial cells, we applied CellPhoneDB to infer potential receptor-ligand interactions between all cell clusters. The analysis revealed COL1A1hi fibroblasts to have the most putative interactions with other stromal and urothelial cell types whereas interactions to, from, and within the immune compartment were rather muted. These highly interactive COL1A1^hi fibroblasts reside just below the basal cell layer, prompting us to examine more closely the potential signaling from this fibroblast cluster to the three basal urothelial clusters and vice versa(各种信号潜能)。

Overall, the putative receptor-ligand communications in both directions showed significant enrichment for WNT, BMP, EGF, and TGFβ-related pathways (likelihood test, p value <0.00001). Such observation is consistent with a postulated importance of SHH expression in basal cells as SHH signaling induces stromal expression of WNT4 as well as the differentiation-associated BMP4 and BMP5. Ligand expression in the COL1A1^hi fibroblast indicated this SHH-induced pattern, with BMP4/5 signaling to the BMP receptors on the proliferative MIK67^hi basal cells, and WNT4 signaling to the frizzled class (FZD) receptors on all three basal cell populations. When we evaluated the reverse signaling from the basal cells to the fibroblasts, WNT7B ligand expression was detected in all three basal cell clusters and could signal to FZD1 and FZD10 expressed on COL1A1^hi fibroblasts. In fact, studies in mice have pointed to an important role for the interaction between urothelial WNT7B and mesenchymal FZD1, working in parallel to SHH signaling, in ureter development. The analysis also revealed Smoothened (SMO), another component of the SHH signaling network, to be expressed by the COL1A1hi fibroblasts, with potential interactions with BMP2 ligand from the JUN/FOS^hi and SHH^hi basal cell clusters. In addition, there were numerous predicted interactions from all three basal cell clusters with EGFR on the COL1A1^hi fibroblasts. Altogether, these results indicated that there are most likely numerous pathways working in parallel with SHH signaling network to mediate proliferation and differentiation of the adult ureter, and that the COL1A1^hi fibroblasts may be a central hub for mediating these processes(总之，这些结果表明，很可能有许多途径与 SHH 信号网络并行工作，以介导成人输尿管的增殖和分化，并且 COL1A1^hi 成纤维细胞可能是介导这些的中心枢纽过程).

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Sonic hedgehog expression predicts ureter organoid generation efficiency(这个分析有点意思)

Ureter organoids have emerged as a potential tool to study ureter development and homeostasis. To start laying the foundation for ureter cell culturing and differentiation ex vivo, we wanted to determine the fidelity of our cell cryopreservation method in maintaining cellular composition and gene expression patterns as well as to establish the ability of these cryopreserved cells to initiate organoid growth. To this end, we performed scRNAseq on freshly dissociated single cells and cryopreserved single cells from the same patient (U2), as well as the organoid derived from these same cryopreserved cells. We detected the major cell types in the immune, stromal, and urothelial compartments as seen in the larger cohort

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Discussion

输尿管缺陷和损伤仍然是一个重大的临床负担，如果不成功修复，会大大降低受其影响的患者的生活质量。组织工程提供了一种独特且有前途的方法来修复这些损伤。传统的组织工程方法需要使用生物材料，理想情况下，可以 (1) 保留组织的解剖结构和功能，(2) 提供适当的结构和机械特性，以及 (3) 概括可以支持适当分化的微环境器官特异性细胞类型和它们自己的细胞外基质蛋白的分泌，最终将取代人工支架。人工支架的使用有一些固有的缺点。例如，在膀胱组织重建过程中，细胞可能无法完全整合到基质支架中。此外，尿路上皮的适当成熟需要上皮细胞和基质之间的相互作用。为了避免这些陷阱，已经提出了基质自组装方法，其中组织来源的基质细胞用于产生细胞类型特异性的细胞外基质。 “自组装”技术已被用于生成各种组织，包括完整的 3D 人体膀胱。尽管这种方法通常更耗时且成本更高，但似乎利大于弊。为了实施输尿管组织工程的成功策略，我们必须了解健康人体输尿管内的细胞群、它们的信号网络以及它们的空间关系。这种详细的理解不仅将指导用于生成组织的方法的选择，而且有助于建立质量控制参数，以确保在工程组织中实现适当的细胞组成和组织结构。我们当前的知识库中缺少有关人类输尿管的这些基本数据，因此我们着手生成健康人类输尿管的细胞图谱。

利用单细胞 RNAseq，我们分析了 30,942 个输尿管细胞，跨越免疫（16 个clusters）、基质（7 个clusters）和尿路上皮（8 个clusters）区室，以描绘输尿管的细胞成分。首次揭示了正常人类输尿管中免疫细胞的丰富多样性，强调需要比较病理条件下的免疫细胞分布，以了解该器官如何自我防御。我们还对人类输尿管进行了首次空间基因表达分析和额外的免疫荧光分析，以验证 scRNAseq 的发现，并在空间上可视化该器官内先前未被重视的细胞类型，包括四种不同类型的成纤维细胞。有趣的是，成纤维细胞亚型之一的特征是 HAS1 的高表达。基于透明质酸的生物材料是组织工程中流行的新兴支架材料。因此，这种成纤维细胞亚型可能是组织工程输尿管未来发展的核心。它还指出考虑将基于透明质酸的材料作为合适的支架。此外，我们对基质隔室的深入分析也可能有助于确定培养基配方，以刺激特定患者来源的成纤维细胞群体的分离和繁殖。这可以加速细胞外基质沉积，这是基质自组装方法的常见障碍。例如，HAS1hi 成纤维细胞表达高水平的 SEMA3C（信号素 3C）。向类器官培养物中添加重组体 SEMA3A（信号素家族蛋白的另一个成员）已被证明可以显着增加类器官的大小。因此，在输尿管类器官培养基中加入 SEMA3C 作为添加剂可能对开发用于移植的自组装输尿管组织感兴趣。

上尿路的尿路上皮，包括输尿管的尿路上皮，来自中胚层，而膀胱尿路上皮则来自内胚层。然而，大多数关于尿路上皮的研究忽视了这种内在差异，并将膀胱和输尿管的尿路上皮视为一个单一的实体。因此，输尿管中更精细的细胞类型和表达模式在历史上一直被忽视。有了这个数据集，我们有机会列举输尿管特异性尿路上皮细胞类型及其各自的基因表达模式。例如，我们发现了一个新的伞状细胞群，它们表达高水平的 FLT、FTH1 和 KRT20。该群体的存在通过 FLT 和 KRT20 蛋白的免疫荧光染色独立证实。铁蛋白在输尿管中的作用尚未得到很好的研究。然而，通过铁蛋白螯合来调节铁的可用性是宿主细胞抵御泌尿病原体的关键。膀胱尿路上皮中过多的铁生物利用度已被证明会增加尿路病原体的生长。因此，这组 FTL^hi 伞状细胞可能通过螯合非生物可利用储库中的过量铁，在尿路上皮防御病原体方面发挥一些独特的作用。看看小鼠输尿管中是否也存在类似的细胞并测试消融是否可能具有功能性后果将会很有趣。同样，输尿管感染患者的单细胞分析可能进一步暗示这种伞状细胞亚型参与尿路上皮防御。最后，这种罕见的终末分化伞状细胞群为评估输尿管工程的成功提供了独特的基准。

研究还表明，尽管具有不同的胚胎起源，输尿管尿路上皮谱系规范与膀胱的谱系具有一些共同的主题。视黄酸 (RA) 信号、过氧化物酶体增殖物激活受体 (PPAR) 信号和声波刺猬 (SHH) 信号都与膀胱尿路上皮的维持、规范和分化有关。类视色素是参与胚胎干细胞和成人稳态尿路上皮分化的有效信号分子。我们的数据揭示了由高水平的细胞视黄酸结合蛋白 2 (CRABP2) 定义的中间簇之一。 CRABP2 促进视黄酸转运至视黄酸受体 (RAR) 以激活其活性。此外，在“维生素消化和吸收”KEGG 途径中，从基底到中间的细胞轨迹之一富含基因，包括视黄醇结合蛋白 2 (RBP2)。 PPAR 信号已被证明是组织修复过程中小鼠膀胱尿路上皮分化的上游调节因子，也从我们的细胞轨迹分析中发现，它是人类输尿管中基底到中间细胞分化的潜在驱动因素。我们的数据还证实了先前的发现，即 SHH 是成人尿路上皮祖细胞维持的关键介质。我们在人类输尿管中检测到表达 SHH 的基底细胞，并确定这些细胞对于体外类器官的形成至关重要。

还确定表皮生长因子受体 (EGFR) 信号是输尿管尿路上皮规范的潜在新参与者。首先，检测到两种含有表达 SHH 的细胞的基底细胞亚型，但一种同时具有高水平的 JUN 和 FOS 表达的特征。 JUN 和 FOS 一起形成激活蛋白 1 (AP-1) 复合物。 AP-1 是 EGFR 通路的下游信号模块，激活 AREG 的转录，进一步刺激 EGF 受体作为正反馈回路的一部分。其次，EGFR 受体-配体相互作用是 COL1A1^hi成纤维细胞和所有基底细胞亚型之间最重要的预测通讯之一。例如，在 COL1A1^hi 成纤维细胞上表达的 EGFR 可能会对基底细胞分泌的 MIF、COPA、TGFB、TGFA、AREG、GRN 和 BTC 产生反应。总之，这些观察结果表明输尿管稳态中 EGFR 和 SHH 信号轴之间存在复杂的相互作用，值得进一步研究。此外，描述四种信号通路（RA、PPAR、SHH 和 EGFR）中的每一种如何促进输尿管修复以应对损伤和感染也很重要。

除了提供人类输尿管的细胞图谱和阐明未来机械研究的关键方向之外，我们的研究还产生了与输尿管组织工程工作直接相关的知识。根据我们的分析，人类输尿管中的成纤维细胞亚型具有不同的表达谱，表明它们在支持尿路上皮细胞分化和生长方面具有独特的功能。特别是COL1A1^hi成纤维细胞和HAS1hi成纤维细胞在组织工程实验中应认真考虑。 COL1A1^hi 成纤维细胞在体内最靠近基底尿路上皮细胞，可以提供关键的信号输入以支持尿路上皮干细胞的维持、生长和分化。另一方面，HAS1^hi 成纤维细胞对于创建输尿管特异性细胞外基质 (ECM) 可能是必不可少的，以确保适当的器官结构。在自组装方法中包含这些患者来源的输尿管成纤维细胞可能是其成功的关键。此外，我们的数据还可用于优化组织培养基，不仅支持干细胞生长，而且促进适当的分化，形成适合移植的人类输尿管。最后但并非最不重要的是，本研究中鉴定的细胞类型标记将帮助我们从患者组织中分离特定细胞群以开始培养、确定目标细胞类型的正确比例以确保生长，并开发工程输尿管的质量控制检测.

In conclusion, our studies offer a comprehensive analysis of the many cell populations within the human ureter, as well as the first spatial transcriptomic analysis of this tissue type. This work will provide an important reference for future research into directing the accurate engineering of ureters in regenerative medicine. In addition, this work will aid in our fundamental understanding of the normal physiology, transcriptional landscape, and signaling networks in the adult ureter, a tissue that has largely been overlooked.

Method(都是一些常规的分析方法，不过多分享了)

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