作者喇聊，追風(fēng)少年i
7月1日恍风，是2022年下半年的開始，單細(xì)胞空間持續(xù)進行時誓篱，細(xì)數(shù)一下發(fā)展朋贬，華大、新格元窜骄、尋因锦募、墨卓、諾禾等等都在布局邻遏，從行業(yè)角度看糠亩，目前國內(nèi)下游為主滔以，標(biāo)準(zhǔn)的微笑曲線做入，上游實力與創(chuàng)新能力不足，同時也意味著發(fā)展?jié)摿薮蟆?/h4>
不過呢钥弯，說實話糊饱，跟我關(guān)系不大垂寥，主要是看國內(nèi)是不是真的有民族企業(yè)家，如果沒有济似，微笑曲線可能波動一下都不可能矫废。
好了，繼續(xù)我們單細(xì)胞空間的分析內(nèi)容砰蠢，今天我們來分享一篇文獻蓖扑，The spatiotemporal program of zonal liver regeneration following acute injury,2022年6月發(fā)表于Cell Stem Cell，不用多說台舱，我們來看看研究內(nèi)容律杠。
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Summary of main events during APAP-induced liver damage and regeneration
肝臟是一個高度異質(zhì)的器官潭流。肝細(xì)胞和支持非實質(zhì)細(xì)胞在被稱為“肝小葉”的重復(fù)六邊形解剖單元中活動。血液進入小葉的角落柜去，稱為“門脈節(jié)點”灰嫉，并通過竇性通道流入引流的中央靜脈 (CVs)。這種極化的血流與肝細(xì)胞的連續(xù)消耗和分泌相結(jié)合嗓奢，產(chǎn)生氧氣讼撒、營養(yǎng)物質(zhì)和激素的梯度分布，導(dǎo)致高度異質(zhì)的微環(huán)境股耽，因此肝臟中不同位置細(xì)胞在基因表達上呈現(xiàn)顯著差異根盒，該現(xiàn)象稱為“肝臟分區(qū)”，目前尚不清楚這種空間異質(zhì)性如何影響肝臟病理和再生過程物蝙。
肝臟具有強大的再生能力炎滞。在急性劑量的藥物作用下，試圖排除這些外來物質(zhì)的中心周圍肝細(xì)胞被poisonous中間體淹沒而死亡诬乞。剩下的肝組織進入再生模式册赛，導(dǎo)致受損的小葉層迅速愈合和替換。分區(qū)再生涉及一系列相互協(xié)調(diào)的過程震嫉，對反應(yīng)有嚴(yán)格的時空控制要求森瘪。死亡的肝細(xì)胞需要被有效清除，防止多種新抗原暴露以誘導(dǎo)適應(yīng)性免疫系統(tǒng)责掏。細(xì)胞外基質(zhì)需要被快速構(gòu)建以支持組織支架柜砾，但一旦新細(xì)胞形成就要分解，以防止持久的纖維化换衬。最重要的是痰驱，起源于小葉區(qū)表達特征截然不同的肝細(xì)胞需要快速生成和重新編程，以接管中心周圍肝細(xì)胞的關(guān)鍵功能瞳浦。目前担映，在整個分區(qū)再生過程中，所有肝細(xì)胞類型的協(xié)調(diào)反應(yīng)的空間和時間動力學(xué)仍亟待探索叫潦。
研究成果
A single-cell atlas of zonal regeneration after APAP
對小鼠不同時間段進行APAP處理蝇完，研究肝臟的變化過程，其中用到了bulk矗蕊、單細(xì)胞以及空間技術(shù)表征肝臟圖譜短蜕。其中bulk轉(zhuǎn)錄組分析了不同時間點的生物學(xué)通路變化、并且利用單細(xì)胞數(shù)據(jù)解卷積bulk數(shù)據(jù)分析細(xì)胞比例的變化傻咖，當(dāng)然了朋魔，空間轉(zhuǎn)錄組分析細(xì)胞的位置變化與空間基因特征。
為了研究分區(qū)重生卿操，他們在小鼠體內(nèi)注射了300 mg/kg的APAP引發(fā)肝臟損傷警检，在不同時間點對肝臟進行采樣孙援，觀察到大量的中心周圍壞死在48小時后達到峰值。損傷的中心周圍區(qū)域在72小時后出現(xiàn)收縮扇雕，出現(xiàn)新的中心周圍肝細(xì)胞拓售，在96h后完全取代壞死組織。作者在多個時間點進行批量和單細(xì)胞RNA-seq镶奉，并進行空間轉(zhuǎn)錄組學(xué)分析以揭示該重生過程的分子機制础淤。bulk RNA-seq的主成分分析 (PCA) 顯示，肝臟基因表達在6腮鞍、24和48 小時出現(xiàn)差異值骇，在96小時回歸到對照水平莹菱。bulk RNA-seq數(shù)據(jù)的通路分析顯示移国，早期免疫信號的誘導(dǎo)，在APAP后6和24小時達到峰值道伟，增殖通路在APAP后48 h達到高峰迹缀。肝臟代謝功能，如谷胱甘肽和細(xì)胞色素P450代謝蜜徽，以及靜止的造血干細(xì)胞功能祝懂，在APAP后72 h重新出現(xiàn)。單細(xì)胞RNA-seq分析表明拘鞋，注射APAP后48 h肝細(xì)胞豐度顯著下降砚蓬，與中心周圍肝細(xì)胞大量壞死相一致，96小時后肝細(xì)胞豐度回歸對照水平盆色。相比之下灰蛙，造血干細(xì)胞、單核細(xì)胞和巨噬細(xì)胞大量增加隔躲，在APAP注射后24-48小時達到峰值摩梧，然后下降到對照組水平宣旱。該分析顯示出肝臟基因表達和多種肝細(xì)胞類型比例的重大變化，在損傷后4天就迅速恢復(fù)到控制水平笙纤。
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圖注：(A) A schematic of the experimental design. Mice were injected with 300 mg APAP/1 kg body weight. Livers were harvested for bulk sequencing at 6, 24, 48, 72, 96 h, 1 week, and 1 month after injection (3 mice per time point), as well as saline injected controls at different time points (2 mice per time point). Livers were dissociated for single-cell sequencing at 24, 48, 72, 96 h, 1 week, and nontreated controls (2–4 mice for each time point, marked with black asterisks). Livers from 24, 48, and 72 h were also taken for Visium spatial transcriptomics (blue asterisks). Mouse injection illustration was created with BioRender.com.
(B) Images of liver lobules at different time points following APAP injection. CV, central vein; PV, portal vein. Yellow dashed lines mark the borders of the damaged areas. Cell nuclei are stained with Dapi (blue). Cell membranes are stained with phalloidin (gray). Scale bars, 20 mm.
(C) Uniform manifold approximation and projection (UMAP) visualization of the integrated data of all 23,944 cells from 6 time points (n = 28 mice). Cells are colored by their cell type annotation.
(D) UMAP visualization of the integrated data. Cells are colored by the time following APAP injection.
Analysis of Visium spatial transcriptomics dataset 圖注：(A) H&E image of Visium slide of liver of 24h after APAP injection. White square marks the inset shown in B and C.
(B) Close up look at the tissue shows distinct regions of pericentral damage and periportal undamaged zones. C - central vein, P - portal vein.
(C) Overlay of the tissue with the Visium spot (spatial clusters). Yellow spots mark spots with fibrogenic regions, which were manually segmented (methods), and grey spots mark non-fibrogenic regions. C - central vein, P - portal vein
(D) Projection of the spots overlaying the tissue, colored by their classification into fibrotic or non-fibrotic regions.
(E) Projection of the spots overlaying the tissue, colored by their distances from the central veins. Each connected region of fibrotic spots was skeletonized (methods) to get to the “central core”. The distance of each spot to the nearest “central core” was then calculated. The “central cores” are marked in black x.
(F) Projection of the spots overlaying the tissue, colored by their expression level of the hepatocyte pericentral gene Cyp2e1, log10 normalized. The “central cores” are marked in black x.
(G) Projection of the spots overlaying the tissue, colored by their expression level of the hepatocyte periportal gene Cyp2f2, log10 normalized. The “central cores” are marked in black x.
(H-J) Scatter plots of log2 of the periportal to pericentral expression ratios of zonated hepatocyte (H), HSC
(I) and endothelial (J) specific genes found in the computationally inferred zonation patterns of the single cell transcriptomics and in the zonation pattern in spatial transcriptomics dataset (methods). Dots are colored by the Visium slide with which the single cell reconstruction was compared
肝細(xì)胞表現(xiàn)出區(qū)域性重編程和跨肝小葉的廣泛增殖
中央周圍和門靜脈周圍肝細(xì)胞在未受干擾的肝臟中表現(xiàn)出明顯不同的基因表達组力。肝細(xì)胞單細(xì)胞數(shù)據(jù)包括來自對照、APAP 注射后 48 和 72 小時的 2,770 個細(xì)胞忿项。使用空間轉(zhuǎn)錄組學(xué)數(shù)據(jù)集蓉冈，確定了肝細(xì)胞標(biāo)志性基因城舞，這些基因在整個再生過程中在門靜脈或中央分區(qū)寞酿。使用這些計算推斷單個測序肝細(xì)胞沿肝小葉軸的坐標(biāo)伐弹。隨后，將每個時間點的細(xì)胞分成三個區(qū)域——中央周圍煌茴、小葉中部和門靜脈周圍——并對它們的表達進行平均以獲得動態(tài)肝細(xì)胞分區(qū)分布日川。正如預(yù)期的那樣，具有周圍中心特征的肝細(xì)胞在 48 小時內(nèi)耗盡回论。分區(qū)坐標(biāo)分布在 72 h 時接近損傷前模式分歇。使用單分子熒光原位雜交 (smFISH) 來證明經(jīng)典的分區(qū)基因职抡，如中央 Glul 和 Cyp2e1 和門靜脈 Ass1 在損傷后 96 小時呈現(xiàn)其損傷前分區(qū)模式。值得注意的是谱净，雖然門靜脈距離沒有顯著變化蹄胰，但新形成的肝細(xì)胞表現(xiàn)出顯著更高的倍性水平，這種增加在中央肝細(xì)胞中更為突出浩蓉。因此捻艳，分析表明庆猫，肝細(xì)胞在急性肝損傷 4 天后恢復(fù)其區(qū)域分子特性。
已顯示帶狀肝細(xì)胞群在穩(wěn)態(tài)以及不同的再生模型中表現(xiàn)出不同的增殖模式嘁字。為了探索 APAP 再生過程中肝細(xì)胞增殖的帶狀動態(tài)纪蜒，分析了不同時間點增殖肝細(xì)胞的帶狀分布。為此随珠，使用 smFISH 量化了表達增殖標(biāo)記 Mki67 的肝細(xì)胞比例猬错。在 APAP 注射后 32 小時倦炒，肝細(xì)胞在整個小葉軸上增殖，有輕微的門靜脈周圍偏差构罗。增殖在 40 和 48 小時增加智玻，顯示出中心偏差吊奢，然后在 72 小時趨于平穩(wěn)纹烹。值得注意的是铺呵，在整個再生過程中，離受損組織區(qū)域較遠(yuǎn)的小葉中區(qū)和門靜脈區(qū)的肝細(xì)胞對 Mki67 呈陽性幻林。在不同小葉區(qū)域的增殖和非增殖肝細(xì)胞之間的差異表達分析揭示了增殖肝細(xì)胞中肝細(xì)胞代謝功能的降低沪饺。最近在肝細(xì)胞中刪除 ZNRF3 和 RNF43 后觀察到增殖肝細(xì)胞中肝細(xì)胞代謝功能的類似下降闷愤。肝細(xì)胞在整個小葉軸上的廣泛增殖可能有助于沿肝板產(chǎn)生增加的有絲分裂壓力讥脐，這可能有助于快速替換受損區(qū)域的細(xì)胞啼器。重要的是镀首，這種有絲分裂壓力將小葉中肝細(xì)胞帶入中央?yún)^(qū)鼠次，需要重新編程它們的轉(zhuǎn)錄狀態(tài)。
- 圖注：(A) Spearman correlation distances between hepatocyte-specific genes for pairs of control samples (n = 4) and mice at different time points (n = 3 mice per time
point) after APAP injection. Horizontal line denotes median distance of control-control pairs. White dots are median distances for each time point.
(B) UMAP visualization of hepatocytes (n = 2,770 cells), colored by time after APAP injection.
(C) UMAP visualization of hepatocytes colored by the expression of the centrally zonated gene Cyp2e1.
(D) UMAP visualization of hepatocytes colored by the expression of the periportal gene Cyp2f2.
(E) UMAP of hepatocytes colored by their inferred lobule spatial coordinate, ranging from CV—cells closest to central vein, to PV—cells closest to portal vein.
(F) Distributions of lobule spatial coordinates of hepatocytes at each time point.
(G and H) smFISH of a liver lobule showing 3 zonated genes: pericentral Glul (red), Cyp2e1 (blue), and periportal Ass1 (green). CV, central vein; PV, portal vein.
Scale bars, 20 mm. Shown are examples of a control lobule (G) and a lobule 96 h after APAP (H).
(I) The fraction of proliferating hepatocytes expressing Mki67+ transcripts out of all hepatocytes located in either pericentral (red), midlobular (yellow), or periportal
(green) zones. The analysis was performed on 2 mice from each time point, at least 5 lobules per mouse were quantified. Significance levels calculated using
Kruskal-Wallis tests. White dots represent the median fraction.
(J) smFISH of a liver lobule 72 h following APAP administration showing Mki67 single transcripts (gray dots). Dashed white line marks the damage border, arrows
point to representative Mki67+ proliferating cells. Cell nuclei are stained with Dapi (blue) and membranes are stained with phalloidin (red). Scale bars, 20 mm.
Interface hepatocytes up-regulate fetal programs and exhibit a mesenchymal shape（he interface between the damaged and nondamaged zones）
發(fā)現(xiàn)界面肝細(xì)胞表現(xiàn)出獨特的表達特征赦役，由在胎兒肝臟和肝細(xì)胞癌中表達但在成人肝細(xì)胞中不表達的基因組成。界面肝細(xì)胞不會簡單地從門靜脈周圍/小葉中狀態(tài)轉(zhuǎn)變?yōu)橹醒胫車鸂顟B(tài)术羔，因為它們被推入中央周圍區(qū)域级历。 相反叭披，它們的細(xì)胞特性變化與胎兒基因的瞬時表達、蛋白質(zhì)翻譯和降解的升高以及細(xì)胞形態(tài)的改變有關(guān)嚼贡。
- 圖注：(A and B) Expression levels of genes in pericentral hepatocytes 48 (A) or 72 h (B) after APAP injection plotted against their expression in control hepatocytes with
matched distribution of lobule spatial coordinates. Gray dots represent all genes. Red/blue dots represent genes upregulated/downregulated respectively in the
regenerating tissue, with mean expression level of above 5 3 10?6, at least 2-fold difference from matched control and FDR threshold of 0.01.
(C) smFISH (top) and insets (bottom) of a liver lobule 48 h after APAP injection. Dashed white line delineate the damage border. CV, central vein; PV, portal vein.
Nuclei and membranes are labeled with Dapi (blue) and phalloidin (green), respectively. Two representative interface hepatocytes outlined in orange shown in the
insets (bottom), together with the smFISH labeling for mRNAs of Afp (left), Cdh17 (middle), and Spp1 (right). Scale bars, 20 mm. Laplacian of Gaussian filter was
applied on the smFISH images.
(D) smFISH of a liver lobule 48 h post-APAP injection for Apoa1 (red) and Actb (gray) mRNA. Nuclei are stained with Dapi (blue). CV, central vein. Scale bars, 20 mm.
White dashed rectangle is the region displayed in (F).
(E) GSEA normalized enrichment scores (NESs) of gene pathways significantly upregulated (red) or downregulated (blue) in interface cells. Dot size corresponds
to number of pathway genes found in the dataset; opacity corresponds to false discovery rate (FDR). Genes sets used for the analysis were taken from Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways dataset.
(F) Magnification of the region marked in (D) showing interface (orange dashed lines) and noninterface (blue dashed lines) hepatocytes. Nuclei and membranes are
labeled with Dapi (blue) and phalloidin (gray), respectively. Scale bars, 20 mm.
(G) Quantification of circularity of interface (orange) and noninterface (blue) hepatocytes. White dots represent group circularity median. Gray boxes mark the 25–
75 percentiles. Significance level was calculated using paired signrank test (n = 60 pairs of interface and adjacent noninterface hepatocytes, taken from 3 mice
48 h after APAP injection and 3 mice 72 h after APAP injection).
HSCs exhibit spatial division of labor（空間分工）
成功的肝再生需要所有肝細(xì)胞類型的緊密協(xié)調(diào)反應(yīng)。 HSC 是肝損傷反應(yīng)和再生的關(guān)鍵參與者叮盘。分析強調(diào)了特定區(qū)域的 HSC 表達程序熊户，這些程序可能促進急性 APAP 損傷后肝臟表現(xiàn)出的免疫募集吭服、ECM 積累和分解的時間協(xié)調(diào)過程。
- 圖注：(A) UMAP visualization of HSCs, colored by time after APAP administration. n = 2,311 cells, at least 2 mice per time point.
(B) UMAP visualization of HSCs, colored by inferred zone
(C–H) Temporal dynamics of selected genes in HSCs, stratified by zone. Lines denote the mean of the normalized expression over cells from the same zone and
time point, patches denote the standard errors of the means (SE). For each gene, the mean and SE for pericentral HSCs (red), mid-lobule HSCs (yellow) and
periportal HSCs (green) are presented. Selected genes belong to various processes: markers of quiescent HSCs (C), activated HSCs (D), ECM collagen genes (E),
proliferation (F), centrally zonated immune modulators (G), and downregulated zonated immune modulators (H).
Endothelial cells exhibit zonated cues along the regeneration process
- 圖注：(A) UMAP visualization of endothelial cells, colored by time after APAP administration. n = 6,527 from 13 mice, at least 2 mice per time point.
(B) UMAP visualization of endothelial cells, colored by expression level of the pericentral Wnt2 (top left), periportal Efnb2 (top right), sinusoidal endothelial cell
marker Kit (bottom left) and vascular endothelial cell marker Vwf (bottom right).
(C) UMAP visualization of endothelial cells, colored by inferred zone (STAR Methods). PC-LVECs, pericentral liver vascular endothelial cells; PC-LSECs, pericentral
liver sinusoidal endothelial cells; mid-LSECs, mid-lobule liver sinusoidal endothelial cells; PP-LSECs, periportal liver sinusoidal endothelial cells; PPLVECs,
periportal liver vascular endothelial cells.
(D) Temporal dynamics of selected genes in endothelial cells, stratified by the zonated endothelial cell populations. Lines denote the mean of the normalized
expression over cells from the same zone and time point, patches denote the standard errors of the means (SE). For each gene, the mean and SE for PC-LVEC
(dark red), PC-LSEC (red), mid-LSEC (yellow), PP-LSEC (green), and PP-LVEC (blue) are presented. Lobule diagram highlights the different zonated endothelial
cell subtypes with their respective colors.
Dynamics of myeloid cell subtypes along the regeneration process
骨髓細(xì)胞有助于急性肝損傷后的再生過程。與肝細(xì)胞和內(nèi)皮細(xì)胞一樣桩匪，活化的kuffer細(xì)胞與fetal kuffer細(xì)胞有顯著相關(guān)性友鼻。 fetal kuffer細(xì)胞的共同趨勢包括抗原呈遞基因減少和脂質(zhì)相關(guān)巨噬細(xì)胞基因增加。
- 圖注：(A) UMAP visualization of myeloid cell populations, colored by time after APAP administration. n = 9,338 cells from 15 mice, at least 2 mice per time point.
(B) UMAP visualization of myeloid cell populations, colored by cell types.
(C) The fractions of different myeloid cell subtypes at each time point.
(D) UMAP visualization of myeloid cells, colored by expression levels of Mmp12.
(E) smFISH scan (top) and zoomed-in insets (bottom) of a liver lobule 24 h after APAP injection. CV, central vein; PV, portal vein. Nuclei and membranes are labeled
with Dapi (blue) and phalloidin (green), respectively. Pericentral (left), mid-lobule (middle), and periportal (right) KCs marked by dashed lines in the scan are
enlarged in the insets (bottom), together with the smFISH for the Kupffer cell gene Marco (green) and Mmp12 (red) mRNAs, together with Dapi (blue). Scale bars,
20 mm for top image, 10 mm for insets. Laplacian of Gaussian filter was applied on the smFISH images.
總的來說，該工作使用空間分辨單細(xì)胞RNA測序 (scRNA-seq) 來研究急性對乙酰氨基酚(APAP)處理后小鼠肝臟再生的動力學(xué)虫碉，發(fā)現(xiàn)肝細(xì)胞在肝小葉內(nèi)增殖敦捧，產(chǎn)生有絲分裂壓力，使壞死的中心區(qū)迅速再生习瑰。位于再生界面的部分肝細(xì)胞會在重新編程到中心周圍狀態(tài)過程中济蝉，短暫上調(diào)胎特異性基因王滤。該研究有助于深刻理解肝分區(qū)再生的協(xié)同程序滓鸠。
強調(diào)一個重點方法（空間為重）
Interaction analysis from spatial transcriptomics dataset
Ligand-receptor interactions were also investigated in the spatial-transcriptomics dataset. Ligand-receptor pairs in each Visium slide were analyzed. Spearman correlation of each ligand-receptor pair expression was calculated, either for across all slide spots, or for each of the three stratified lobule zones (discretized by growing distances from the central cores). In addition to correlations, interaction potential was also calculated as the mean product of the expression of the ligand and receptor over all spots, either across the whole slide, or across each zone.
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