众所周知，哺乳动物肝脏具有显着的再生能力。目前已被描述的两种损伤反应模式为：（1）当所有肝细胞都受到慢性肝病的影响时，干细胞的响应从胆管树中消失。（2）大量的成熟肝细胞促生命反应发生在急性肝损伤时，例如部分肝切除术（PHx）。虽然干细胞应答已被从胆管细胞中的生长类器官体外捕获，但肝细胞增殖反应尚未在培养中重现。来自荷兰和中国的数名研究人员联合进行实验，建立起一个长期3D器官培养系统，用于研究小鼠和人原发性肝细胞，并将一系列研究成果发表于顶级期刊《Cell》杂志上。
(J) Violin plot comparing the expression of markers in undamaged hepatocytes, post-PHx hepatocytes isolated and Hep-Orgs. Expression of cell-cycle-/growthrelated genes is given in the top row. Markers were selected for their specific expression in regenerating liver, but unrelated to cell cycling (bottom-three rows). Transcript counts are provided on log10 scale.
Hubrecht Organoid Technology 作为类器官培养的顶级研究机构，在本次研究中，研究人员探讨了成熟小鼠肝细胞和人肝细胞的直接增殖能力，并描述了一种与胆管细胞衍生的类器官不同的增殖性肝细胞类器官（Hep Orgs）的长期培养系统。本研究可以达到以单个肝细胞建立类器官并生长数月，同时保留关键的形态学，功能和基因表达特征。类似物的转录谱类似于PHx后增殖的肝细胞的转录谱。植入小鼠后，人肝细胞类器官大量繁殖，从而概括了肝细胞的增殖损伤反应。
研究人员从野生型成体C57BL / 6小鼠肝脏中分离出原代肝细胞，并不断调整改善培养条件，从能能够促进类器官生长。结果表明小鼠Hep-Orgs起源于单个成熟肝细胞。然后通过免疫荧光和免疫组织化学染色分析Hep-Orgs。Hep-Orgs显示出强烈的albu min表达（绿色），但胆管标记物Krt19或Krt7呈阴性。值得注意的是，参与肝细胞功能的基因如细胞色素P450活性，糖原代谢，脂质代谢，类固醇代谢，尿素循环和补体活化都在Hep-Orgs和原代肝细胞之间显示出相似的表达谱。
Characterization of Mouse Hepatocyte Organoids
(A) Confocal z stack (left) and single plane (right) images of Hep-Orgs. Albumin (green), E-cadherin (red, right panel), and DAPI (blue). Scale bar = 20 mm.
(B) Confocal z stack (left) and single plane (right) images of mouse Chol-Orgs. Krt7 (blue), Krt19 (Red), and DAPI (white). Scale bar = 20 mm.
(C and D) qRT-PCR analysis of gene expression of hepatocyte markers
(C) and cholangiocyte/progenitor markers (D) in Hep-Orgs and Chol-Orgs relative to primary hepatocytes. Graph presents mean results from 4 replicates from three independent mice. Data are represented as mean ± SEM. ** indicates p < 0.01, *** indicates p < 0.001.
(E) Glycogen accumulation evaluated by Periodic-Acid Schiff (PAS) staining (dark pink) in Hep-Orgs. Nuclei were stained with hematoxylin (blue). Scale Bar = 20 mm.
(F) Low density lipoprotein (LDL) uptake was analyzed by Dil-ac-LDL fluorescent staining (Red) in cultured Hep-Orgs. Nuclei were stained with DAPI (blue). Scale bar = 20 mm.
(G) Albumin secretion measured after 24h culturing of primary hepatocytes, Hep-Orgs of Passage 0 (P0) day 15 and Passage 3 (P3) and Chol-Orgs in expansion medium (EM) or differentiation medium (DM). Results are indicated as picograms of albumin per cell. Data are represented as mean ± SEM.
(H) Measurement of cytochrome activity (Cyp1a2) in cultured primary hepatocytes, Hep-Orgs of p0 day 15 and p3. Relative light units (RLU) per ml per million cells is indicated. Data are represented as mean ± SEM.
(I) Heatmap of liver gene expression determined by mRNA sequencing comparing three independent Hep-Orgs, (p1) with primary hepatocytes (n = 1), and three independent Chol-Orgs (p8-p12) in expansion medium (EM) (full gene list in Figure S2K)
接下来，研究人员在HepOrgs和Chol-Orgs上进行单细胞mRNA测序。对来自每个细胞的384个细胞进行了测序，并通过了RaceID2分析结果。并对来自Hep-Orgs和Chol-Orgs的组合数据集进行了分析，并通过t-SNE对可视化的基因表达进行了分析。结果一致地显现，Alb，Ahsg，Afp，Fgg和Gc仅在Hep-Orgs群体中高度表达，而Epcam和Krt7在Chol-Orgs中高度表达，两种类器官类型都含有增殖细胞。然后科研人员开始进行人肝细胞Hep-Orgs的建立与鉴定。Hep-Org培养基针对人肝细胞的克隆形成和扩增进行了优化（Hep-Medium），从妊娠11-20周的供体胚胎中分离出人胎肝细胞。还从婴幼儿和成人原代人肝细胞中建立了Hep-Orgs（PHH-Orgs）（PHHs）。
Single-Cell Transcriptome Analysis of Hep-Orgs
(A) Overview of single-cell sequencing experiment of Hep-Orgs and Chol-Orgs and hepatocytes isolated from Albumin-CreERT2; Rosa26-LSL-tdTomato mice (‘‘undamaged control’’) or 3 days after 2/3 partial hepatectomy (‘‘regeneration’’).
(B) t-SNE maps indicating origin of individual cells: Hep-Org cells (green), Chol-Org cells (blue).
(C-D) t-SNE plot showing the expression of Alb (C) and Krt7 (D) in single cells derived from Hep-Orgs and Chol-Orgs. Expression is given as normalized log2 value.
(E) t-SNE map of all cell clusters from Hep-Orgs obtained by RaceID2 algorithm.
(F-H) t-SNE plot showing the expression levels of Alb (F), Pcna (G), and Krt7 (H) in Hep-Orgs. Expression is given as normalized log2 value.
(I) GSEA of genes in Hep-Orgs versus primary hepatocytes. Expression enrichment was compared to a gene set of differentially expressed genes generated by comparing mouse liver three days after partial hepatectomy compared with control non-damaged liver. Enrichment of upregulat d genes at 3 days post-PHx: upper panel; enrichment of downregulated genes at 3 days of post-PHx (lower panel).
最后，研究人员讨论了人类Hep-Orgs是否能够移植和重新填充受损的肝脏组织。成熟的原代细胞和类器官在植入水平和移植物增殖方面明显优于其胎儿对应物，这证明了Hep-Orgs的再生能力和在器官培养中忠实保护基本组织特征（例如，可移植性）。总之，研究数据显示Hep-Orgs能够成功地重建受损的肝脏并显示移植后显着的移植物扩张。
在整个研究过程中，随处可见小分子试剂的身影，尤其是购于AbMole的Y-27632，更是在类器官培养的过程中不可或缺。
参考文献：
Huili Hu, et al. Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids. Cell. 2018 Nov 19.
https://www.ncbi.nlm.nih.gov/pubmed/30500538
