作者感谢 U19 AI157984、U01 DK103168、U19 AI144297、P30 DK56338、P01 AI057788、U19 AI116497 赠款和 NASA 合作协议通知/TRISH NNX16AO69A。

此处使用的类器官系来自德克萨斯医学中心消化疾病中心 GEMS Core。简而言之，为了最初建立类器官系，对供体组织样品进行洗涤和酶消化以释放肠隐窝。隐窝包埋在基底膜中并在培养基中培养。贝勒医学院机构审查委员会批准了获取组织样本的研究方案，从中建立类器官系，并获得所有供体的知情同意，以从捐赠组织中建立类器官系。
1. 3D HIO 的传代以扩展以进行分化</s...

将单细胞转录组学与人肠道类器官整合

<ol>
	<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Blutt, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+microphysiological+systems.">Gastrointestinal microphysiological systems.</a> Exp Biol Med. 242 (16), 1633-1642 (2017).</li><li>VanDussen, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+enhanced+human+gastrointestinal+epithelial+culture+system+to+facilitate+patient-based+assays.">Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.</a> Gut. 64 (6), 911-920 (2015).</li><li>Elmentaite, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+of+the+human+intestinal+tract+mapped+across+space+and+time.">Cells of the human intestinal tract mapped across space and time.</a> Nature. 597 (7875), 250-255 (2021).</li><li>Hickey, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+human+intestine+at+single-cell+resolution.">Organization of the human intestine at single-cell resolution.</a> Nature. 619 (7970), 572-584 (2023).</li><li>Burclaff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proximal-to-distal+survey+of+healthy+adult+human+small+intestine+and+colon+epithelium+by+single-cell+transcriptomics.">A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics.</a> Cell Mol Gastroenterol Hepatol. 13 (5), 1554-1589 (2022).</li><li>Triana, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+reveals+immune+response+of+intestinal+cell+types+to+viral+infection.">Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection.</a> Mol Syst Biol. 17 (7), e9833 (2021).</li><li>Triana, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analyses+reveal+SARS-CoV-2+interference+with+intrinsic+immune+response+in+the+human+gut.">Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut.</a> Mol Syst Biol. 17 (4), e10232 (2021).</li><li>Fujii, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+intestinal+organoids+maintain+self-renewal+capacity+and+cellular+diversity+in+niche-inspired+culture+condition.">Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition.</a> Cell Stem Cell. 23 (6), 787-793 (2021).</li><li>Beumer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+gradient+along+the+intestinal+villus+axis+controls+zonated+enterocyte+and+goblet+cell+states.">BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.</a> Cell Rep. 38 (9), 110438 (2022).</li><li>Beumer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+mRNA+and+secretome+atlas+of+human+enteroendocrine+cells.">High-resolution mRNA and secretome atlas of human enteroendocrine cells.</a> Cell. 182 (4), 1062-1064 (2020).</li><li>Busslinger, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gastrointestinal+epithelia+of+the+esophagus,+stomach,+and+duodenum+resolved+at+single-cell+resolution.">Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.</a> Cell Rep. 34 (10), 108819 (2021).</li><li>Luecken, M. D., Theis, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+best+practices+in+single-cell+RNA-seq+analysis:+a+tutorial.">Current best practices in single-cell RNA-seq analysis: a tutorial.</a> Mol Syst Biol. 15 (6), e8746 (2019).</li><li>Heumos, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+for+single-cell+analysis+across+modalities.">Best practices for single-cell analysis across modalities.</a> Nat Rev Genet. 24 (8), 550-572 (2023).</li></ol>

作者没有利益冲突。

使用单细胞基因组学平台，可以分解复杂的生物系统，例如模拟肠上皮的组织来源的 HIO 培养物，从而产生对整体生物反应的单个细胞贡献 4,5,6。细胞异质性和稀有细胞群也可以被识别和询问。需要优化细胞输入，以使用基于单细胞转录组学的平台最大限度地提高输出。在这里，我们描述了将产生高度活力和高质量单细胞悬液?...

小肠上皮有两个不同的区域：容纳肠道干细胞 （ISC） 的隐窝和由分泌和吸收谱系的分化细胞组成的绒毛。上皮的区域特异性增加了这种复杂性，它在小肠区域之间提供了独特的功能特性。开创性工作建立了培养条件，其中人类小肠隐窝和绒毛区都可以从手术组织或组织活检中 离体 产生1。这些培养物弥合了动物研究和临床试验之间的差距，并揭示了以前未被认识的胃肠道细胞生物学、生物化学和生理学。HIO 使用含有促进干细胞活力的生长因子和细胞外支持基质的培养基以 3D 球形结构繁殖。这些条件导致主要由祖细胞和干细胞组成的隐窝样 HIO 模型。去除生长因子促进 ISC 分化（绒毛样模型）和成熟肠上皮细胞（杯状、肠内分泌、簇状、肠上皮细胞）的产生，以及细胞增殖和分化、极化、屏障完整性、区域特异性特征和适当的生理反应2.HIO 培养物在遗传上是稳定的，可以在其隐窝样状态下无限增殖。允许以单层形式生长的培养条件提供了易于进入这些培养物顶端表面的机会 3。HIO 还允许在生物分析中考虑宿主个体的变异性，例如遗传、年龄、性别、种族和疾病状态。分析和功能评估工具与以转化细胞系为中心的方法中使用的工具相同，包括多种分子技术，如流式细胞术、显微镜、转录组学、蛋白质组学和代谢组学。
单细胞转录组学通过深入了解每种细胞类型对生物过程的个体贡献，正在彻底改变我们对小肠生物学和生理学的理解。使用这项技术的开创性工作提供了天然人类肠道中存在的细胞类型的景观 4,5,6。单细胞转录分析平台允许探索单个细胞的转录景观，使细胞异质性成为科学探索的最前沿。在一些单细胞转录分析平台中，微流控分配和条形码用于从每个样品中多达 10,000 个细胞获得的细胞多聚腺苷酸化 mRNA 生成 cDNA 文库。在该平台上，含有单细胞、条形码寡核苷酸、逆转录试剂和油的液滴形成反应囊泡，导致来自单个细胞的所有 cDNA 具有相同的条形码。然后，可以使用下一代测序对条形码 cDNA 进行高效测序。生成的数据可以通过软件和可视化工具进行处理，这些工具将条形码序列转换为可视化图谱和单细胞转录图谱。可以使用公开可用的人类小肠上皮数据库来识别细胞群 4,5,6。尽管许多研究利用该平台在单细胞水平上询问小鼠肠道类器官，但对 HIO 的单细胞转录反应的分析落后于 7,8,9,10,11,12。
在这里，我们提供了从小肠 HIO 中分离活细胞以在单细胞转录分析平台上进行处理的分步指南（图 1）。我们提供培养和分化指南以及针对上皮分化优化的培养基成分。我们概述了三种不同培养形式的细胞回收方法：在塑料或膜细胞培养小室上进行三维 （3D） 和单层培养。我们提供使用开源软件获得的样本聚类数据，以得出每个聚类中的差异表达基因。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Leu15]-Gastrin I</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan blue</td>
			<td>Millipore-Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS Ca- Mg-</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Transwell</td>
			<td>Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Nunclon delta surface tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Flowmi tip strainer</td>
			<td>SP Bel-Art Labware</td>
			<td>H13680-0040</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Flowmi tip strainer</td>
			<td>SP Bel-Art Labware</td>
			<td>H13680-0070</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>StemCell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#8217; GEM, Library &#38; Gel Bead Kit v3.1</td>
			<td>10x Genomics</td>
			<td>PN-1000128</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5533-5MG</td>
			<td>33 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Corning Cell Recovery Solution&#160;</td>
			<td>VWR</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Mg2-, Ca2-)</td>
			<td>Invitrogen</td>
			<td>14190-136</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>L-WRN conditioned media</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, phenol free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse recombinant EGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>500 &#181;M</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3413</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Stem Cell Technologies</td>
			<td>72308</td>
			<td>10 &#181;M</td>
		</tr>
	</tbody>
</table>

using human intestinal organoids to understand the small intestine epithelium at the single cell transcriptional level

单细胞转录组学彻底改变了我们对人体细胞生物学的理解。最先进的人小肠类器官培养物提供了 离体 模型系统，弥合了动物模型和临床研究之间的差距。单细胞转录组学在人肠道类器官 （HIO） 模型中的应用揭示了以前未被认识的胃肠道细胞生物学、生物化学和生理学。先进的单细胞转录组学平台使用微流控分配和条形码生成 cDNA 文库。这些条形码 cDNA 可以通过下一代测序平台轻松测序，并被各种可视化工具用于生成图谱。在这里，我们描述了以不同形式培养和区分人小肠 HIO 的方法和程序，用于从这些形式中分离活细胞，这些形式适用于单细胞转录分析平台。这些协议和程序有助于使用小肠 HIO，以加深对在各种不同环境背景下人类肠上皮细胞在转录水平上的细胞反应的理解。

The small intestinal epithelium has two distinct zones: the crypt that houses the intestinal stem cell (ISC) and the villus, which is comprised of differentiated cells of the secretory and absorptive lineages. Adding to this complexity is the regional specificity of the epithelium that provides unique functional properties between the regions of the small intestine. Pioneering work established culture conditions in which both the human small intestinal crypt and villus zones can be generated ex vivo from surgical tissues or tissue biopsies1. These cultures are bridging the gap between animal studies and clinical trials and are revealing previously unrecognized cell biology, biochemistry, and physiology of the gastrointestinal tract. The HIOs are propagated as 3D spherical structures using a media with growth factors that promote stem cell viability and extracellular support matrices. These conditions result in a crypt-like HIO model that consists mostly of progenitors and stem cells. Removal of the growth factors promotes ISC differentiation (villus-like model) and production of mature intestinal epithelial cells (goblet, enteroendocrine, tuft, enterocyte) in appropriate ratios along with cell proliferation and differentiation, polarization, barrier integrity, regional specific features, and appropriate physiological responses2. HIO cultures are genetically stable and can be propagated indefinitely in their crypt-like state. Easy access to the apical surface of these cultures is provided by culture conditions that allow growth in a monolayer format3. HIOs also allow considerations of host individual variability such as genetics, age, sex, ethnicity, and disease status to be included in biological analyses. Analytic and functional assessment tools are identical to those used in approaches centered on transformed cell lines and include a variety of molecular techniques such as flow cytometry, microscopy, transcriptomics, proteomics, and metabolomics.
Single cell transcriptomics is revolutionizing our understanding of the biology and physiology of the small intestine by providing insight into the individual contributions of each cell type to a biological process. Pioneering work using this technology has provided a landscape of the cell types present in the native human intestine4,5,6. Single cell transcriptional profiling platforms allow exploration of the transcriptional landscape of individual cells, allowing cell heterogeneity to come to the forefront of scientific exploration. In some single cell transcriptional profiling platforms, microfluidic partitioning and barcoding are used to generate cDNA libraries from cellular polyadenylated mRNAs obtained from up to 10,000 cells per sample. On this platform, droplets containing single cells, barcoded oligonucleotides, reverse transcription reagents, and oil form a reaction vesicle that results in all cDNAs from a single cell having the same barcode. The barcoded cDNAs can then be efficiently sequenced using next generation sequencing. Data generated can be handled through software and visualization tools, which convert the barcoded sequences into visualization maps and single cell transcriptional profiles. Cell populations can be identified using publicly available databases of human small intestinal epithelium4,5,6. Although many studies have utilized this platform to interrogate murine intestinal organoids at the single cell level, the analysis of single cell transcriptional responses of HIOs has lagged behind7,8,9,10,11,12.
Here, we provide a step-by-step guide to isolating viable cells from small intestinal HIOs for processing on single cell transcriptional profiling platforms (Figure 1). We provide culturing and differentiation guidelines along with media components that have been optimized for epithelial differentiation. We outline cell recovery methods for three different culture formats: three dimensional (3D) and monolayer cultures either on plastic or on membrane cell culture inserts. We provide sample clustering data obtained using open-source software to derive differentially expressed genes in each cluster.

作者聚焦：将单细胞转录组学与类器官培养相结合，以获得高级研究和治疗见解

使用人类肠道类器官在单细胞转录水平上了解小肠上皮

We are integrating single-cell transcriptomics and organoid technology to better understand the biology of the small intestine. These technologies are allowing us deeper insight into the cellular heterogeneity that exists within that tissue. The tools that improve the throughput, sensitivity, and the resolutions are the keys that advanced research in the field.Some examples are improved computational methods and the technological innovations. Cost reductions also a major focus. The process of preparing and sequencing the libraries of samples can cause transcription variations, which is one challenge.For organoids, specific challenges include limited spatial context or information about temporal dynamics. Single-cell analysis of the human intestinal organoids provides previously unattainable information into the cell types that make up the intestinal epithelium. This, thereby, opens the window to the transcriptional landscape of those cells.We are using these technologies to identify biomarkers and mechanisms of human intestinal diseases. These biomarkers and mechanisms will advance our ability to utilize personalized medicine-based approaches to treat human disease and improve human health overall.

从 2-3 个膜细胞培养插入室、单层和 3D HIO 孔中混合单细胞悬液，以确保足够的细胞产量并减少孔间差异。使用单细胞转录分析平台专用试剂制备单细胞文库。并在下一代测序平台上使用配对末端读数进行测序，30,000 个读数/细胞。使用单细胞基因组学分析工具对读数进行定位、计数和分析。线粒体读取超过 20% 或计数基因少于 200 个的低质量细胞被排除在分析之外。为了评估细胞的质量，我们绘制...

该方案将人类肠道类器官技术与单细胞转录组学分析相结合，为以前未曾探索的肠道生物学提供了重要的见解。

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

using-human-intestinal-organoids-to-understand-small-intestine

Research

JoVE Journal

Biology

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

339 Views.  Baylor College of Medicine. We are integrating single-cell transcriptomics and organoid technology to better understand the biology of the small intestine. These technologies are allowing us deeper insight into the cellular heterogeneity that exists within that tissue. The tools that improve the throughput, sensitivity, and the resolutions are the keys that advanced research in the field.Some examples are improved computational methods and the technological innovations. Cost reductions also a major focus. The pro...