从小鼠子宫建立3D子宫内膜类器官
Summary
该协议描述了建立小鼠子宫内膜上皮类器官以进行基因表达和组织学分析的方法。
Abstract
子宫内膜组织排列在子宫内腔，并受到雌激素和孕激素的周期性控制。它是一种由管腔和腺体上皮、基质隔室、血管网络和复杂免疫细胞群组成的组织。小鼠模型一直是研究子宫内膜的有力工具，揭示了控制植入、胎盘和癌症的关键机制。3D子宫内膜类器官培养的最新发展提供了一个最先进的模型来剖析子宫内膜生物学的信号通路。从基因工程小鼠模型中建立子宫内膜类器官，分析其转录组并以单细胞分辨率可视化其形态是研究子宫内膜疾病的关键工具。本文概述了建立小鼠子宫内膜上皮3D培养的方法，并描述了量化基因表达和分析类器官组织学的技术。目标是提供可用于建立、培养和研究子宫内膜上皮类器官的基因表达和形态特征的资源。
Introduction
子宫内膜 – 子宫腔的内衬粘膜组织 – 是一种独特且高度动态的组织，在女性的生殖健康中起着关键作用。在生殖寿命期间，子宫内膜有可能经历数百个增殖，分化和分解的循环，由卵巢激素 – 雌激素和孕激素的协调作用协调。对基因工程小鼠的研究揭示了支撑子宫内膜对激素反应和控制胚胎植入、基质细胞蜕膜化和怀孕的基本生物学机制1。然而，由于难以在传统的2D细胞培养物中维持未转化的原代小鼠子宫内膜组织，体外研究受到限制2，3。子宫内膜组织培养为3D器官系统或类器官的最新进展为研究控制子宫内膜细胞再生和分化的生物学途径提供了新的机会。小鼠和人子宫内膜类器官系统已由封装在各种基质中的纯子宫内膜上皮4，5开发而人子宫内膜已培养为无支架上皮/基质共培养物6，7，最近作为胶原封装的上皮/基质组合8 .上皮类器官培养物的生长和再生潜力得到了生长因子和小分子抑制剂的定义混合物的支持，这些生长因子和小分子抑制剂已被经验确定为最大化类器官的生长和再生4，5，9。此外，冷冻和解冻子宫内膜类器官的能力允许长期储存来自小鼠和人类的子宫内膜类器官，用于未来的研究。
基因工程小鼠揭示了控制早期怀孕和蜕膜化的复杂信号通路，并已被用作妊娠丢失，子宫内膜癌和子宫内膜异位症的模型。这些遗传研究主要是通过使用在女性生殖组织中特别活跃的cre重组酶对loxP侧翼等位基因（“floxed”）的细胞特异性缺失来实现的。这些小鼠模型包括广泛使用的黄体酮受体-cre10，其在子宫内膜上皮和基质组织中具有很强的重组酶活性，乳铁蛋白i-cre，诱导成年小鼠子宫内膜上皮重组11，或Wnt7a-cre，在Müllerian衍生组织中触发上皮特异性缺失12.将基因工程小鼠模型中的子宫内膜组织培养为3D类器官为研究子宫内膜生物学提供了绝佳的机会，并促进了控制子宫内膜细胞更新和分化的生长因子和信号通路的鉴定13，14。文献中描述了小鼠子宫内膜组织的分离和培养方法，并报道了使用各种酶促策略分离子宫上皮以随后培养子宫内膜上皮类器官4。虽然以前的文献为子宫内膜上皮类器官培养方案提供了关键框架4，5，6，但本文为生成，维持，处理和分析这些类器官提供了一种清晰，全面的方法。这些技术的标准化对于加速妇女生殖生物学领域的进步非常重要。在这里，我们报告了一种酶促和机械纯化小鼠子宫内膜上皮组织的详细方法，用于随后在凝胶基质支架中培养子宫内膜类器官。我们还描述了凝胶基质封装的小鼠子宫内膜上皮类器官的下游组织学和分子分析方法。
Protocol
小鼠处理和实验研究是根据贝勒医学院机构动物护理和使用委员会（IACUC）批准的协议以及NIH实验动物护理和使用指南制定的指南进行的。 1.使用酶和机械方法从小鼠中分离子宫上皮 注意：本节描述了使用凝胶基质支架建立，传代，冷冻和解冻小鼠上皮子宫内膜类器官所需的步骤。先前的研究已经确定，小鼠子宫内膜类器官的最佳培养物是在?…
Representative Results
小鼠子宫内膜类器官的相衬图像我们从WT小鼠子宫内膜上皮建立了类器官，如所附方案所述（见 图1中的图表）。在对小鼠子宫内膜上皮进行酶解离后，将上皮片与子宫基质细胞机械分离，并进一步与胶原酶解离以产生单细胞悬液。如果正确执行，这种上皮和基质细胞分离方法应产生污染不超过相反细胞类型10%-15%的样品（参见 图2中的免…
Discussion
在这里，我们描述了从小鼠子宫内膜生成子宫内膜上皮类器官的方法以及常规用于其下游分析的方案。子宫内膜类器官是研究控制子宫内膜相关疾病（如子宫内膜异位症、子宫内膜癌和植入失败）的机制的有力工具。2017年发表的具有里程碑意义的研究报告了长期和可再生培养小鼠和人类上皮子宫内膜类器官的条件4，5。尽管类器官已被广泛用于研究其?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢Stephanie Pangas博士和Martin M. Matzuk博士（M.M.M.）对我们手稿的批判性阅读和编辑。研究得到了 Eunice Kennedy Shriver 国家儿童健康与人类发展研究所拨款R00-HD096057（D.M.），R01-HD105800（D.M.），R01-HD032067（M.M.M.）和R01-HD110038（M.M.M.）以及NCI-P30癌症中心支持补助金（NCI-CA125123）的支持。Diana Monsivais博士持有Burroughs Wellcome Fund颁发的下一代怀孕奖。
Materials
| Organoid Media Formulation | |||
| Name | Company | Catalog Number | Final concentration |
| Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-free | Corning | 354230 | 100% |
| Trypsin from Bovine Pancreas | Sigma Aldrich | T1426-1G | 1% |
| Advanced DMEM/F12 | Life Technologies | 12634010 | 1X |
| N2 supplement | Life Technologies | 17502048 | 1X |
| B-27™ Supplement (50X), minus vitamin A | Life Technologies | 12587010 | 1X |
| Primocin | Invivogen | ant-pm-1 | 100 µg/mL |
| N-Acetyl-L-cysteine | Sigma Aldrich | A9165-5G | 1.25 mM |
| L-glutamine | Life Technologies | 25030024 | 2 mM |
| Nicotinamide | Sigma Aldrich | N0636-100G | 10 nM |
| ALK-4, -5, -7 inhibitor, A83-01 | Tocris | 2939 | 500 nM |
| Recombinant human EGF | Peprotech | AF-100-15 | 50 ng/mL |
| Recombinant human Noggin | Peprotech | 120-10C | 100 ng/mL |
| Recombinant human Rspondin-1 | Peprotech | 120-38 | 500 ng/mL |
| Recombinant human FGF-10 | Peprotech | 100-26 | 100 ng/mL |
| Recombinant human HGF | Peprotech | 100-39 | 50 ng/mL |
| WNT3a | R&D systems | 5036-WN | 200 ng/mL |
| Other supplies and reagents | |||
| Name | Company | Catalog Number | Final concentration |
| Collagenase from Clostridium histolyticum | Sigma Aldrich | C0130-1G | 5 mg/mL |
| Deoxyribonuclease I from bovine pancreas | Sigma Aldrich | DN25-100MG | 2 mg/mL |
| DPBS, no calcium, no magnesium | ThermoFisher | 14190-250 | 1X |
| HBSS, no calcium, no magnesium | ThermoFisher | 14170112 | 1X |
| Falcon Polystyrene Microplates (24-Well) | Fisher Scientific | #08-772-51 | |
| Falcon Polystyrene Microplates (12-Well) | Fisher Scientific | #0877229 | |
| Falcon Cell Strainers, 40 µm | Fisher Scientific | #08-771-1 | |
| Direct-zol RNA MiniPrep (50 µg) | Genesee Scientific | 11-331 | |
| Trizol reagent | Invitrogen | 15596026 | |
| DMEM/F-12, HEPES, no phenol red | ThermoFisher | 11039021 | |
| Fetal Bovine Serum, Charcoal stripped | Sigma Aldrich | F6765-500ML | 2% |
| Estratiol (E2) | Sigma Aldrich | E1024-1G | 10 nM |
| Formaldehyde 16% in aqueous solution, EM Grade | VWR | 15710 | 4% |
| Epredia Cassette 1 Slotted Tissue Cassettes | Fisher Scientific | 1000961 | |
| Epredia Stainless-Steel Embedding Base Molds | Fisher Scientific | 64-010-15 | |
| Ethanol, 200 proof (100%) | Fisher Scientific | 22-032-601 | |
| Histoclear | Fisher Scientific | 50-899-90147 | |
| Permount Mounting Medium | Fisher Scientific | 50-277-97 | |
| Epredia Nylon Biopsy Bags | Fisher Scientific | 6774010 | |
| HistoGel Specimen Processing Gel | VWR | 83009-992 | |
| Hematoxylin solution Premium | VWR | 95057-844 | |
| Eosin Y (yellowish) solution Premium | VWR | 95057-848 | |
| TBS Buffer, 20X, pH 7.4 | GenDEPORT | T8054 | 1X |
| TBST (10X), pH 7.4 | GenDEPORT | T8056 | 1X |
| Citric acid | Sigma Aldrich | C0759-1KG | |
| Sodium citrate tribasic dihydrate | Sigma Aldrich | S4641-500G | |
| Tween20 | Fisher Scientific | BP337-500 | |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A2153-100G | 3% |
| DAPI Solution (1 mg/mL) | ThermoFisher | 62248 | 1:1000 dilution |
| VECTASHIELD Antifade Mounting Medium | Vector Labs | H-1000-10 | |
| Clear Nail Polish | Fisher Scientific | NC1849418 | |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 22037246 | |
| VWR Micro Cover Glasses | VWR | 48393-106 | |
| SuperScript VILO Master Mix | ThermoFisher | 11755050 | |
| SYBR Green PCR Master Mix | ThermoFisher | 4364346 | |
| Krt8 Antibody (TROMA-I) | DSHB | TROMA-I | 1:50 dilution |
| Vimentin Antobody | Cell Signaling | 5741S | 1:200 dilution |
| Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 |
ThermoFisher | A-21209 | 1:250 dilution |
| Donkey anti-Rabbin IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
ThermoFisher | A-21206 | 1:250 dilution |
| ZEISS Stemi 508 Stereo Microscope | ZEISS | ||
| ZEISS Axio Vert.A1 Inverted Routine Microscope with digital camera | ZEISS | ||
| Primer Sequence | Forward (5'-3') | Reverse (5'-3') | _ |
| Lipocalin 2 (Lcn2) | GCAGGTGGTACGTTGTGGG | CTCTTGTAGCTCATAGATGGTGC | |
| Lactoferrin (Ltf) | TGAGGCCCTTGGACTCTGT | ACCCACTTTTCTCATCTCGTTC | |
| Progesterone (Pgr) | CCCACAGGAGTTTGTCAAGCTC | TAACTTCAGACATCATTTCCGG | |
| Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) | CAATGTGTCCGTCGTGGATCT | GCCTGCTTCACCACCTTCTT | |
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Citazione di questo articolo
Tang, S., Parks, S. E., Liao, Z., Cope, D. I., Blutt, S. E., Monsivais, D. Establishing 3D Endometrial Organoids from the Mouse Uterus. J. Vis. Exp. (191), e64448, doi:10.3791/64448 (2023).