破骨细胞与人诱导多能干细胞的分化和表征
Summary
该方案介绍了人破骨细胞与诱导多能干细胞（iPSCs）的分化，并描述了破骨细胞和破骨细胞前体的表征方法。
Abstract
该方案详细介绍了人iPSC的繁殖和传代以及它们分化为破骨细胞。首先，将iPSC解离成单细胞悬浮液，以进一步用于胚状体诱导。在中胚层诱导后，胚状体发生造血分化，产生漂浮的造血细胞群。随后，收获的造血细胞经历巨噬细胞集落刺激因子成熟步骤，最后进行破骨细胞分化。破骨细胞分化后，破骨细胞的特征是结合甲基绿色核染色进行 TRAP 染色。破骨细胞被观察到为多核 TRAP+ 多核子。组织蛋白酶 K 染色可以进一步支持它们的鉴定。骨和矿物质吸收测定可以进行功能表征，确认真正的破骨细胞的身份。该协议展示了一种区分人破骨细胞和iPSCs的稳健且通用的方法，并允许在需要大量功能性人破骨细胞的应用中轻松采用。可以设想在骨骼研究、癌症研究、组织工程和内假体研究领域的应用。
Introduction
破骨细胞 （OC） 是造血来源的 1,2 多功能细胞类型，通常被研究人员用于骨病研究 3,4、癌症研究 5,6、组织工程 7,8 和内假体研究 9,10 等领域.然而，OC 分化可能具有挑战性，因为单核前体融合成多核 OC 对于产生功能性 OC11 是必要的。一些生物学因素，如NF-κB配体受体激活剂（RANKL）和巨噬细胞集落刺激因子（M-CSF），是OC分化所必需的。据报道，M-CSF 对细胞增殖、细胞存活和 RANK 表达有积极影响 12,13,14。另一方面，RANKL 与 RANK 结合，后者激活诱导破骨细胞生成的下游信号级联反应。激活通过 TNF 受体相关因子 6 （TRAF6） 介导，这导致 B 细胞抑制剂 α （IκB-α） 中 κ 轻多肽基因增强子的核因子降解，α 是一种结合 NF-kB 二聚体的结合蛋白16,17。因此，IκB-α降解释放NF-kB二聚体，然后转位到细胞核中并诱导转录因子c-Fos和活化T细胞核因子1（NFATc1）的表达。这反过来又触发了大量 OC 分化相关蛋白的转录 15,18。上调蛋白（如 DC-Stamp 和 Atp6v0d2）介导 OC 前体的细胞间融合，导致合胞体形成 19,20,21。
就人原代细胞而言，CD34+ 和 CD14+ PBMC 是目前用于分化为 OCs22 的最广泛使用的细胞类型。然而，这种方法受到来自供体23 的收获细胞 CD34+ 群体内的异质性及其有限的扩增性的限制。人 iPSC 是 OC 的替代来源。由于它们可以无限繁殖24，因此它们允许 OC 生产的可扩展性和升级。这允许区分大量的OC，从而促进OC研究。
已经发表了几种将 iPSC 分化为 OC 的方案 25,26,27。整个分化过程可分为iPSC增殖部分、中胚层和造血分化部分以及OC分化。在分化过程之前繁殖 iPSC 允许在分化之前扩大 OC 产量。关于中胚层和造血分化存在几种方法。传统上，胚状体 （EB） 形成已被用于分化造血细胞，但基于单层的方法代表了另一种不需要 EB 诱导的造血分化策略。然而，基于单层的系统似乎需要进一步优化，因为我们和其他人发现基于EB的方法对于OC的区分更可靠。
在这里，我们描述了使用基于EB的方案将OC与人iPSCs区分开来。该方案改编自Rössler等人26 ，并进行了修改以提高稳健性并允许在分化过程中进行冷冻保存。首先，我们在分化 10 天后仅收获一次造血细胞。然后对造血细胞进行冷冻保存，以便在分化过程中具有更大的灵活性。此外，我们将造血细胞接种密度从 1 x 105 增加到 2 x 105 个细胞/cm2 用于 OC 分化。使用较新的人 iPSC 无血清培养基（hiPSC-SFM，见 材料表），并用 200-300 μg/mL 的基础膜提取物（见 材料表）代替 0.1% 明胶进行孔包被。青霉素/链霉素未添加到培养基中。
Rössler等人26的方案最初从iPSC改编为巨噬细胞分化方案28，该方案使用EB形成进行造血分化。虽然研究人员已经将 EB 的形成用于造血分化很长一段时间29,30，但文献中已经描述了几种 EB 诱导方法，例如自发聚集、圆底孔板离心、悬滴培养、生物反应器培养、锥形管培养、慢转侧血管和微模凝胶培养31.该方案使用在圆底孔板中离心解离的iPSC，使单个iPSC细胞彼此靠近并允许球体（EB）形成，如下所述。
Protocol
注意：本协议中使用的所有试剂都可以在 材料表中找到。除非另有说明，否则所有介质在使用前都预平衡至37°C。所有离心步骤均在37°C下进行，并使用最慢的加速/减速模式。除非另有说明，否则上清液始终使用一次性巴斯德玻璃移液器去除。 1. 人iPSCs的解冻和繁殖 在解冻 iPSC 前一天，用浓度为 200-300 μg/mL 的 1 mL 基础膜提取物包覆 6 孔板的?…
Representative Results
在整个分化过程中监测细胞形态下面描述的所有结果均使用 MCND-TENS2 iPSC 系进行 OC 分化生成。该 iPSC 系之前已用于多项研究32,33。尽管如此，其他iPSC细胞系也已成功用于该分化方案。 定期的视觉评估揭示了iPSCs在整个分化过程中与OCs的不同和不同的形态学特征（图2）。将iPSC菌落（<strong class="xf…
Discussion
该协议提供了一种可靠且稳健的方法来将 iPSC 区分为 OC。然而，在整个差异化过程中可能会遇到一些陷阱。使用该协议33 成功分化了来自不同组织来源的细胞生成的人 iPSC 细胞系。冷冻iPSCs时（参见方案步骤“3.冷冻 iPSCs“），在传代点将一个孔冷冻回一个冷冻管中。解冻时（参见协议步骤“1.人iPSCs的解冻和繁殖“），将一个冷冻管解冻到6孔板的单个孔中。不同的iPSC细胞系会表…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢Giachelli实验室成员的技术帮助和支持。我们感谢 W. M. Keck 显微镜中心和 Keck 中心经理 Nathanial Peters 博士在获取共聚焦显微镜和宽场显微镜图像方面提供的帮助。我们还要感谢 UW Flow 核心设施和 Flow 核心设施经理 Aurelio Silvestroni 的技术支持和帮助。最后，我们感谢 Hannah Blümke 对插图和平面设计的支持。
资金由美国国立卫生研究院拨款 R35 HL139602-01 提供。我们还感谢 NIH S10 赠款 S10 OD016240用于 WM Keck 中心的仪器资助，以及 NIH 资助 1S10OD024979-01A1 用于 UW Flow 核心设施的仪器资助。
Materials
| 2-Mercaptoethanol | Sigma Aldrich | M6250-10ML | |
| Antibody – Anti-Cathepsin K | Abcam | ab19027 | |
| Antibody – APC-conjugated Anti-Human CD45 | BD | 555485 | |
| Antibody – APC-conjugated Mouse IgG1, κ Isotype Control | BD | 555751 | |
| Antibody – BV711-conjugated Anti-Human CD14 | BD | 563372 | |
| Antibody – BV711-conjugates Mouse IgG2b, κ Isotype Control | BD | 563125 | |
| Antibody – Goat Anti-Rabbit IgG H&L Alexa Fluor® 647 | Abcam | ab150079 | |
| Antibody – PE-conjugated Anti-Human CD14 | R&D Systems | FAB3832P-025 | |
| Antibody – PE-conjugated Anti-Human Integrin alpha M/CD11b | R&D Systems | FAB16991P-025 | |
| Antibody – PE-Cy7-conjugated Anti-Human CD34 | BD | 560710 | |
| Antibody – PE-Cy7-conjugated Mouse IgG1 κ Isotype Control | BD | 557872 | |
| Antibody – PE/Cyanine5-conjugated Anti-Human CD11b | Biolegend | 301308 | |
| Antibody – PE/Cyanine5-conjugated Mouse IgG1, κ Isotype Ctrl | Biolegend | 400118 | |
| Antibody – PerCP-Cy5.5-conjugated Mouse IgG1 κ Isotype Control | BD | 550795 | |
| Antibody – PerCpCy5.5-conjugated Anti-Human CD43 | BD | 563521 | |
| Bone Resorption Assay Kit | CosmoBioUSA | CSR-BRA-24KIT | |
| Countess 3 Automated Cell Counter | ThermoFisher | 16812556 | |
| Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract | R&D Sytems | 3434-010-02 | Basal membrane extract |
| DAPI | R&D Systems | 5748/10 | |
| Dispase (5 U/mL) | STEMCELL Technologies | 7913 | |
| DMEM/F-12 with 15 mM HEPES | Stem Cell | 36254 | |
| DMSO | Sigma Aldrich | D2650 | |
| DPBS | Sigma Aldrich | D8537-500ML | |
| Human Bone Morphogenetic Protein 4 (hBMP4) | STEMCELL Technologies | 78211 | |
| Human IL-3 | STEMCELL Technologies | 78146.1 | |
| Human Macrophage Colony-stimulating Factor (hM-CSF) | STEMCELL Technologies | 78150.1 | |
| Human Soluble Receptor Activator of Nuclear Factor-κB Ligand (hsRANKL) | STEMCELL Technologies | 78214.1 | |
| Human Stem Cell Factor (hSCF) | STEMCELL Technologies | 78155.1 | |
| Human TruStain FcX (Fc Receptor Blocking Solution) | Biolegend | 422301 | |
| Human Vascular Endothelial Growth Factor-165 (hVEGF165) | STEMCELL Technologies | 78073 | |
| Invitrogen Rhodamine Phalloidin | Invitrogen | R415 | |
| MEM α, nucleosides, no phenol red | ThermoFisher | 41061029 | |
| mFreSR | STEMCELL Technologies | 05855 | Serum free cryopreservation medium |
| mTeSR Plus medium | STEMCELL Technologies | 100-0276 | Human iPSC-serum free medium (hiPSC-SFM) |
| Nunclon Sphera 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate | Thermo Scientific | 174925 | Round bottom ultra-low attachment 96-well plate |
| P1000 Wide Bore Tips | ThermoFisher | 2079GPK | |
| ROCK-Inhibitor Y-27632 | STEMCELL Technologies | 72304 | |
| StemSpan SFEM | StemCell | 09650 | Hematopoietic cell culture medium |
| TrypLE Select Enzyme (1X), no phenol red | Thermo Fisher | 12563011 | Single-cell dissociation reagent |
| Ultraglutamine | Bioscience Lonza | BE17-605E/U1 | |
| X-VIVO 15 Serum-free Hematopoietic Cell Medium | Bioscience Lonza | 04-418Q | Hematopoietic basal medium |
| µ-Slide 8 Well High | Ibidi | 80806 |
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Cite This Article
Blümke, A., Simon, J., Leber, E., Scatena, M., Giachelli, C. M. Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells. J. Vis. Exp. (205), e66527, doi:10.3791/66527 (2024).