本协议显示了如何在3D和2D培养条件下区分具有轻度细胞毒性的CD3 – / CD45 + CD56 +细胞与人扩增潜在干细胞(hEPSC)。这允许在不破坏复杂微环境的情况下进行常规表型验证。
自然杀伤(NK)细胞与人类多能干细胞的分化允许研究和制造用于免疫治疗的临床级细胞产品。这里描述的是一种两相方案,它使用无血清商业培养基和细胞因子(白细胞介素 [IL]-3、IL-7、IL-15、干细胞因子 [SCF] 和 FMS 样酪氨酸激酶 3 配体 [Ftl3L])将人扩增电位干细胞 (hEPSC) 分化为具有 NK 细胞特性的细胞通过三维 (3D) 和二维 (2D) 培养技术在 体外 。按照该方案,CD3 – CD56 +或CD45 + CD56 + NK细胞持续产生。当与肿瘤靶标共培养3小时时,与IL-2非依赖性永久性细胞系NK92mi细胞相比,分化的产物显示出轻度细胞毒性。该协议通过生成3D结构保留了分化微环境的复杂性,从而促进了免疫细胞与其生态位之间空间关系的研究。同时,2D培养系统可以在不损害精细分化生态位的情况下对细胞分化进行常规表型验证。
与人类胚胎干细胞(hESCs)和人类诱导多能干细胞(hiPSCs)等传统多能干细胞相比,人类扩增潜能干细胞(hEPSC)更接近全能性状态,因为它们可以分化为胚胎外和胚胎谱系1。例如,hEPSC可以分化为滋养层1 和卵黄囊样细胞2。为了实现hEPSC的独特效力,在含有几种抑制谱系承诺信号1的小分子的培养基中培养单个卵裂球,该培养基称为EPSC培养基(EPSCM)。在EPSCM中培养hESC和hiPSC可以扩大其先前受限的效力,以将它们分化为滋养层细胞1。
多能干细胞是试验新型基因改造的宝贵研究工具。由于多能干细胞的自我更新和分化能力,多能干细胞的一个转化克隆可以产生在同一位点具有相同基因修饰的分化细胞产物。Zhu和Kaufman建立了NK细胞与传统多能干细胞(hESC和hiPSCs)分化的标准3。首先,他们从源自多能干细胞的胚状体诱导造血干细胞(HSC)。干细胞因子(SCF),FMS样酪氨酸激酶3配体(Ftl3L),白细胞介素(IL)-7,IL-15和IL-3的早期补充剂的添加使HSC偏向发展为自然杀伤(NK)细胞。随后,他们用人工抗原呈递细胞(aAPC)扩增NK细胞,这些细胞呈递膜结合IL-21,并不断补充IL-2。研究人员已将这种方法应用于免疫疗法,以产生赋予嵌合抗原受体(CAR-iNK)的iPSC衍生的自然杀伤细胞4。
人NK细胞被定义为外周血中的CD3−CD56+白细胞。它们是对抗病毒感染细胞和肿瘤细胞的效应器5。NK细胞上的一些抑制受体识别正常细胞中普遍表达的分子。例如,在NK细胞上表达的NKG2A / CD94异二聚体识别MHC I类分子5。同时,NK细胞上的激活受体识别应激诱导的配体,就像NKG2D识别转化诱导的MHC I类多肽相关序列A(云母/MIB)6一样。一些转化细胞下调其“自配体”以逃避免疫监视并上调异常配体,从而触发NK细胞执行其裂解机制。NK细胞的内在抗肿瘤能力引起了人们对这种免疫细胞类型的关注。
同时产生胚胎和胚胎外谱系的能力可能使hEPSCs比传统的多能干细胞更忠实地概括胚胎发育生态位。根据经验,维持hEPSCs的效力比hiPSC更容易。在本研究中,开发了一种方案(图1)以偏向hEPSCs发育为造血谱系,然后在 体外将 祖细胞分化为自然杀伤(NK)细胞。在该方案中,使用商业培养基以避免血清不一致,然后逐步添加细胞因子以偏向分化为淋巴系。该协议有两个系统来保存复杂的3D微环境,同时衍生出表型和功能类似于人类NK细胞的CD3 – CD56 + / CD45 + CD56 +细胞。
该协议可能有助于研究免疫细胞与其分化生态位的相互作用,并且可能具有纯化细胞产物用于免疫治疗用途的潜力。
有几个关键步骤可以确保CD45 + CD56 +细胞从hEPSC中成功分化。预分化(步骤1.2)至关重要,因为EPSC培养基含有谱系承诺1的抑制剂。预分化后,hEPSC的圆顶形菌落变平(图2B)。在hEPSC形成EBs的过程中(步骤1.3)添加Y27632,一种Rho激酶抑制剂(ROCKi),对于hEPSC解离后的存活是必不可少的。另一个重要的考虑因素是每个跨孔上种植的EB数量。由于它是一个小规模的系统,每个跨孔两到三个EB是防止介质过度消耗的最佳密度。在3D系统的NK细胞分化的整个过程中保持气液界面,据信可以保持基质细胞的极性并支持AGM衍生造血祖细胞的扩增14,15。
如前所述,Transwell上EB的密度是成功分化的决定性因素。其关键标志是在Transwell上植入EB后1天培养基的颜色。如果颜色迅速变黄,则表明污染或过度拥挤。为了解决这个问题,应使用1 mL移液器手动拾取额外的EB并将其转移到另一个Transwell中。
该协议的一个主要限制是IVD产物中CD3 – / CD45 + CD56 +细胞的纯度,与纯NK92mi细胞相比,这被认为是较差的细胞毒性的部分原因。采用3D培养系统通过产生类似于三个胚胎胚层的 EB来 诱导造血祖细胞。该策略模拟骨髓微环境中血细胞的发育,从而消除了对基质细胞支持分化的需要3。如果没有纯化造血祖细胞的分选步骤,IVD产品的纯度预计将降低。保留复杂分化生态位同时提高终点产物纯度的策略是开发一种用于分选CD3−CD56 + IVD产物的扩增方法。例如,分选的CD3−CD56 + IVD产物可以在人工抗原呈递细胞(aAPC)上培养,例如用膜结合IL-21改造的辐照K562细胞。随着培养物中IL-2的供应,该方法可以使NK细胞数量增加1,000倍3。
另一个限制是作为阳性参考的真正的人NK细胞的获取有限。NK92mi是共培养测定中使用的阳性参考,但它不够准确。NK92mi细胞由NK92细胞(一种表达活化的NK细胞表型16的永久性细胞系)改造而成,可自主产生IL-2以满足培养要求。NK92细胞在 体外 对K562细胞表现出比人原代NK细胞更好的杀伤活性17。通过平行测定外周血NK细胞的细胞毒性,可以实现IVD产物肿瘤杀伤能力的更公平比较,但不幸的是,缺乏血库的途径。
这种双系统培养方案旨在从hEPSC产生CD3− / CD45 + CD56 +细胞。FACS对表面标志物和细胞毒性的评估可以在不破坏分化生态位的情况下常规进行。尽管CD3-CD56 +细胞的百分比存在差异,但3D和2D系统可以衍生出CD3-CD56 +细胞,CD56表达变化相似(图3),并且2D系统反映了3D培养条件下淋巴祖细胞的存在。
利用3D培养系统的意义在于,它允许使用高分辨率分析分析,例如空间转录组学或成像质谱法,来研究复杂分化生态位内的空间关系和细胞间相互作用。
The authors have nothing to disclose.
我们要感谢曹汉迪博士、高三星、向大卫、赵一鸣、丽蒂卡·乔加尼、欧文·陈、斯蒂芬妮·张和陈席琳的技术援助和有益的讨论。这项工作得到了平台技术基金的支持。 图 1 是使用 BioRender.com 创建的。
30 w/v% Albumin Solution, from Bovine Serum(BSA), Fatty Acid Free | Wako Chemicals | 017-22231 | For resuspending cytokines. |
ACCUMAX | STEMCELL Technologies | 07921 | |
Anti-human-CD159a-PE | BD | 375104 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 5% FBS) |
Anti-human-CD3-APC antibodies | BD | 555355 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 2% FBS) |
Anti-human-CD45-APC antibodies | BD | 555485 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 3% FBS) |
Anti-human-CD56-PE antibodies | BD | 555516 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 4% FBS) |
Anti-human-CD56-PEcy7 antibodies | BD | 335826 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 5% FBS) |
Anti-human-CD94-APC | BD | 559876 | During antibody staining, 0.5 µL of antibodies is added into the staining buffer (PBS + 5% FBS) |
Bemis Parafilm M Laboratory Wrapping Film | Thermo Scientific | 11772644 | |
Cd244 Monoclonal Antibody (eBioC1.7 (C1.7)), Functional Grade, eBioscience | Thermo Scientific | 16-5838-85 | |
Costar 6.5 mm Transwell, 0.4 µm Pore Polyester Membrane Inserts | STEMCELL Technologies | 3470 | |
Dapi Solution, 1 mg/mL | BD | 564907 | It is resuspended with ddH2O so the concentration is 10 µM/mL. Add 1 µL of the aliquot in 300 µL of FACS buffer (PBS + 2% FBS) |
DMEM | CPOS-Bioreagent core | 11965092 | |
DMEM/F12 | Thermo Scientific | 11320033 | |
FcX, human | Biolengend | 422302 | |
Fetal Bovine Serum(FBS) qualified E.U.-approved South America | CPOS-Bioreagent core | 10270106 | |
FlowJo v10.8.0 | BD | ||
Gibco PBS (10x) pH 7.4 | CPOS-Bioreagent core | 70011044 | 10x concentrated PBS is manufactured as follows: without calcium, magnesium or phenol red |
K562 cells | ATCC | CCL-243 | |
Knockout Serum Replacement | Thermo Scientific | 10828-028 | |
MyeloCult H5100 medium | STEMCELL Technologies | 5150 | |
NK92mi cells | ATCC | CRL-2408 | Lot: 70045208. Once thawed, they are cultured in MyeloCult H5100 medium, and maintained the density between 2 x 105 to 1 x 106 cell/mL. |
Nunc Cell-Culture Treated Multidishes 12 well plate | Thermo Scientific | 150628 | flat bottom |
Nunc Cell-Culture Treated Multidishes 24 well plate | Thermo Scientific | 142475 | |
Nunc EasYDish Dishes | Thermo Scientific | 150460 | |
pLenti-GFP Lentiviral Control Vector | CELL BIOLABS | LTV-400 | It is packaged as the manufactuer suggested (https://www.cellbiolabs.com/sites/default/files/LTV-400-gfp- lentiviral-plasmid.pdf). 40 μL of 50x lentivirus is added with 1 μL 10 mg/mL polybrene per 1 mL of cell suspension. Complete medium change is performed 24 h after the addition of lentivirus. Cells are incubated undisputedly for 3 days at 37 °C, 5% CO2. |
Polybrene | EMD Millipore | TR-1003-G | |
Recombinant Human Flt-3 Ligand/FLT3L Protein | R&D Systems | 308-FK-005 | It is resuspend with PBS + 0.2% BSA. Thr working concentration is 10 ng/mL or 12 IU/mL. |
Recombinant Human IL-15 Protein | R&D Systems | 247-ILB-005 | It is resuspended with PBS + 0.2% BSA. The working concentration is 10 ng/mL or 4500 IU/mL. |
Recombinant Human IL-18/IL-1F4 Protein | R&D Systems | 9124-IL-010 | It is resuspended with PBS + 0.2% BSA. The working concentration is 50 ng/mL. The IU is not provided by the company. |
Recombinant Human IL-2 Protein | R&D Systems | 202-IL-010 | It is resuspended with PBS + 0.2% BSA. The working concentration is 50 ng/mL or 455 IU/mL. |
Recombinant Human IL-3 Protein, 50ug | PeproTech | 200-03 | It is resuspended with PBS + 0.2% BSA. The working concentration is 5 ng/mL or 14 IU/mL. |
Recombinant Human IL-7 Protein | R&D Systems | 207-IL-010 | It is resuspended with PBS + 0.2% BSA. The working concentration is 20 ng/mL or 8800 IU/mL. |
Recombinant Human SCF Protein | R&D Systems | 255-SC-010 | It is resuspended with PBS + 0.2% BSA. The working concentration is 20 ng/mL or 26 IU/mL. |
STEMdiff Hematopoietic Kit | STEMCELL Technologies | 5310 | Medium A: 45 mL STEMdiff Hematopoietic Basal Medium + STEMdiff Hematopoietic Supplement A ; Medium B: 75 STEMdiff Hematopoietic Basal Medium + STEMdiff Hematopoietic Supplement B |
StemSpan lymphoid differentiation coating material | STEMCELL Technologies | 9950 | It is resuspended in PBS (100x) |
StemSpan NK cell differentiation medium | STEMCELL Technologies | 9960 | It is prepared by adding 500 µL StemSpan NK Cell Differentiation Supplement (100x) into 49.5 mL of SFEM II. Both are provided in StemSpan NK Cell Generation Kit. |
StemSpan lymphoid expansion medium | STEMCELL Technologies | 9960 | It is prepared by adding 5 mL StemSpan Lymphoid Progenitor Expansion Supplement (10x) into 45 mL of StemSpan SFEM II. Both are provided in StemSpan NK Cell Generation Kit. |
StemSpan NK Cell Generation Kit | STEMCELL Technologies | 9960 | Thaw the medium and materials in room temperature and the medium is stored in 4 °C once thawed. |
The BD LSRFortessa cell analyzer | BD | ||
Trypsin-EDTA (0.05%), phenol red | Thermo Scientific | 25300054 | |
Y-27632 dihydrochloride | Tocris | 1254 | working concentration: 10 µM |