脐带血单个核细胞诱导多能干细胞来源自然杀伤细胞的生物学特性

doi:10.3877/cma.j.issn.2095-1221.2021.06.002

中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (06) : 329 -336. doi: 10.3877/cma.j.issn.2095-1221.2021.06.002

论著

脐带血单个核细胞诱导多能干细胞来源自然杀伤细胞的生物学特性

杜为¹, 崔丽娟², 徐迎², 张华², 杜宏伟², 张金美², 刘容志², 王征宇³, 杨文玲⁴, 张宇²^,^†(

)

^1. 300384　天津，协和干细胞基因工程有限公司；300371　天津，南开大学医学院；300384　天津，天津市血液细胞治疗技术企业重点实验室；300384　天津，国家干细胞产品产业化基地
^2. 300384　天津，协和干细胞基因工程有限公司；300384　天津，天津市血液细胞治疗技术企业重点实验室；300384　天津，国家干细胞产品产业化基地
^3. 300384　天津，天津市血液细胞治疗技术企业重点实验室；300020　天津，实验血液学国家重点实验室
^4. 300384　天津，协和干细胞基因工程有限公司；300384　天津，国家干细胞产品产业化基地

收稿日期:2021-03-16 出版日期:2021-12-01
通信作者: 张宇
基金资助:
天津市科技计划创新平台专项(18PTSYJC00070); 天津市博士后择优资助计划项目(TJQYBSH2018030)

Biological characteristics of natural killer cells from cord blood mononuclear cell-derived induced pluripotent stem cells

Wei Du¹, Lijuan Cui², Ying Xu², Hua Zhang², Hongwei Du², Jinmei Zhang², Rongzhi Liu², Zhengyu Wang³, Wenling Yang⁴, Yu Zhang²^,^†()

^1. Union Stem Cell&Gene Engineering Co., LTD, Tianjin 300384, China; School of Medicine, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Blood Cell Therapy Technology, Tianjin 300384, China; National Stem Cell Product Industrialization Base, Tianjin 300384, China
^2. Union Stem Cell&Gene Engineering Co., LTD, Tianjin 300384, China; Tianjin Key Laboratory of Blood Cell Therapy Technology, Tianjin 300384, China; National Stem Cell Product Industrialization Base, Tianjin 300384, China
^3. Tianjin Key Laboratory of Blood Cell Therapy Technology, Tianjin 300384, China; State Key Laboratory of Experimental Hematology, Tianjin 300020, China
^4. Union Stem Cell&Gene Engineering Co., LTD, Tianjin 300384, China; National Stem Cell Product Industrialization Base, Tianjin 300384, China

Received:2021-03-16 Published:2021-12-01
Corresponding author: Yu Zhang

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

杜为, 崔丽娟, 徐迎, 张华, 杜宏伟, 张金美, 刘容志, 王征宇, 杨文玲, 张宇. 脐带血单个核细胞诱导多能干细胞来源自然杀伤细胞的生物学特性[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(06): 329-336.

Wei Du, Lijuan Cui, Ying Xu, Hua Zhang, Hongwei Du, Jinmei Zhang, Rongzhi Liu, Zhengyu Wang, Wenling Yang, Yu Zhang. Biological characteristics of natural killer cells from cord blood mononuclear cell-derived induced pluripotent stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(06): 329-336.

目的

利用脐带血单个核细胞（CBMCs）获得诱导多能干细胞（iPSCs）并进一步诱导分化为自然杀伤（NK）细胞。通过与其他来源NK细胞的比较，探讨iPSCs来源NK细胞的生物学特性。

方法

获取CBMCs并使用非整合质粒电转法对其进行重编程，利用免疫荧光染色和流式细胞术鉴定获得的iPSCs的多能性因子的表达水平。将CBMCs来源iPSCs（CB-iPSCs）诱导分化为NK细胞，并利用流式细胞术对NK细胞表型进行鉴定。利用肿瘤细胞体外杀伤实验，比较CB-iPSCs来源NK（CB-iPSCs-NK）细胞、脐带血来源NK（CB-NK）细胞和外周血来源NK（PB-NK）细胞的杀伤活性。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

CBMCs经过携带重编程因子的非整合质粒转染后，启动多能性基因表达，成功地获得iPSCs克隆，经扩增和筛选，最后获得了可稳定增殖的CB-iPSCs。免疫荧光染色和流式细胞术检测结果显示，获得的CB-iPSCs表达高水平的多能性标志物。然后，利用CB-iPSCs成功分化NK细胞。流式细胞术检测NK受体，包括抑制性受体CD94、细胞毒性受体CD16和Nkp46；与CB-NK和PB-NK比较，CB-iPSCs-NK中CD94（94.17%±1.80%，94.10%±1.32%比87.60%±2.10%）和CD16表达（64.53%± 2.80%，92.40%±2.00%比9.23%±3.37%）较低，Nkp46表达（85.30%±2.36%，29.43%±9.08%比88.00%±3.60%）较高（P均< 0.05）。肿瘤细胞体外杀伤实验结果显示，三种来源NK细胞对K562、MDA-MB-231细胞均具有较好的杀伤作用；但是，对Raji细胞的杀伤作用，CB-iPSCs-NK与CB-NK相似，均弱于PB-NK（P均< 0.05）。

结论

免疫表型和对不同肿瘤细胞杀伤效果的差异，提示CB-iPSCs-NK细胞具有不同的生物学特性。

关键词: 脐带血, 诱导多能干细胞, 自然杀伤细胞, 生物学特性

Objective

To obtain induced pluripotent stem cells (iPSCs) from cord blood mononuclear cells (CBMCs) and further induce them to generate natural killer (NK) cells, then to investigate the biological characteristics of CB-iPSCs-derived NK (CB-iPSCs-NK) cells and compare key traits between CB-iPSCs-NK cells and the other-derived NK cells.

Methods

CBMCs were obtained and transduced with reprogramming factors by non-integrated plasmids to generate iPSCs. The pluripotency factors expressions of CBMCs-derived iPSCs (CB-iPSCs) were determined by immunofluorescence staining and flow cytometry. CB-iPSCs were induced to generate NK cells, and phenotypes of NK cells were identified by flow cytometry. The cytotoxic effects of CB-iPSCs-NK cells, compared with cord blood derived NK (CB-NK) cells and peripheral blood derived NK (PB-NK) cells, were tested by killing tumor cell assay in vitro. One-way ANOVA was used for comparison of variables among multiple groups, and LSD-t test was used for comparison of variables between groups.

Results

After transfection with reprogramming factors by non-integrated plasmid, pluripotent gene expression of CBMCs were initiated, and clones of iPSCs were successfully obtained. After proliferation and selection, the stable CB-iPSCs were finally obtained. The results of immunofluorescence staining and flow cytometry showed that CB-iPSCs expressed high levels of pluripotency markers. Then, CB-iPSCs-NK cells were successfully obtained from CB-iPSCs. NK receptors including inhibitory receptor CD94, cytotoxic receptor CD16 and Nkp46 were detected by flow cytometry. Compared with CB-NK and PB-NK, CD94 expression of CB-iPSCs-NK was lower (94.17%±1.80%, 94.10%±1.32% vs 87.60%±2.10%) , CD16 expression was lower (64.53% ± 2.80%, 92.40%±2.00% vs 9.23%±3.37%) , Nkp46 expression was higher (85.30%±2.36%, 29.43%±9.08% vs 88.00%±3.60%) (All P < 0.05) . The results of killing tumor cell assays in vitro showed that three kindsof NK cells hadexcitingcytotoxic effects to K562 and MDA-MB-231 cells. However, compared with PB-NK cells, cytotoxic effects of CB-iPSCs-NK cells and CB-NK cells to Raji cells were weaker (All P < 0.05) . Cytotoxic effect of CB-iPSCs-NK cells to Raji cells was similar to that of CB-NK cells.

Conclusion

The differences of immunophenotypes and cytotoxic effects suggested that CB-iPSCs-NK cells have different biological characteristics for killing tumor cells.

Key words: Cord blood, Induced pluripotent stem cells, Natural killer cells, Biological characteristics

图1 iPSCs多能性标志物分析注：a图为倒置相差显微镜下观察的AP染色结果（×100）；b ~ d图为多能性标志物流式检测结果：e ~ i图为倒置荧光显微镜下观察多能性标志物免疫荧光结果（×200）；e图为TRA-1-60（红色）：f图为SSEA4（红色）：g图为NANOG（绿色）：h图为OCT3/4（绿色）：i图为SOX2（绿色），DAPI（蓝色）

图2 倒置荧光显微镜下观察iPSCs三胚层分化的免疫荧光结果（×100）注：a图为内胚层标志蛋白AFP3（绿色）；b图为中胚层标志蛋白1A4（绿色）；c图为外胚层标志蛋白10C2（绿色），DAPI（蓝色）

图3 iPSC三胚层分化的qPCR Array检测结果注：未分化的iPSC作为对照（Ectoderm为外胚层，Mesoderm为中胚层，Endoderm为内胚层，Pluripotent/Undifferentiation为未分化的iPSC）

图4 CB-iPSC分化成NK细胞过程中不同时间点的细胞状态（×100）注：a图为当CB-iPSCs生长到60%~70%融合时状态良好（第0天）；b图为消后细胞加入3D培养基，培养2d iPS球体直径可达50 ~ 150 μm（第2天）；c图为继续培养2 d拟胚体直径达到150 ~ 250 μm（第4天）；d图为更换APEL培养基进入拟胚体分化，继续培养至第15天；e图为更换AEL培养基继续培养至第29天；f图为培养至第43天，培养基中出现大量的悬浮细胞

图5 FACS分析3种自然杀伤细胞的表面标志物注：a ~ e图为FACS分析CB-NK细胞表面标志物；f ~ j图为FACS分析PB-NK细胞表面标志物；k ~ o图为分析CB-iPSC-NK细胞表面标志物（包括CD3、CD56、CD94、Nkp46和CD16）

表1 3种NK细胞表面标志物比例（%，±s，n = 3）

分组		CD3	CD16	NKp46	CD94
CB-NK		17.03±0.80	64.53±2.80	85.30±2.36	94.17±1.80
PB-NK		4.32±0.83^a	92.40±2.00^a	29.43±9.08^a	94.10±1.32
CB-iPSC-NK		0.67±0.26^ab	9.23±3.37^ab	88.00±3.60^ab	87.60±2.10^ab
	F值	472.28	695.90	97.45	13.65
	P值	< 0.001	< 0.001	< 0.001	0.006

表1 3种NK细胞表面标志物比例（%，±s，n = 3）

分组		CD3	CD16	NKp46	CD94
CB-NK		17.03±0.80	64.53±2.80	85.30±2.36	94.17±1.80
PB-NK		4.32±0.83^a	92.40±2.00^a	29.43±9.08^a	94.10±1.32
CB-iPSC-NK		0.67±0.26^ab	9.23±3.37^ab	88.00±3.60^ab	87.60±2.10^ab
	F值	472.28	695.90	97.45	13.65
	P值	< 0.001	< 0.001	< 0.001	0.006

表2 三种NK细胞不同效/靶比对k562细胞的杀伤比例（%，±s，n = 3）

分组		0.5∶1	1∶1	5∶1	10∶1
CB-NK		0.45±0.05	0.68±0.12	0.95±0.04	0.98±0.01
PB-NK		0.54±0.01	0.82±0.06	0.97±0.02	0.99±0.01
CB-iPSC-NK		0.47±0.07	0.74±0.06	0.96±0.02	0.98±0.01
	F值	2.98	1.94	0.68	0.89
	P值	0.13	0.22	0.54	0.46

表2 三种NK细胞不同效/靶比对k562细胞的杀伤比例（%，±s，n = 3）

分组		0.5∶1	1∶1	5∶1	10∶1
CB-NK		0.45±0.05	0.68±0.12	0.95±0.04	0.98±0.01
PB-NK		0.54±0.01	0.82±0.06	0.97±0.02	0.99±0.01
CB-iPSC-NK		0.47±0.07	0.74±0.06	0.96±0.02	0.98±0.01
	F值	2.98	1.94	0.68	0.89
	P值	0.13	0.22	0.54	0.46

表3 三种NK细胞不同效/靶比对MDA-MB231细胞的杀伤比例（%，±s，n = 3）

分组		0.5∶1	1∶1	5∶1	10∶1
CB-NK		0.14±0.04	0.25±0.02	0.77±0.08	0.90±0.06
PB-NK		0.23±0.05	0.32±0.04	0.86±0.03	0.95±0.02
CB-iPSC-NK		0.15±0.03	0.29±0.06	0.81±0.07	0.92±0.07
	F值	4.55	2.05	1.67	0.60
	P值	0.06	0.21	0.26	0.59

表3 三种NK细胞不同效/靶比对MDA-MB231细胞的杀伤比例（%，±s，n = 3）

分组		0.5∶1	1∶1	5∶1	10∶1
CB-NK		0.14±0.04	0.25±0.02	0.77±0.08	0.90±0.06
PB-NK		0.23±0.05	0.32±0.04	0.86±0.03	0.95±0.02
CB-iPSC-NK		0.15±0.03	0.29±0.06	0.81±0.07	0.92±0.07
	F值	4.55	2.05	1.67	0.60
	P值	0.06	0.21	0.26	0.59

表4 3种NK细胞不同效/靶比对Raji细胞的杀伤比例（%，±s， n = 3）

分组		0.5:1	1:1	5:1	10:1
CB-NK		0.05±0.03	0.14±0.03	0.21±0.03	0.26±0.03
PB-NK		0.19±0.03^a	0.33±0.05^a	0.64±0.05^a	0.79±0.04^a
CB-iPSC-NK		0.04±0.02^b	0.11±0.03^b	0.16±0.02^b	0.19±0.03^b
	F值	32.08	35.95	169.38	338.70
	P值	< 0.001	< 0.001	< 0.001	< 0.001

表4 3种NK细胞不同效/靶比对Raji细胞的杀伤比例（%，±s， n = 3）

分组		0.5:1	1:1	5:1	10:1
CB-NK		0.05±0.03	0.14±0.03	0.21±0.03	0.26±0.03
PB-NK		0.19±0.03^a	0.33±0.05^a	0.64±0.05^a	0.79±0.04^a
CB-iPSC-NK		0.04±0.02^b	0.11±0.03^b	0.16±0.02^b	0.19±0.03^b
	F值	32.08	35.95	169.38	338.70
	P值	< 0.001	< 0.001	< 0.001	< 0.001

1	Shimasaki N, Jain A, Campana D. NK cells for cancer immunotherapy[J]. Nat Rev Drug Discov, 2020, 19(3):200-218.
2	Zeng J, Tang SY, Toh LL, et al. Generation of "Off-the-Shelf" natural killer cells from peripheral blood cell-derived induced pluripotent stem cells[J]. Stem Cell Reports, 2017, 9(6):1796-1812.
3	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4):663-676.
4	Glicksman M. Induced pluripotent stem cells: the most versatile source for stem cell therapy[J]. Clin Ther, 2018, 40(7):1060-1065.
5	Haase A, Olmer R, Schwanke K, et al. Generation of induced pluripotent stem cells from human cord blood[J]. Cell Stem Cell, 2009, 5(4):434-441.
6	Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy[J]. Nat Rev Clin Oncol, 2021, 18(2):85-100.
7	Oberschmidt O, Morgan M, Huppert V, et al. Development of automated separation, expansion, and quality control protocols for clinical-scale manufacturing of primary human NK cells and alpharetroviral chimeric antigen receptor engineering[J]. Hum Gene Ther Methods, 2019, 30(3):102-120.
8	Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells[J]. Nat Immunol, 2008, 9(5):503-510.
9	Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells in cancer immunotherapy[J]. 2016, 17(9):1025-1036.
10	Robinson MW, Harmon C, O'Farrelly C. Liver immunology and its role in inflammation and homeostasis[J]. Cell Mol Immunol, 2016, 13(3):267-276.
11	Sungur CM, Murphy WJ. Positive and negative regulation by NK cells in cancer[J]. Crit Rev Oncog, 2014, 19(1-2):57-66.
12	Chester C, Fritsch K, Kohrt HE. Natural killer cell immunomodulation: targeting activating, inhibitory, and co-stimulatory receptor signaling for cancer immunotherapy[J]. Front Immunol, 2015, 6:601.
13	Knorr DA, Kaufman DS. Pluripotent stem cell-derived natural killer cells for cancer therapy[J]. Transl Res, 2010, 156(3):147-154.
14	Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy[J]. Nat Rev Clin Oncol, 2021, 18(2):85-100.
15	Carlsten M, Childs R W. Genetic manipulation of NK cells for cancer immunotherapy: techniques and clinical implications[J]. Front Immunol, 2015, 6:266. doi: 10.3389/fimmu.2015.00266.
16	Zhu H, Kaufman DS. An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells[J]. Methods Mol Biol, 2019, 2048:107-119.
17	Clynes RA, Desjarlais JR. Redirected t cell cytotoxicity in cancer therapy[J]. Annu Rev Med, 2019, 70:437-450.
18	Bloushtain N, Qimron U, Bar-Ilan A, et al. Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46[J]. J Immunol, 2004, 173(4):2392-2401.
19	Diefenbach A, Raulet DH. Innate immune recognition by stimulatory immunoreceptors[J]. Curr Opin Immunol, 2003, 15(1):37-44.
20	Knorr DA, Ni Z, Hermanson D, et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy[J]. Stem Cells Transl Med, 2013, 2(4):274-283.
21	Hermanson DL, Bendzick L, Pribyl L, et al. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer[J]. Stem Cells, 2016, 34(1):93-101.

[1]	柳成林, 荀文兴, 杨海珍, 范素萌, 刘宇博, 张红梅. 程序性坏死特异性抑制剂-1对高糖环境下牙周膜干细胞增殖和成骨分化的影响[J]. 中华口腔医学研究杂志(电子版), 2022, 16(03): 160-167.
[2]	钟勇辉, 谢福川, 魏宜胜. 结肠癌来源外泌体对自然杀伤细胞miR-18a和NKG2D表达的影响[J]. 中华普通外科学文献(电子版), 2021, 15(03): 172-177.
[3]	曹玲莉, 涂平华, 吴展陵, 李新军. NK细胞、Treg细胞、T淋巴亚群在NSCLC外周血中表达及临床意义[J]. 中华肺部疾病杂志(电子版), 2023, 16(01): 55-57.
[4]	刘泽, 郝言, 赵铎, 徐辉. 复方甘草酸苷对小儿哮喘T淋巴细胞及NK细胞的影响[J]. 中华肺部疾病杂志(电子版), 2022, 15(01): 97-99.
[5]	崔燕妮, 任颜, 梁凤婷, 覃艳红, 王宏伟. 脐带血源性产品Omidubicel的临床研发进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 178-182.
[6]	符莞孟, 王晓黎, 刘玉, 张潍, 张菊. 干细胞治疗多囊卵巢综合征的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 108-114.
[7]	胡敏洁, 王思贤, 王永煜. 人诱导多能干细胞及其在血管相关疾病模型中的应用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 167-175.
[8]	李华, 李晓帆, 李乃农. 间充质干细胞促进脐带血干细胞体外扩增的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(01): 34-38.
[9]	杨慧, 姚浩, 陈丹. 急性髓系白血病的细胞治疗进展[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(05): 311-316.
[10]	马恩奇, 张倩, 陈雪梅, 刘韬. iPSCs诱导的造血干细胞对内皮细胞血管生成作用的影响及相关作用机制研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 193-199.
[11]	薛玲玲, 陈锦阳, 庄盼, 刘军权. 人羊膜上皮细胞和人羊膜间充质干细胞的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(03): 184-188.
[12]	谭天华, 宋京海. 肝细胞癌NK细胞及其相关免疫治疗研究进展[J]. 中华肝脏外科手术学电子杂志, 2023, 12(02): 243-246.
[13]	蔡惠宁, 钱师宇, 陈伟材, 黄梦淳, 陈文捷, 卢建溪. 体外扩增外周血NK细胞对不同肿瘤细胞系的细胞毒作用[J]. 中华肝脏外科手术学电子杂志, 2022, 11(04): 411-415.
[14]	汪东生, 吴理达, 顾雨春. 细胞基因疗法在视网膜退行性疾病中的应用和挑战[J]. 中华眼科医学杂志(电子版), 2022, 12(03): 129-133.
[15]	陈婷婷, 江学良, 余佳丽, 柯剑林. 干细胞治疗炎症性肠病的安全性[J]. 中华消化病与影像杂志(电子版), 2023, 13(04): 193-198.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Biological characteristics of natural killer cells from cord blood mononuclear cell-derived induced pluripotent stem cells

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Biological characteristics of natural killer cells from cord blood mononuclear cell-derived induced pluripotent stem cells