Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation.

Q1 Biochemistry, Genetics and Molecular Biology
Maria Siemionow, Sonia Brodowska, Klaudia Różczka, Claire Roesler
{"title":"Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation.","authors":"Maria Siemionow,&nbsp;Sonia Brodowska,&nbsp;Klaudia Różczka,&nbsp;Claire Roesler","doi":"10.21037/sci-2022-026","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Cell-based and chimerism-based therapies represent a promising approach for tolerance induction in transplantation. We propose a new cell therapy of the <i>ex vivo</i> created human hematopoietic chimeric cells (HHCC) as an alternative approach to bone marrow (BM)-based therapies in support of solid organ and vascularized composite allotransplantation (VCA). This study aimed to characterize <i>in vitro</i> the phenotype, genotype, clonogenic, and tolerogenic properties of HHCC.</p><p><strong>Methods: </strong>Thirty <i>ex vivo</i> fusions of CD34<sup>+</sup> cells from two unrelated human BM donors were performed. CD34<sup>+</sup> cells were stained separately with PKH26 and PKH67 membrane dyes and fused using polyethylene glycol (PEG). Creation of human HHCC and chimeric state was confirmed by flow cytometry (FC), confocal microscopy (CM) and electron microscopy (EM). HHCC's phenotype (CD34, CD133, CD117, CD4, CD19, CD4/CD25) was assessed by FC, viability by Trypan Blue, LIVE/DEAD and apoptosis by AnnexinV/Sytox Blue and TUNEL assay, while mixed lymphocyte reaction (MLR) assay assessed HHCC's immunogenicity and tolerogenic properties. HHCC differentiation, proliferation and clonogenic potential were assessed by the colony forming unit (CFU). Polyploidy was evaluated by fluorescence in situ hybridization (FISH), whereas polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeats-polymerase chain reaction (STR-PCR) assessed HHCC's genotype, and chimerism. Reverse transcription polymerase chain reaction (RT-PCR) analyzed cytokines secretion [interleukin (IL)-10, transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α)] up to 14 days post-fusion.</p><p><strong>Results: </strong>FC and CM confirmed creation of HHCC by fusion of CD34<sup>+</sup> cells from two unrelated human donors. After fusion, maintenance of hematopoietic markers and increased expression of Treg-cells (CD4/CD25) was confirmed. Moreover, high HHCC viability (99%) and a low apoptosis rate (1.2%) were revealed HHCC presented decreased immunogenicity by MLR, and significant, 40-fold increase of IL-10 the pro-tolerogenic cytokine at 21 days after fusion (RT-PCR) P<0.0001. The number of polyploid cells was negligible (0.48%). PCR-rSSOP of HHCC after fusion confirmed presence of human leukocyte antigen (HLA) class I and class II-alleles and presence of the loci specific for both CD34<sup>+</sup> cells BM donors by STR-PCR.</p><p><strong>Conclusions: </strong>We have created a new hematopoietic cell line of HHCC from two unrelated human donors, and have successfully characterized <i>in vitro</i>, viability, phenotype, genotype, clonogenic, and tolerogenic properties of HHCC. These unique immunomodulatory and tolerogenic properties introduce HHCC as a novel therapeutic approach for tolerance induction in VCA and solid organ transplantation.</p>","PeriodicalId":21938,"journal":{"name":"Stem cell investigation","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/79/b8/sci-09-2022-026.PMC9813662.pdf","citationCount":"2","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Stem cell investigation","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21037/sci-2022-026","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 2

Abstract

Background: Cell-based and chimerism-based therapies represent a promising approach for tolerance induction in transplantation. We propose a new cell therapy of the ex vivo created human hematopoietic chimeric cells (HHCC) as an alternative approach to bone marrow (BM)-based therapies in support of solid organ and vascularized composite allotransplantation (VCA). This study aimed to characterize in vitro the phenotype, genotype, clonogenic, and tolerogenic properties of HHCC.

Methods: Thirty ex vivo fusions of CD34+ cells from two unrelated human BM donors were performed. CD34+ cells were stained separately with PKH26 and PKH67 membrane dyes and fused using polyethylene glycol (PEG). Creation of human HHCC and chimeric state was confirmed by flow cytometry (FC), confocal microscopy (CM) and electron microscopy (EM). HHCC's phenotype (CD34, CD133, CD117, CD4, CD19, CD4/CD25) was assessed by FC, viability by Trypan Blue, LIVE/DEAD and apoptosis by AnnexinV/Sytox Blue and TUNEL assay, while mixed lymphocyte reaction (MLR) assay assessed HHCC's immunogenicity and tolerogenic properties. HHCC differentiation, proliferation and clonogenic potential were assessed by the colony forming unit (CFU). Polyploidy was evaluated by fluorescence in situ hybridization (FISH), whereas polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeats-polymerase chain reaction (STR-PCR) assessed HHCC's genotype, and chimerism. Reverse transcription polymerase chain reaction (RT-PCR) analyzed cytokines secretion [interleukin (IL)-10, transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α)] up to 14 days post-fusion.

Results: FC and CM confirmed creation of HHCC by fusion of CD34+ cells from two unrelated human donors. After fusion, maintenance of hematopoietic markers and increased expression of Treg-cells (CD4/CD25) was confirmed. Moreover, high HHCC viability (99%) and a low apoptosis rate (1.2%) were revealed HHCC presented decreased immunogenicity by MLR, and significant, 40-fold increase of IL-10 the pro-tolerogenic cytokine at 21 days after fusion (RT-PCR) P<0.0001. The number of polyploid cells was negligible (0.48%). PCR-rSSOP of HHCC after fusion confirmed presence of human leukocyte antigen (HLA) class I and class II-alleles and presence of the loci specific for both CD34+ cells BM donors by STR-PCR.

Conclusions: We have created a new hematopoietic cell line of HHCC from two unrelated human donors, and have successfully characterized in vitro, viability, phenotype, genotype, clonogenic, and tolerogenic properties of HHCC. These unique immunomodulatory and tolerogenic properties introduce HHCC as a novel therapeutic approach for tolerance induction in VCA and solid organ transplantation.

Abstract Image

Abstract Image

Abstract Image

建立人造血嵌合细胞(HHCC)系作为移植耐受诱导的新策略。
背景:基于细胞和嵌合的治疗是一种很有前途的移植耐受诱导方法。我们提出了一种新的细胞疗法,即体外培养的人造血嵌合细胞(HHCC),作为骨髓(BM)基础治疗的替代方法,支持实体器官和血管化复合异体移植(VCA)。本研究旨在体外鉴定HHCC的表型、基因型、克隆性和耐受性。方法:对来自两名无亲缘关系的人骨髓供体的30例CD34+细胞进行体外融合。CD34+细胞分别用PKH26和PKH67膜染料染色,聚乙二醇(PEG)融合。通过流式细胞术(FC)、共聚焦显微镜(CM)和电镜(EM)证实了人HHCC的形成和嵌合状态。采用FC法检测HHCC的表型(CD34、CD133、CD117、CD4、CD19、CD4/CD25),台泛蓝法检测细胞活力,AnnexinV/Sytox Blue和TUNEL法检测细胞凋亡,混合淋巴细胞反应(MLR)法检测HHCC的免疫原性和耐受性。采用菌落形成单位(colony forming unit, CFU)评价HHCC的分化、增殖和克隆潜能。采用荧光原位杂交(FISH)评估多倍体,聚合酶链反应-反序列特异性寡核苷酸探针(PCR-rSSOP)和短串联重复聚合酶链反应(STR-PCR)评估HHCC的基因型和嵌合性。逆转录聚合酶链反应(RT-PCR)分析融合后14天细胞因子的分泌[白细胞介素(IL)-10,转化生长因子-β (TGF-β)和肿瘤坏死因子-α (TNF-α)]。结果:FC和CM证实了两名无亲缘关系的人类供者的CD34+细胞融合产生HHCC。融合后,证实造血标志物维持,treg细胞(CD4/CD25)表达增加。此外,高HHCC活力(99%)和低凋亡率(1.2%)显示,MLR显示HHCC免疫原性下降,融合(RT-PCR)后21天(STR-PCR) P+细胞BM供者的IL-10显着增加40倍。结论:我们从两个无亲缘关系的人类供体中建立了一种新的HHCC造血细胞系,并成功地在体外鉴定了HHCC的活力、表型、基因型、克隆性和耐受性。这些独特的免疫调节和耐受性特性使HHCC成为VCA和实体器官移植中耐受性诱导的新治疗方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Stem cell investigation
Stem cell investigation Biochemistry, Genetics and Molecular Biology-Developmental Biology
CiteScore
5.80
自引率
0.00%
发文量
9
期刊介绍: The Stem Cell Investigation (SCI; Stem Cell Investig; Online ISSN: 2313-0792) is a free access, peer-reviewed online journal covering basic, translational, and clinical research on all aspects of stem cells. It publishes original research articles and reviews on embryonic stem cells, induced pluripotent stem cells, adult tissue-specific stem/progenitor cells, cancer stem like cells, stem cell niche, stem cell technology, stem cell based drug discovery, and regenerative medicine. Stem Cell Investigation is indexed in PubMed/PMC since April, 2016.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信