新型人类脐带双嵌合(HUDC)细胞移植治疗无终身免疫抑制。

Q1 Biochemistry, Genetics and Molecular Biology
Maria Siemionow, Joanna Cwykiel, Lucile Chambily, Stephanie Gacek, Sonia Brodowska
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引用次数: 1

摘要

背景:基于细胞的疗法在骨髓(BM)、实体器官和血管化复合异体移植(VCA)的耐受性诱导方面很有前景。骨髓移植(BMT)方案的毒性阻碍了这种方法的常规临床应用。为了解决这一问题,我们开发了一种新的治疗方法,即人类脐带双嵌合细胞(HUDC)用于移植耐受诱导。本研究建立了构建的HUDC细胞的体外表征。方法:我们进行了16次体外聚乙二醇(PEG)介导的人脐带血(UCB)细胞融合,这些细胞来自两个不相关的供者。通过体外流式细胞术(FC)和共聚焦显微镜(CM)证实融合的可行性。采用淋巴细胞毒性试验和短串联重复聚合酶链反应(STR-PCR)分析HUDC细胞的基因型,FC检测表型,LIVE/DEAD®检测细胞活力,Annexin V染色检测细胞凋亡水平。我们采用COMET法评估融合后HUDC细胞的遗传毒性。采用集落形成单位(CFU)法评价HUDC细胞的克隆特性。混合淋巴细胞反应(MLR)试验评估了HUDC细胞的免疫原性和耐受性。结果:我们通过FC和CM证实了两个不相关的人UCB细胞供体产生HUDC细胞。人类白细胞抗原(HLA) I类和II类分型,以及对HUDC细胞的STR-PCR分析证实了来自两个无亲缘关系的UCB供者的等位基因和位点的存在(供者嵌合:49%±8.3%,n=4)。FC证实了HUDC细胞的造血表型。我们证实,与融合前pkh染色的UCB细胞阳性对照(20.4%)相比,融合后的HUDC细胞存活率高(死亡细胞的0.47%),凋亡水平低(15.9%)。HUDC细胞的COMET检测显示没有DNA损伤。CFU实验证实了HUDC细胞的克隆原性,MLR实验显示HUDC细胞的免疫原性较低。结论:本研究证实了一种新的HUDC细胞系是通过离体peg介导的来自两个不相关供体的UCB细胞融合而产生的。创建HUDC细胞系的独特概念,代表了移植供体和受体的基因型和表型,为BM、实体器官和VCA移植引入了一种有前途的耐受诱导方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Background: Cell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells.

Methods: We performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells' genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells' genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells.

Results: We confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells' viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells.

Conclusions: This study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

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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.
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