Lombard Catherine A, Fabre Alexandre, Ambroise Jérôme, Ravau Joachim, André Floriane, Jazouli Nawal, Najimi Mustapha, Stéphenne Xavier, Smets Françoise, Vaerman Jean-Luc, Sokal Etienne M
Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200 Brussels, Belgium.
Service de Pédiatrie Multidisciplinaire, Hôpital de la Timone, APHM, Marseille, France.
Stem Cells Int. 2019 Jun 12;2019:8129797. doi: 10.1155/2019/8129797. eCollection 2019.
Cell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used. The use of a droplet digital PCR (ddPCR) assay based on mismatch of null alleles is a promising alternative.
We selected genes with a high frequency of null genotype in the general population (SRY, RHD, TRY6, LEC3C, GSTM1, and GSTT1) and investigated their expression by liver progenitor cell donors and liver cell therapy recipients, in order to identify genes of interest for each donor/recipient couple. We first validated the detection of microchimerism by ddPCR and then used these assays to detect and quantify microchimerism in pre- and postinfusion liver biopsies.
We validated the ddPCR detection of the selected genes based on linearity, precision, lack of inhibition, and accuracy, and we established limits of blank, limits of detection, and limits of quantification to ensure the reliability of the results. After genotyping donors and recipients, we were able to identify at least one gene of interest for each donor/recipient couple. We detected donor cells in the three patients posttransplantation. However, analysis of several biopsies taken at the same timepoint revealed a heterogeneous cell distribution. In addition, the values obtained remained below the limit of quantification. Therefore, the actual quantification of microchimerism may not be entirely accurate.
Overall, our study demonstrates that the detection of microchimerism post-liver cell transplantation can be performed using ddPCR amplification of null allele genes expressed by the donor but absent from the recipient. However, this technique can be extended to other cell types and target organs in cell transplantation.
细胞移植正在临床开发中，用于治疗包括获得性和先天性肝脏疾病在内的各种疾病。输注后供体细胞的检测和定量仍然困难。传统方法（基于性别的荧光原位杂交、人类白细胞抗原错配和短串联重复序列聚合酶链反应）只能达到较低的灵敏度水平（1%），因此很少使用。基于无效等位基因错配的液滴数字聚合酶链反应（ddPCR）检测是一种有前途的替代方法。
我们选择了在普通人群中无效基因型频率较高的基因（SRY、RHD、TRY6、LEC3C、GSTM1和GSTT1），并研究了肝祖细胞供体和肝细胞治疗受者对这些基因的表达情况，以便为每对供体/受者确定感兴趣的基因。我们首先通过ddPCR验证了微嵌合体的检测，然后使用这些检测方法来检测和定量输注前和输注后肝活检中的微嵌合体。
我们基于线性、精密度、无抑制性和准确性验证了ddPCR对所选基因的检测，并建立了空白限、检测限和定量限，以确保结果的可靠性。在对供体和受体进行基因分型后，我们能够为每对供体/受者确定至少一个感兴趣的基因。我们在三名患者移植后检测到了供体细胞。然而，对同一时间点采集的多个活检样本的分析显示细胞分布不均一。此外，获得的值仍低于定量限。因此，微嵌合体的实际定量可能并不完全准确。
总体而言，我们的研究表明，肝细胞移植后微嵌合体的检测可以通过对供体表达但受体不存在的无效等位基因进行ddPCR扩增来实现。然而，这项技术可以扩展到细胞移植中的其他细胞类型和靶器官。
