Biodistribution and retention of locally administered human mesenchymal stromal cells: Quantitative polymerase chain reaction-based detection of human DNA in murine organs

详细信息    查看全文

• 作者：Michael Creane ; Linda Howard ; Timothy O'Brien ; Cynthia M. Coleman ; ^{cynthia.coleman@nuigalway.ie}
• 关键词：biodistribution ; cell therapy ; genomic DNA ; human Alu sequence ; mesenchymal stromal cell ; polymerase chain reaction ; translational stem cell research
• 刊名：Cytotherapy
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：19
• 期：3
• 页码：384-394
• 全文大小：1177 K
• 卷排序：19

文摘

Determining the distributive fate and retention of a cell therapy product after administration is an essential part of characterizing it's biosafety profile. Therefore, regulatory guidelines stipulate that biodistribution assays are a requirement prior to advancing a cell therapy to the clinic. Here the development of a highly sensitive quantitative polymerase chain reaction (qPCR)-based method of tracking the biodistribution and retention of human mesenchymal stromal cells (hMSCs) in mice, rats or rabbits is described.MethodsA primer-probe–based qPCR assay was developed to detect and quantify human Alu sequences in a heterogeneous sample of human DNA (hDNA) and murine DNA from whole organ genomic DNA extracts. The assay measures the amount of genomic hDNA by amplifying a 31–base pair sequence of the human Alu (hAlu) repeat sequence, thus enabling the detection of 0.1 human cells in 1.5 × 106 heterogeneous cells.ResultsUsing this assay we investigated the biodistribution of 3 × 105 intramuscularly injected hMSCs in Balb/c nude mice. Genomic DNA was extracted from murine organs and hAlu sequences were quantified using qPCR analysis. After 3 months, hDNA ranging from 0.07%–0.58% was detected only at the injection sites and not in the distal tissues of the mice.DiscussionThis assay represents a reproducible, sensitive a method of detecting hDNA in rodent and lapine models. This manuscript describes the method employed to generate preclinical biodistribution data that was accepted by regulatory bodies in support of a clinical trial application.