title: Biodistribution and retention of locally administered human mesenchymal stromal cells: Quantitative polymerase chain reaction-based detection of human DNA in murine organs.
abstract: 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.
  
  
  
  A 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 × 106heterogeneous cells.
  
  
  
  Using this assay we investigated the biodistribution of 3 × 105intramuscularly 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.
  
  
  
  This 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.
authors.affiliation: Regenerative Medicine Institute, National University of Ireland, Galway, County Galway, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: Creane
authors.affiliation: Regenerative Medicine Institute, National University of Ireland, Galway, County Galway, Ireland.
authors.forename: Linda
authors.initials: L
authors.lastname: Howard
authors.affiliation: Regenerative Medicine Institute, National University of Ireland, Galway, County Galway, Ireland.
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Brien
authors.affiliation: Regenerative Medicine Institute, National University of Ireland, Galway, County Galway, Ireland. Electronic address: cynthia.coleman@nuigalway.ie.
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Coleman
mesh_terms: D020087:Alu Elements; D000818:Animals; D002465:Cell Movement; D058948:Cell Tracking / Q000379:methods*; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D005260:Female; D006801:Humans; D007273:Injections, Intramuscular; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016133:Polymerase Chain Reaction; D051381:Rats; D013045:Species Specificity; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
chemical_list: D004247:DNA
keywords: biodistribution; cell therapy; genomic DNA; human Alu sequence; mesenchymal stromal cell; polymerase chain reaction; translational stem cell research