Overestimation of hematopoietic stem cell frequencies in human liver grafts
Authors: Sean R.R. Hall, Alexander Pedroza-Gonzalez, Qiuwei Pan, Hugo W. Tilanus, Jeroen de Jonge, Gerard Wagemaker, Luc J.W. van der Laan
Journal: Hepatology (2012)

We read with great interest the article by Wang et al.1 demonstrating evidence of blood chimerism of donor origin after liver transplantation potentially as a result of donor liver-derived hematopoietic stem cells (HSCs). The adult liver harbors progenitor cells that enable hematopoiesis.2, 3 Our group identified liver-derived CD34+ cells with hematopoietic potential, in agreement with earlier published work,2 which we presented at the American Association for the Study of Liver Diseases (AASLD) annual meeting in 2008. The field of human HSC biology has progressed considerably since the discovery that the majority of HSCs are found within the CD34+ compartment.4 To date, cells with the marker profile Lin−CD34+CD38−CD90+CD45RA− satisfy the most stringent criteria for HSC appellation.2 Wang et al.1 report surprisingly high levels of HSCs within donor livers based on the antigenic profile Lin−CD34+CD38−CD90+. However, besides omitting CD45RA, the authors fail to include a stringent gate in the side scatter (SSC) versus forward scatter (FSC) dotplot or a viability dye, which can safeguard against including debris and dead cells that autofluoresce and nonspecifically bind antibodies, a common event following tissue digestion. We have previously detected CD34+ cells in the preservation fluid (perfusates) of human liver grafts.5 Upon further investigation of the perfusates, we can detect rare Lin−CD34+CD38−CD90+CD45RA− based on a stringent gating and antigenic criterion4 (mean 2.2 × 10−5% ± 0.8 × 10−5 standard error of the mean [SEM], n = 8), as shown in Fig. 1. We also were surprised at the poor level of human hematopoietic chimerism observed after engraftment in immunocompromised mice, which may be related to the authors' use of both impure Lin−CD45+ or CD45+ liver cells and the genetic strain of the recipient mouse. Flow cytometric analysis of rare HSCs from human liver grafts. Representative flow plots of unfractioned cells obtained from liver graft preservation fluid (perfusate)5 stained with a panel of antibodies containing lineage (LIN), CD34, CD38, CD90, and CD45RA and analyzed by flow cytometry. Cells were analyzed using a sequential gating strategy. An initial gate (R1) was set on SSC versus FSC to exclude debris, which was then displayed on an SSC versus 7-AAD plot. A gate (R2) was drawn to include only live (7AAD−) cells, which was then displayed on an SSC versus lineage plot. A third gate (R3) was drawn to include live Lin− cells, which was displayed on a CD38 versus CD34 dotplot. A fourth gate (R4) was drawn around a cluster of CD34+CD38− cells. Following this, live (7-AAD−), Lin-CD34+CD38− viable cells were further applied on a fifth plot of CD90 versus CD45RA. In this plot, we can detect a rare population of Lin−CD34+CD38−CD90+CD45RA− cells (gate R5). Compensation was carried out using fluorescent beads, and gates were confirmed using a fluorescent minus one (FMO) strategy, as well as unstained cells. A minimum of 1.0 × 106 events were collected for analysis. Shown is one representative result of eight. In conclusion, we agree that the human (donor) liver contains a subset of rare HSCs. However, we disagree that the level of HSCs are comparable to that found in human cord blood,4 which to date, is the richest source.