Circulating adult stem and progenitor cell numbers—can results be trusted?

Abstract

Background Within the last years, the interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown. Cell estimates are often derived by determining the subgroup of CPC as percent lymphocytes (LYM) or mononuclear cells (MNC) via flow cytometry and back calculation over whole blood (WB) cell counts. However, results might depend on the used cell isolation technique and/or gating strategy. We aimed to investigate MNC loss and apoptosis during the flow cytometry sample preparation process preceded by either density gradient centrifugation (DGC) or red blood cell lysis (RBCL) and the potential difference between results derived from back calculation at different stages of cell isolation and from WB. Methods Human blood was subjected to DGC and RBCL. Samples were stained for flow cytometry analysis of CPC (CD34+/CD45dim) and apoptosis analysis (Annexin V) of MNC and CPC subsets. MNC and LYM gating strategies were compared. Results Both DGC as well as RBCL yielded comparable CPC concentrations independent of the gating strategy when back calculated over WB values. However, cell loss and apoptosis differed between techniques, where after DGC LYM, and monocyte (MONO) concentrations significantly decreased (p < 0.01 and p < 0.05, respectively), while after RBCL LYM concentrations significantly decreased (p < 0.05) and MONO concentrations increased (p < 0.001). LYM apoptosis was comparable between techniques, but MONO apoptosis was higher after DGC than RBCL (p < 0.001). Conclusions Investigated MNC counts (LYM/MONO ratio) after cell isolation and staining did not always mimic WB conditions. Thus, final CPC results should be corrected accordingly, especially when reporting live CPC concentrations after DGC; otherwise, the CPC regenerative potential in circulation could be biased. This is of high importance in the context of non-invasively induced CPC mobilization such as by acute physical exercise, since these cell changes are small and conclusions drawn from published results might affect further applications of physical exercise as non-invasive therapy.

Acute Exercise-Induced Oxidative Stress Does Not Affect Immediate or Delayed Precursor Cell Mobilization in Healthy Young Males

“…Cells were considered to be apoptotic when they stained positively for Annexin V -early apoptotic (Annexin V + /Aqua − ) and late apoptotic (Annexin V + /Aqua + ). Lymphocyte to monocyte ratios (LYM/MONO) resulting from both the hemocytometer analysis of the whole blood samples and the flow cytometry analysis of the processed MNC samples were compared, as it has previously been reported that unaccounted changes due to sample preparation might confound results if reporting live cells/ml after density gradient centrifugation (Kröpfl et al, 2019). However, due to the nature of our gating strategy where all cells were pre-gated on total single MNCs (FSC-A vs. FSC-H and FSC-A vs. SSC-A; Finak et al, 2016), a correction was not necessary as LYM/MONO changes from whole blood to flow cytometry analysis were negligible.…”

Exercise-Induced Circulating Hematopoietic Stem and Progenitor Cells in Well-Trained Subjects

“…Our study extends these findings by showing that a single acute exercise session had a comparable effect on HPC proportions but simultaneously increased HPC concentrations in well-trained participants. It is important to note that HPC concentrations are calculated estimates from mature immune cell numbers (Kropfl et al, 2019), and that acute exercise-in contrast to exercise training-induced lymphocytosis and monocytosis and therefore also a postexercise HPC concentration increase. Moreover, an animal study also found an exercise training-induced increase of HPC proportions in the bone marrow, indicating increased HPC maintenance and preservation (Baker et al, 2011).…”

“…Relative HPC content (proportion) was given as %MNCs. Estimates of HPC concentrations were given as cells/µl calculated by multiplying respective total HPC proportions by the absolute number of MNCs in peripheral blood measured by a standard hematology analyzer (Kropfl et al, 2019). In addition, median fluorescent intensity (MFI) of CD31 expressed on all detected HPCs per sample was extracted (Niemiro et al, 2018).…”

Changes in Circulating Stem and Progenitor Cell Numbers Following Acute Exercise in Healthy Human Subjects: a Systematic Review and Meta-analysis

“…3 (continued) means of 0.66 for ESCs and 0.47 for HSCs, reflecting the true effects of exercise on stem cell mobilization directly after exercise, represent an increase of 0.15% and 0.005% MNCs, respectively. Assuming an estimate of approximately 2 × 10 6 MNCs/ml blood [86], this equals an absolute increase of 3000 cells/ml for ESCs and 100 cells/ml for HSCs.…”