Characterization of Hematopoietic and Non-Hematopoietic Stem/Progenitor Cells in Freshly Isolated Adult Human Bone Marrow Using an 8-Color Flow Cytometric Assay.
Authors: Tekinturhan Ferda , Zimmerlin Ludovic , Donnenberg Vera S. , Pfeifer Melanie E. , Monlish Darlene A. , Guvenc Birol , Bradley James , Donnenberg Albert D. 
Published: October 2019

Abstract
Bone marrow (BM) contains hematopoietic stem cells (HSCs), which can give rise to all mature blood cells and marrow stromal cells as well. Recently, it has been shown that non-hematopoietic stem/progenitor cells which can differentiate into non-hematopoietic tissues also reside in the BM. Although culture expanded cells have been studied in great detail, little is known about the phenotype and quantity of these cells in freshly harvested adult human BM. The aim of this study is to isolate and characterize hematopoietic and non-hematopoietic stem/progenitor cells in adult human BM by comparing two different isolation techniques and their effects on the yield of hematopoietic, mesenchymal and endothelial stem/progenitor cell populations. BM samples were collected mechanically from isolated rib specimens obtained during lung resection (n=10), or from BM aspirates harvested from the humerus of orthopedic patients (n=17). BM mononuclear cells were purified on a Ficoll/Hypaque density gradient and stained simultaneously using CD105 FITC, CD73 PE, CD34 ECD, CD90 PE.Cy5, CD117 PE.Cy7, CD133 APC, CD45 APC.Cy7 and DAPI as a marker of nucleated cells. 2–15 million cells per sample were acquired on a Dako CyAn cytometer and the data were analyzed offline using prototype analytical software (Venturi, Applied Cytometry Systems). The significant difference in the percentage of the CD45 − singlets (non-hematopoietic cells) between BM aspirates and rib-derived samples indicates hemodilution in the bone marrow aspirates. Although we have observed a slight difference in the mean of hematopoietic stem cell content between samples, it was not statistically significant. According to our results, the quantity of mesenchymal stem cells was higher in rib-derived BM than BM aspirates (p value=0.028). The expression of some stem/progenitor cell markers, such as CD90 (Thy-1), CD117 (c-Kit) and CD133 remained similar for all cell types. Our results are shown in the table below.
Surface Antigens RibBM (n=10)¥ BMA (n=17)¥ p Value % % Total Cells CD45- of nucleated cells 15.3 ± 7.9 5.7 ± 5.2 0.004 CD34+ Hematopoietic Stem Cells (HSCs)* CD34 of CD45+ 1.7 ± 1.48 2.6 ± 2.0 0.883 CD117 74.6 ± 31.3 53.3 ± 18.8 0.073 CD90 60.3 ± 44.5 35.9 ± 36.5 0.134 CD133 70.3 ± 31.8 62.3 ± 21.4 0.443 Endothelial Progenitor Cells (EPCs)* EPCs of nucleated cells 0.05 ± 0.03 0.12 ± 0.2 0.323 CD117 81.3 ± 29.8 78.1 ± 20.2 0.746 CD90 66.7 ± 39.7 53.7 ± 31.4 0.356 CD133 45.9 ± 32.7 33.9 ± 22.0 0.265 Mesenchymal Stem Cells (MSCs)* MSCs of nucleated cells 0.086 ± 0.14 0.008 ± 0.01 0.028 CD117 60.2 ± 36.8 49.8 ± 34.3 0.471 CD90 66.0 ± 27.7 65.7 ± 29.1 0.981 CD133 37.8 ± 27.4 39.9 ± 28.9 0.857
RibBM: Rib-derived BM, BMA: Bone Marrow Aspirate ¥Data are given as mean ± SD. *CD90, CD117 and CD133 expressions are shown for each stem/progenitor fraction: Hematopoietic stem cells (CD34 + CD45 + and light scatter properties according to the ISHAGE protocol), endothelial progenitor cells (CD34bright CD45 − CD105 +) and mesenchymal stem cells (CD34 − CD45 − CD73 + CD105 +).