A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow

Abstract

BackgroundIsolation of bone marrow cells, including hematopoietic stem cells, is a commonly used technique in both the research and clinical settings. A quantitative and qualitative assessment of cell populations isolated from mouse and human bone marrow was undertaken with a focus on the distribution of hematopoietic cells between the central bone marrow (cBM) and endosteal bone marrow (eBM).MethodsTwo approaches to cBM isolation from the hind legs were compared using the C57BL/6J and BALB/cJ strains of laboratory mice. The content of hematopoietic stem cells in eBM was compared to cBM from mice and human fetal bone marrow using flow cytometry. Enzymatic digestion was used to isolate eBM and its effects on antigen expression was evaluated using flow cytometry. Humanized immunodeficient mice were used to evaluate the engraftment of human precursors in the cBM and eBM and the effects of in vivo maturation on the fetal stem cell phenotype were determined.ResultsThe two methods of mouse cBM isolation yielded similar numbers of cells from the femur, but the faster single-cut method recovered more cells from the tibia. Isolation of eBM increased the yield of mouse and human stem cells. Enzymatic digestion used to isolate eBM did, however, have a detrimental effect on detecting the expression of the human HSC-antigens CD4, CD90 and CD93, whereas CD34, CD38, CD133 and HLA-DR were unaffected. Human fetal HSCs were capable of engrafting the eBM of immunodeficient mice and their pattern of CD13, CD33 and HLA-DR expression partially changed to an adult pattern of expression about 1 year after transplantation.ConclusionsA simple, rapid and efficient method for the isolation of cBM from the femora and tibiae of mice is detailed. Harvest of tibial cBM yielded about half as many cells as from the femora, representing 6.4 % and 13 %, respectively, of the total cBM of a mouse based on our analysis and a review of the literature. HSC populations were enriched within the eBM and the yield of HSCs from the mouse and human long bones was increased notably by harvest of eBM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-015-0031-7) contains supplementary material, which is available to authorized users.

Bioluminescence Imaging Enhances Analysis of Drug Responses in a Patient-Derived Xenograft Model of Pediatric ALL

“…To provide a reference for highly engrafted BM, this calculation was also performed on control mice, which exhibited a median of 3.1 × 10 7 cells in femoral BM (data not shown). These values strongly reflect cell counts performed using trypan blue exclusion following femoral BM harvest of highly engrafted ALL-11-GL mice (median = 2.2 × 10 7 , data not shown), as well as cell numbers observed via isolation of murine femoral BM by Mahajan et al (2016) (35). …”

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis

“…To focus on the role of endothelial cell IQGAP1 specifically, bone marrow chimeras were used for the animal studies described here using standard protocols (Duran-Struuck and Dysko, 2009; Mahajan et al, 2015). WT and IQGAP1 knockout mice were lethally irradiated with a 1,000-cGy dose using a Gammacell 40 Exactor 137 Cs irradiator and reconstituted with bone marrow from C57BL/6 WT mice (or LysM +/GFP heterozygous mice for the intravital studies).…”

Comparison of Human Hematopoietic Reconstitution in Different Strains of Immunodeficient Mice

“…Samples were 20 and 21 weeks' gestation, estimated based on foot-length, obtained from elective abortions at San Francisco General Hospital. Central and endosteal bone marrow were isolated from long bones as previously described and cells pooled [14]. Washed cells were counted with a hemocytometer and either prepared directly for transplantation or suspended in 10% dimethyl sulfoxide (Sigma-Aldrich) and 40% fetal bovine serum (Life Technologies) and cryopreserved.…”

“…Tubes used to collect blood contained ethylenediaminetetraacetic acid powder (Eppendorf North America). Femoral central bone marrow, spleen, and liver were collected and prepared for antibody staining as previously described [13,14]. A small portion of blood collected was separated, added to blocking buffer, and reserved for antibody staining as whole blood.…”

“…Bone marrow cell counts were preformed on a Cellometer Auto X4, version 1.1.1 (Nexcelom Bioscience). The total number of bone marrow cells in a mouse was estimated based on the bone marrow of two femurs representing 13% of total bone marrow cells [14].…”