Hematological deficiencies increase with aging including anemias, reduced responses to hematopoietic stress and myelodysplasias. This investigation tested the hypothesis that increased bone marrow (BM) fat content in humans with age, was associated with decreased numbers of side population (SP) hematopoietic stem cells (HSC) and this decrease correlated with changes in cytokine levels. BM was obtained from the femoral head and trochanteric region of the femur removed at surgery for total hip replacement (N=100 subjects). In addition, BM from cadavers (N=36), with no evidence of hip disease, was evaluated for fat content. Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and lipid content determined. Marrow cells were stained with Hoechst dye and SP profiles were acquired. Plasma levels of IGF-1, SDF-1 and IL-6 were measured using ELISA. Fat content in the BM of human subjects and cadavers increased with age. The numbers of SP stem cells in BM and as well as plasma IGF-1 and SDF-1 levels decreased in correlation with increased BM fat. IL-6 had no relationship to changes in marrow fat. These data suggest that increased BM fat may be associated with decreased number of SP stem cells and IGF- 1 and SDF-1 levels with aging. This data further raises a more general question as to the role of adipose cells in the regulation of tissue stem cells.

Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N = 100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean = 30.7, SEM = 2) decreased and IL-6 levels (mean = 4.4, SEM = 1) increased with age as did marrow fat (mean = 1.2 mm fat/g, SEM = 0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive.

The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher concentrations of the inflammatory marker C‐reactive protein (CRP) (P = 0.03) and lower concentrations of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45−/CD34+) in the bone marrow (r = −0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45−/31+) in the blood (r = −0.17; P = 0.02). In addition, there was a significant correlation between BMI‐ and endothelial‐related cells of hematopoietic origin (CD133+/VEGFR‐2+) in the bone marrow (r = −0.26; P = 0.0007). Patients with higher plasma IL‐10 and insulin‐like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti‐inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.

Identification of mouse cell lines with properties of primary multipotential mesenchymal stromal cells (MSC) is required to facilitate the use of mouse models for evaluation of mechanisms in bone formation, hematopoiesis and cellular therapies for regenerative medicine. Primary murine MSC vary between strains, are difficult to grow in vitro and have inconsistent properties. The main aim of the study was to establish OMA-AD cells as an appropriate model system to conduct studies on MSC, bone formation and hematopoiesis. OMA-AD cells were isolated by differential trypsinization of C57BL/6J mouse bone marrow (BM) cells. The cells were then repassaged, cloned and characterized. OMA-AD cells were immortal and non-tumorigenic, differentiated readily to all mesenchymal cell types including bone, supported mouse and human hematopoiesis and were immunosuppressive. Our results demonstrated that OMA-AD cells possessed the properties of primary MSC. In addition, these cells grew readily and consistently, thereby facilitating future studies of bone formation, hematopoiesis and mesenchymal cells for regenerative medicine

This study enumerated CD45hi/CD34+ and CD45hi/CD133+ human hematopoietic stem cells (HSC) and granulocyte-monocyte colony forming (GM-CFC) progenitor cells in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined by multi-parameter flow cytometry employing an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood but less than 5% for all values. The frequency of CD45hi/CD133+ cells correlated highly with the frequency of CD45hi/CD34+ cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow GM-CFC progenitor cells showed no significant trends with age, but femoral marrow GM-CFC trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, that include the precursors of CD45hi/CD133+ and CD45hi/CD34+, decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy.