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• W1980421076 abstract "This study enumerated CD45hi/CD34+ and CD45hi/CD133+ human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using 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 progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs 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, which includes 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. This study enumerated CD45hi/CD34+ and CD45hi/CD133+ human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using 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 progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs 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, which includes 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. In man, steady state hematopoiesis is generally successfully maintained with age [1Van Zant G. Liang Y. The role of stem cells in aging.Exp Hematol. 2003; 31: 659-672Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 2Liang Y. Van Zant G. Genetic control of stem-cell properties and stem cells in aging.Curr Opin Hematol. 2003; 10: 195-202Crossref PubMed Scopus (25) Google Scholar], but compensatory responses to stresses such as bleeding or cancer chemotherapy can be compromised [3Janzen V. Forkert R. Fleming H.E. et al.Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.Nature. 2006; 443: 421-426Crossref PubMed Scopus (904) Google Scholar], and stem cells are implicated [4Rossi D.J. Jamieson C.H. Weissman I.L. Stems cells and the pathways to aging and cancer.Cell. 2008; 132: 681-696Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 5Roobrouck V.D. Ulloa-Montoya F. Verfaillie C.M. Self-renewal and differentiation capacity of young and aged stem cells.Exp Cell Res. 2008; 314: 1937-1944Crossref PubMed Scopus (218) Google Scholar]. Because of this situation, and because of increased comorbidities, chemotherapy doses used for older patients in general oncology are often reduced; unfortunately, this can compromise therapy outcomes [6Dixon D.O. Neilan B. Jones S.E. et al.Effect of age on therapeutic outcome in advanced diffuse histiocytic lymphoma: The southwest oncology group experience.J Clin Oncol. 1986; 4: 295-305Crossref PubMed Scopus (329) Google Scholar, 7Balducci L. Hardy C.L. Lyman G.H. Hemopoietic reserve in the older cancer patient: clinical and economic considerations.Cancer Control. 2000; 7: 539-547Crossref PubMed Scopus (79) Google Scholar]. These data, along with increased risks of anemias, hematologic diseases relating to myelodysplasia, and malignancies, suggest that the human hematopoietic stem cell (HSC) compartment can be compromised with aging, similar to the mouse [8Pearce D. Bonnet D. Ageing within the hematopoietic stem cell compartment.Mech Ageing Dev. 2009; 130: 54-57Crossref PubMed Scopus (13) Google Scholar]. The effects of aging on the HSC compartment have been extensively studied in the mouse but, until recently, less so in man [8Pearce D. Bonnet D. Ageing within the hematopoietic stem cell compartment.Mech Ageing Dev. 2009; 130: 54-57Crossref PubMed Scopus (13) Google Scholar, 9Pang W.W. Price E.A. Sahoo D. et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc Natl Acad Sci U S A. 2011; 108: 20012-20017Crossref PubMed Scopus (527) Google Scholar]. In the mouse, the effects of aging are strain dependent and the number of proliferating HSC has been related to lifespan [8Pearce D. Bonnet D. Ageing within the hematopoietic stem cell compartment.Mech Ageing Dev. 2009; 130: 54-57Crossref PubMed Scopus (13) Google Scholar, 10De Haan G. Van Zant G. Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span.FASEB J. 1999; 13: 707-713PubMed Google Scholar]. HSC numbers in most mouse strains decline with age; however, HSC numbers increase in the long-lived C57Bl mouse, especially side population (SP), and these SP HSCs in older mice have an increased expression of antiapoptotic genes [8Pearce D. Bonnet D. Ageing within the hematopoietic stem cell compartment.Mech Ageing Dev. 2009; 130: 54-57Crossref PubMed Scopus (13) Google Scholar, 11Pearce D.J. Anjos-Afonso F. Ridler C.M. Eddaoudi A. Bonnet D. Age-dependent increase in side population distribution within hematopoiesis: implications for our understanding of the mechanism of aging.Stem Cells. 2007; 25: 828-835Crossref PubMed Scopus (57) Google Scholar]. In addition to changes in numbers, the differentiation potential of HSCs shifts toward myeloid cells (myeloid bias) [9Pang W.W. Price E.A. Sahoo D. et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc Natl Acad Sci U S A. 2011; 108: 20012-20017Crossref PubMed Scopus (527) Google Scholar] and away from B lymphoid cells, in mice and humans [12Muller-Sieburg C.E. Sieburg H.B. Bernitz J.M. Cattarossi G. Stem cell heterogeneity: implications for aging and regenerative medicine.Blood. 2012; 119: 3900-3907Crossref PubMed Scopus (101) Google Scholar]. The make-up of the T cell pool also shifts with a significant decrease in numbers and functions of new thymic emigrant cells [13Aw D. Silva A.B. Palmer D.B. Is thymocyte development functional in the aged?.Aging (Albany NY). 2009; 1: 146-153PubMed Google Scholar]. It has been shown that the functions of HSCs are compromised by aging [14Chambers S.M. Shaw C.A. Gatza C. Fisk C.J. Donehower L.A. Goodell M.A. Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation.PLoS Biol. 2007; 5: e201Crossref PubMed Scopus (582) Google Scholar, 15Vijg J. Campisi J. Puzzles, promises and a cure for ageing.Nature. 2008; 454: 1065-1071Crossref PubMed Scopus (279) Google Scholar, 16Waterstrat A. Van Zant G. Effects of aging on hematopoietic stem and progenitor cells.Curr Opin Immunol. 2009; 21: 408-413Crossref PubMed Scopus (61) Google Scholar] because of cellular senescence [17Campisi J. d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells.Nat Rev Mol Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (2942) Google Scholar, 18Wagner W. Bork S. Horn P. et al.Aging and replicative senescence have related effects on human stem and progenitor cells.PLoS One. 2009; 4: e5846Crossref PubMed Scopus (373) Google Scholar], impaired DNA repair [19Rossi D.J. Bryder D. Seita J. Nussenzweig A. Hoeijmakers J. Weissman I.L. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age.Nature. 2007; 447: 725-729Crossref PubMed Scopus (848) Google Scholar, 20Nijnik A. Woodbine L. Marchetti C. et al.DNA repair is limiting for haematopoietic stem cells during ageing.Nature. 2007; 447: 686-690Crossref PubMed Scopus (433) Google Scholar, 21Ruzankina Y. Brown E.J. Relationships between stem cell exhaustion, tumour suppression and ageing.Br J Cancer. 2007; 97: 1189-1193Crossref PubMed Scopus (35) Google Scholar, 22Sharpless N.E. DePinho R.A. How stem cells age and why this makes us grow old.Nat Rev Mol Cell Biol. 2007; 8: 703-713Crossref PubMed Scopus (674) Google Scholar], or telomere erosion [23Widmann T. Kneer H. Konig J. Herrmann M. Pfreundschuh M. Sustained telomere erosion due to increased stem cell turnover during triple autologous hematopoietic stem cell transplantation.Exp Hematol. 2008; 36: 104-110Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 24Gadalla S.M. Savage S.A. Telomere biology in hematopoiesis and stem cell transplantation.Blood Rev. 2011; 25: 261-269Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar]. Although the consequences of aging on HSC function in humans are uncertain [25Klapper W. Shin T. Mattson M.P. Differential regulation of telomerase activity and TERT expression during brain development in mice.J Neurosci Res. 2001; 64: 252-260Crossref PubMed Scopus (116) Google Scholar], this likely influences physical abilities [26Bendix L. Gade M.M. Staun P.W. et al.Leukocyte telomere length and physical ability among danish twins age 70+.Mech Ageing Dev. 2011; 132: 568-572Crossref PubMed Scopus (28) Google Scholar] and marrow failure [27Calado R.T. Telomeres and marrow failure.Hematology Am Soc Hematol Educ Program. 2009; : 338-343Crossref PubMed Scopus (52) Google Scholar]. Homing and engraftment of murine HSC is less efficient with aging [2Liang Y. Van Zant G. Genetic control of stem-cell properties and stem cells in aging.Curr Opin Hematol. 2003; 10: 195-202Crossref PubMed Scopus (25) Google Scholar, 28Morrison S.J. Wandycz A.M. Akashi K. Globerson A. Weissman I.L. The aging of hematopoietic stem cells.Nat Med. 1996; 2: 1011-1016Crossref PubMed Scopus (653) Google Scholar], and decreases in mitochondrial function with aging have been described [29Lee H.C. Wei Y.H. Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging.Exp Biol Med (Maywood). 2007; 232: 592-606PubMed Google Scholar]. In addition, it has been increasingly recognized that the HSC compartment exhibits heterogeneity [12Muller-Sieburg C.E. Sieburg H.B. Bernitz J.M. Cattarossi G. Stem cell heterogeneity: implications for aging and regenerative medicine.Blood. 2012; 119: 3900-3907Crossref PubMed Scopus (101) Google Scholar, 30Sharp J.G. Jackson J.D. Hematopoietic stem cells and cytokines.in: Armitage J.O. Atlas of Clinical Hematology. 2nd ed. Current Medicine, Philadelphia2007Google Scholar] and that the mosaic of functional clones changes with aging [12Muller-Sieburg C.E. Sieburg H.B. Bernitz J.M. Cattarossi G. Stem cell heterogeneity: implications for aging and regenerative medicine.Blood. 2012; 119: 3900-3907Crossref PubMed Scopus (101) Google Scholar, 31Cho R.H. Sieburg H.B. Muller-Sieburg C.E. A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells.Blood. 2008; 111: 5553-5561Crossref PubMed Scopus (251) Google Scholar] and potentially differs among individuals. Overall, HSC heterogeneity would account for variations in HSC numbers and functions with aging and, potentially, in sensitivities to chemotherapy. Recent studies of changes in the frequency of human hematopoietic stem cells with aging are not all comparable, potentially because different markers (e.g., SP versus lineage negative CD34+ or CD133+ cells) [9Pang W.W. Price E.A. Sahoo D. et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc Natl Acad Sci U S A. 2011; 108: 20012-20017Crossref PubMed Scopus (527) Google Scholar, 32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar] have been used to identify HSC populations. Transplantation of these HSC populations to immunodeficient mice shows that their ability to produce circulating human mononuclear cells varies [30Sharp J.G. Jackson J.D. Hematopoietic stem cells and cytokines.in: Armitage J.O. Atlas of Clinical Hematology. 2nd ed. Current Medicine, Philadelphia2007Google Scholar]. There are differing potentialities even within the HSC hierarchy, with SP cells having greater potential than CD34+ cells, which in turn have greater potential than CD34– cells to produce human mononuclear cells [30Sharp J.G. Jackson J.D. Hematopoietic stem cells and cytokines.in: Armitage J.O. Atlas of Clinical Hematology. 2nd ed. Current Medicine, Philadelphia2007Google Scholar]. SP HSC numbers decline with age, although the surviving cells are of high quality [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar]. In contrast, aldehyde dehydrogenase high, CD34+, and CD133+ human HSCs in bone marrow have been reported to show no age-related declines, although circulating progenitors did decline [33Povsic T.J. Zhou J. Adams S.D. Bolognesi M.P. Attarian D.E. Peterson E.D. Aging is not associated with bone marrow-resident progenitor cell depletion.J Gerontol A Biol Sci Med Sci. 2010; 65: 1042-1050Crossref PubMed Scopus (24) Google Scholar]. However, several other studies showed increases in bone marrow CD34+ and CD133+ HSC numbers in older subjects [9Pang W.W. Price E.A. Sahoo D. et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc Natl Acad Sci U S A. 2011; 108: 20012-20017Crossref PubMed Scopus (527) Google Scholar, 34Taraldsrud E. Grogaard H.K. Solheim S. et al.Age and stress related phenotypical changes in bone marrow CD34+ cells.Scand J Clin Lab Invest. 2009; 69: 79-84Crossref PubMed Scopus (25) Google Scholar, 35Kuranda K. Vargaftig J. de la Rochere P. et al.Age-related changes in human hematopoietic stem/progenitor cells.Aging Cell. 2011; 10: 542-546Crossref PubMed Scopus (113) Google Scholar, 36Matsuoka S. Ebihara Y. Xu M. et al.CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages.Blood. 2001; 97: 419-425Crossref PubMed Scopus (105) Google Scholar], indicating that additional data are needed to settle this question. In 2003, we hypothesized at a National Institutes of Aging Workshop on stem cells that the more primitive, undifferentiated, and likely quiescent SP HSCs might be lost or used up during the aging process. This loss could be compensated for by an increase in more differentiated HSCs, and more of these cells are likely proliferating and sensitive to chemotherapy; this might explain the increase in sensitivity of older patients to chemotherapeutic agents. This hypothesis predicted that SP HSC numbers would decrease with aging [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar], but that there would be a compensatory increase in CD34+ and CD133+ stem cells in the HSC compartment. Whether these changes would be evident only in bone marrow, or also observed in blood, was unclear because the relationship between these two HSC compartments is dynamic, its regulation is uncertain, and the role of the microenvironment in HSC aging is unclear [16Waterstrat A. Van Zant G. Effects of aging on hematopoietic stem and progenitor cells.Curr Opin Immunol. 2009; 21: 408-413Crossref PubMed Scopus (61) Google Scholar, 37Rando T.A. Stem cells, ageing and the quest for immortality.Nature. 2006; 441: 1080-1086Crossref PubMed Scopus (564) Google Scholar, 38Wagner W. Horn P. Bork S. Ho A.D. Aging of hematopoietic stem cells is regulated by the stem cell niche.Exp Gerontol. 2008; 43: 974-980Crossref PubMed Scopus (74) Google Scholar, 39Woolthuis C.M. de Haan G. Huls G. Aging of hematopoietic stem cells: intrinsic changes or micro-environmental effects?.Curr Opin Immunol. 2011; 23: 512-517Crossref PubMed Scopus (45) Google Scholar], although age-related changes in stem cell niches in Drosophila testis cause a decline in germ cell self-renewal [40Boyle M. Wong C. Rocha M. Jones D.L. Decline in self-renewal factors contributes to aging of the stem cell niche in the drosophila testis.Cell Stem Cell. 2007; 1: 470-478Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar]. To test the hypothesis, 233 human subjects (21–88 years old) undergoing hip replacement surgery were enrolled in a study approved by an institutional review board. The study enumerated the SP, CD34+, and CD133+ HSCs by flow cytometry and myeloid-colony forming cells (i.e., granulocyte-macrophage forming cells [GM-CFCs]) in culture from the bone marrow of the trochanteric region of the femoral diaphysis and femoral head as well as in blood. The results of a study of SP HSCs, showing a decline in numbers with age but an increase in quality of the surviving cells, have been described previously [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar]. This report presents the data on the changes in numbers of the intermediate compartment of CD34+ and CD133+ stem cells and progenitor cells, assayed as colony-forming cells (i.e., GM-CFC) cells with age and with sites in bone marrow and blood as well as the correlations between these cell populations and aging. The results indicated differences in the frequencies of different HSC populations and differential changes based on the site of origin of HSC (i.e., blood, trochanter marrow, or femoral head marrow) with age. The National Institute of Aging supported this study describing the relationship of stem cell number and quality to age and health status, but had no role in data analysis or interpretation. Institutional review board approval was received to consent and enroll up to 240 individuals undergoing total hip replacement. Exclusion criteria included a diagnosis of avascular necrosis, any abnormal bone marrow condition, a history of malignancy, or any previous chemotherapy or radiation therapy. Peripheral blood samples, along with bone marrow from both the femoral head and trochanteric region, were collected from each subject at the time of surgery and processed within 6 hours. A detailed description of the technique used employed to collect femoral and trochanteric bone marrow samples has been described previously [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar, 41Garvin K. Feschuk C. Sharp J.G. Berger A. Does the number or quality of pluripotent bone marrow stem cells decrease with age?.Clin Orthop Relat Res. 2007; 465: 202-207PubMed Google Scholar]. Peripheral blood mononuclear cells (PBMCs) were obtained using lymphocyte separation medium (Mediatech, Manassas, VA, USA). Cells were harvested from femoral head and trochanter bone marrow samples by gently crushing the bone using a mortar and pestle and washing with HBSS without Ca or Mg (Invitrogen, Carlsbad, CA), containing 20% fetal bovine serum (Hyclone, Logan, UT, USA), 13.5 μg/mL DNAse (Sigma-Aldrich, St. Louis, MO, USA), and 10 U/mL sodium heparin (Elkins-Sinn, Cherry Hill, NJ, USA). Mononuclear cells (MNCs) from the trochanter and femoral head bone marrow mixture were harvested using a density gradient. Each sample was photographed digitally, and the depth of the supernatant fat layer was measured along with the total depth of the samples. This measurement allowed for the calculation of the amount of fat (millimeters per gram) in the sample, which was plotted against the age of the subject. One million PBMCs and bone marrow samples (i.e., MNCs) were stained with fluorochrome-conjugated antibodies CD45-FITC plus CD133-PE or CD34-PE using standard phenotyping techniques (15 min, 4°C). Analysis was performed with a FACSAria (Becton-Dickinson, San Jose, CA, USA); results were evaluated with FlowJo software (Tree Star, Ashland, OR, USA). Hematopoietic stem cells were defined and identified as CD45hi/CD133+ and CD45hi/CD34+ and reported as percent PBMCs (peripheral blood samples) or percent MNCs (bone marrow samples). The numbers of GM-CFCs in the collections were determined using a previously described culture technique [30Sharp J.G. Jackson J.D. Hematopoietic stem cells and cytokines.in: Armitage J.O. Atlas of Clinical Hematology. 2nd ed. Current Medicine, Philadelphia2007Google Scholar]. Briefly, 3 × 105 MNCs isolated from the bone marrow samples were plated in 35-mm Petri dishes containing Iscove's modified Dulbecco's medium, 20% fetal bovine serum, 50 μmol/L 2-mercaptoethanol, 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.3% agar supplemented with 200 U/mL interleukin-3, 200 U/mL GM-CSF, and 200 U/mL G-CSF. Cultures were incubated at 37°C in a fully humidified atmosphere of 5% CO2 in air for 14 days, at which time the plates were stained with Wright's Giemsa and colony numbers were enumerated. Colonies were defined as groups of 30 or more cells. A small number of assays failed quality control requirements; therefore, there were minor variations in the number of subjects in various comparisons. An internal control was included to quantify technically derived variance. The CD34 antibody was included in two separate tubes (Samples A and B), and these results were compared for equivalency and the frequency of outliers (discrepancies of +/− 20%) noted. A scatter plot was produced to look at the association of the cell count for each cell population (CD34+, CD133+, and GM-CFC) and age for trochanteric and femoral bone marrow and peripheral blood. Spearman Rank Correlation and linear regression were used to test for significant trends between various stem and progenitor cell phenotypes, age, and anatomic sites (blood, trochanter, and femur). In addition, prior data indicate that CD133+ cells are a subpopulation of CD34+ cells; therefore, the frequency of both of these populations was compared based on the slope of the relationship for trochanteric and femoral bone marrow and peripheral blood. Based on predetermined criteria, comparisons were considered significant at p ≤ 0.05 and a trend at p ≤ 0.1. Independent staining of CD45/CD34 in two separate tubes (Samples A and B, Fig. 1) was used to determine the inherent variance of the staining and analysis procedures. The plot of CD34+ cells in two separate tubes (Samples A and B) showed a straight line at slightly less than a 45-degree angle, with a statistically significant p value less than 0.0001. This result indicated that, methodologically, the data were internally consistent, with generally less than 5% of values presenting as significant high or low outliers. Blood had the highest proportion of high and low outliers. CD45hi/CD34+ and CD45hi/CD133+ cell populations (Fig. 2) were highly correlated in peripheral blood and trochanteric and femoral head bone marrow samples (p < 0.0001; Fig. 2). However, the relative proportion of CD45hi/CD133+ cells in peripheral blood compared with CD45hi/CD34+ cells (33%) was lower than in trochanteric and femoral bone marrow (∼55% ± 5%). These data suggest site-specific variations in the relationships of CD45hi/CD133+ to CD45hi/CD34+ cells. Similarly, trochanteric marrow GM-CFC numbers correlated highly with femoral GM-CFC numbers (Fig. 1D), although slightly more (by ∼10%) GM-CFC per nucleated cell were observed in femoral bone marrow.Figure 2Comparison of the proportion of CD45hi/CD34+ cells with the less differentiated CD45hi/CD133+ cells. Cells are represented as a percentage of peripheral blood mononuclear cells (A), trochanteric bone marrow mononuclear cells (B), or femoral bone marrow mononuclear cells (C). For all figures, the linear regression line is visually depicted and the Spearman correlation (r) noted.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In peripheral blood, CD45hi/CD34+ cell populations showed no significant trend with age (p = 0.57), whereas, CD45hi/CD133+ cell populations increased slightly with age (p = 0.026; Fig. 3A and D). In trochanteric bone marrow, both CD45hi/CD34+ and CD45hi/CD133+ cell populations increased significantly with age (p = 0.0003 and p < 0.0001, respectively; Fig. 3B and E). CD45hi/CD34+ and CD45hi/CD133+ populations also increased significantly with age (p = 0.0002 and p < 0.0001, respectively) in femoral head bone marrow (Fig. 3C and F). Although the trochanteric bone marrow GM-CFC numbers correlated highly with femoral bone marrow GM-CFC numbers (Fig. 1D), trochanteric bone marrow GM-CFC numbers did not change with age (Fig. 4A), whereas femoral bone marrow GM-CFCs declined moderately but significantly with age (p = 0.047; Fig. 4B). This finding potentially reflects the lower age at which femoral bone marrow converts from active red bone marrow inactive fatty bone marrow compared to trochanteric bone marrow [42Tuljapurkar S.R. McGuire T.R. Brusnahan S.K. et al.Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging.J Anat. 2011; 219: 574-581Crossref PubMed Scopus (77) Google Scholar]. Consequently, the number of femoral bone marrow GM-CFCs was related to the amount of fat in the bone marrow (millimeters of fat per gram of marrow per tube; Fig. 4C). The number of femoral bone marrow GM-CFC declined as the amount of fat increased; however, this decline did not attain statistical significance (p = 0.067). The relationship between fat and hematopoietic progenitor cell numbers was opposite to that of the GM-CFCs. There was a positive relationship between fat and CD45hi/CD34+ cell numbers in the trochanter (r = 0.16; p = 0.03); however, no relationship was found in the femur. CD45hi/CD133+ cells in both the trochanter and femur demonstrated a positive trend with amount of bone marrow fat. These data indicate that the frequency of HSC and progenitors with age varied for CD34, CD133, and GM-CFC populations as well as with sites of harvest. Differences were observed between CD34+ and CD133+ cells in blood with age and effects with age were less evident than in bone marrow. In bone marrow CD34+ and CD133+ HSC increased with age, which is compatible with the hypothesis and most, but not all, previous reports. Changes with age were less evident in the GM-CFC compartment. The internal controls of this experiment showed that the methods were valid. The data also confirm that the frequency of CD133+ and CD34+ cells correlated highly in blood and trochanteric and femoral bone marrow, but the ratio (CD133+/CD34+) varied with site, ranging from approximately 33% in blood to 55% in femoral marrow and trochanteric marrow, suggesting different dynamics of these cell populations at various sites with aging especially between blood and marrow. The basis of these differences is not known. The primary finding in this study was that the compartment of hematopoietic stem cells with intermediate potential, in this study defined as CD45hi/CD133+ and CD45hi/CD34+ cells, increased significantly in trochanteric and femoral head bone marrow in humans with aging. Myeloid progenitor cells (GM-CFC) in femoral bone marrow declined somewhat, but in trochanteric marrow showed little change with age; therefore, this compensation shown by the intermediate compartment [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar] did not extend to the progenitor compartment. We have shown previously that high potential SP HSC in the blood and bone marrow decreased in frequency in humans with aging [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar]. The results of the present analyses support the hypothesis that the CD45hi/CD133+ and CD45hi/CD34+ populations exhibited a compensatory increase related to the decrease in the high potential multipotential SP HSC compartment of the stem cell population with aging. The increase in HSC was most obvious in bone marrow, but not significantly reflected in blood. Overall, the balance of other studies also favors an increase in the intermediate potential compartment of HSC in bone marrow with age. Because such cells likely have greater proliferative activity, this could explain why hematopoiesis in the elderly shows increased chemosensitivity. Furthermore, these observations are compatible with the description of the effects of inhibiting autophagy of high potential (SP) stem cells with aging [43Mortensen M. Watson A.S. Simon A.K. Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation.Autophagy. 2011; 7: 1069-1070Crossref PubMed Scopus (85) Google Scholar]. Autophagy is required for maintenance of HSCs [44Warr M.R. Binnewies M. Flach J. et al.FOXO3A directs a protective autophagy program in haematopoietic stem cells.Nature. 2013; 494: 323-327Crossref PubMed Scopus (425) Google Scholar]. Loss of autophagy in HSCs leads to the expansion of progenitor cells in bone marrow [43Mortensen M. Watson A.S. Simon A.K. Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation.Autophagy. 2011; 7: 1069-1070Crossref PubMed Scopus (85) Google Scholar], as observed in this study. In support of this postulate, administration of rapamycin, which promotes autophagy, counters the effects of aging and extends lifespan in mice [45Harrison D.E. Strong R. Sharp Z.D. et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.Nature. 2009; 460: 392-395Crossref PubMed Scopus (2657) Google Scholar]. In conclusion, we have shown that CD34+/CD133+ HSCs increase with age in bone marrow from humans undergoing hip replacement surgery. Although the relative and absolute increase in CD34+ cells in human bone marrow has been reported previously [34Taraldsrud E. Grogaard H.K. Solheim S. et al.Age and stress related phenotypical changes in bone marrow CD34+ cells.Scand J Clin Lab Invest. 2009; 69: 79-84Crossref PubMed Scopus (25) Google Scholar], Povsic et al. [33Povsic T.J. Zhou J. Adams S.D. Bolognesi M.P. Attarian D.E. Peterson E.D. Aging is not associated with bone marrow-resident progenitor cell depletion.J Gerontol A Biol Sci Med Sci. 2010; 65: 1042-1050Crossref PubMed Scopus (24) Google Scholar] found no relationship between progenitor cells in human bone marrow and aging. In addition, the data indicate that the dynamics of stem cells in blood versus bone marrow are differentially regulated and not necessarily correlated, so that blood stem cell numbers cannot be used reliably as surrogate for bone marrow HSC behaviors. The results of this study combined with prior studies suggest that likely quiescent HSC, with greater proliferative potential are used up with aging [32Brusnahan S.K. McGuire T.R. Jackson J.D. et al.Human blood and marrow side population stem cell and stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.Mech Ageing Dev. 2010; 131: 718-722Crossref PubMed Scopus (40) Google Scholar] and there is a compensatory increase in intermediate potential HSC (CD34+, CD133+, or both). As noted earlier, this intermediate potential HSC population most probably includes a greater proportion of proliferating HSC. It is unclear whether more clones of HSC are involved in proliferative activities or whether the number of progeny produced by individual clones is increased (or both). The amplification of clones, or of clonal progeny, especially of cells that might have accumulated unrepaired or incompletely repaired DNA damage with aging, would also explain the increase in the frequency of myelodysplastic clones and malignancies with aging [46Henry C.J. Marusyk A. DeGregori J. Aging-associated changes in hematopoiesis and leukemogenesis: What’s the connection?.Aging (Albany NY). 2011; 3: 643-656PubMed Google Scholar, 47Jan M. Snyder T.M. Corces-Zimmerman M.R. et al.Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia.Sci Transl Med. 2012; 4: 149ra118Crossref PubMed Scopus (540) Google Scholar]. Potential mechanisms by which intermediate HSC numbers or proliferation increase with age are unclear. The levels of inflammatory cytokines increase with aging, and these might stimulate stem cell production by feedback mechanisms. In addition, there is evidence of decreased blood flow with aging to at least some areas of HSC production (e.g., proximal femur and pelvis) [42Tuljapurkar S.R. McGuire T.R. Brusnahan S.K. et al.Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging.J Anat. 2011; 219: 574-581Crossref PubMed Scopus (77) Google Scholar]. This evidence might indicate that local hypoxia is associated with increases in production of cytokines, potentially by mesenchymal cells that contribute to the stem cell niche and whose secretome is altered by hypoxia including increased production of hepatocyte growth factor and VEGF [48Chung-Ching C. Ching-Ping C. Mao-Tsun L. Hypoxic preconditioning enhances the potentially therapeutic secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury.Science. 2012; 338: 1485Google Scholar]. Hypoxia has also been associated with increases in stem cell populations in breast cancer [49Lin Q. Yun Z. Impact of the hypoxic tumor microenvironment on the regulation of cancer stem cell characteristics.Cancer Biol Ther. 2010; 9: 949-956Crossref PubMed Scopus (85) Google Scholar] and neural cells [50Ling-Ling Z. Li-Ying W. Kui-Wu W. Ming F. Mild hypoxia regulates the properties and functions of neural stem cells in vitro.Science. 2012; 338: 1485Google Scholar]. Consequently, a plausible hypothesis to explain the observations of this study is that declines in blood flow, to at least some areas of bone marrow, result in local hypoxia which then promotes mesenchymal stem cell production of VEGF and other cytokines/chemokines that then amplify, proliferating intermediate potential HSCs. This hypothesis also might have relevance to the site specific alterations in HSC numbers with aging that were observed in this study. Confirmation of these potential interactions would offer the opportunity to intervene and modify the changes of HSCs with aging, perhaps by promoting blood flow by increasing physical activity [51Wardyn G.G. Rennard S.I. Brusnahan S.K. et al.Effects of exercise on hematological parameters, circulating side population cells, and cytokines.Exp Hematol. 2008; 36: 216-223Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar] or by promoting autophagy. Alternatively, it may be possible to decrease the sensitivity of HSC to radiation or chemotherapy in patients with cancer, many of whom are older, by altering relevant cytokine/chemokine levels to decrease the proportion of proliferating HSC [52Davis T.A. Mungunsukh O. Zins S. Day R.M. Landauer M.R. Genistein induces radioprotection by hematopoietic stem cell quiescence.Int J Radiat Biol. 2008; 84: 713-726Crossref PubMed Scopus (67) Google Scholar, 53Winkler I.G. Barbier V. Nowlan B. et al.Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self-renewal and chemoresistance.Nat Med. 2012; 18: 1651-1657Crossref PubMed Scopus (295) Google Scholar]. The authors thank Dr. Greg Perry (Creighton University Flow Cytometry) and Dr. Charles Kuszinsky (UNMC Cell Analysis Core Facilities) for their advice and assistance, the patients who consented to donate their tissues, and the National Institutes of Ageing, especially Dr. Chandra Dutta and Dr. Evan Hadley for their support of this research." @default.
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