Generation time (GT) of human bone marrow cells cultured in the CFC‐gm assay

Abstract

Generation time ( G T ) of normal human bone marrow cells cultured in the C FC-gm assay was measured by using bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. Cells were cultured in methylcellulose for 72 hr prior to the addition of BrdU and then harvested at 6-12 hr intervals for up to 72 hr. The time interval between the appearance of second and third division metaphases at the 50% level gave mean GTs which ranged from 32 to 43 hr. These values are longer than those reported for myeloblasts and promyelocytes but shorter than those previously reported for myelocytes.Human bone marrow functions as a continuously renewing cell system in which pluripotent stem cells give rise by proliferation and differentiation to functionally mature haematopoietic elements. Studies of the generation time of normal human bone marrow precursor cells in uiuo and in uifro are few, due both to ethical considerations (i.e. injecting normal individuals with [3H]TdR) and to difficulties in obtaining adequate cell numbers in uitro to apply traditional cytokinetic techniques.Bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining has proved to be a rapid means for identifying successive cell divisions in a proliferating population (Tice, Schneider & Rary, 1976; Crossen & Morgan, 1977a,b;Crossen et al., 1985). In this method, cells were cultured in medium that contained the thymidine analogue 5-bromodeoxyuridine (5-BrdU) which is readily incorporated into chromosomal DNA. Metaphases which have completed two complete cell cycles in medium containing BrdU have chromosomes in which the DNA of one chromatid has both strands substituted with BrdU while the other chromatid has only one strand substituted with BrdU. When stained by the sister chromatid exchange technique, the doubly-substituted chromosome appears pale staining while the singly substituted is dark staining. Cells that have completed one cell cycle have both chromatids dark staining while metaphases that have completed three cell cycles have three quarters of their chromatin pale staining. By harvesting the cultures at various time intervals, a detailed analysis of the proliferative characteristics of the culture can be obtained. Recent studies (Trent et af., 1986) indicate that a modification of the BrdU technique

Effect of Plantago major on cell proliferation in vitro

“…Possible explanations for these differences are the following: Only 30-40% of bone marrow cells are capable of multiplying, while 70-80% of spleen cells are B or T lymphocytes, with high proliferation capability (Popp and Popp, 1979). Bone marrow cells cell cycle lasts 15-32 h, while spleen cell cycle takes 15 h even reduced to 6 h in the presence of mitogens or certain antigens (Croseen et al, 1986;Salmi and Jalkanen, 1997).…”

The journey of neutropoiesis: how complex landscapes in bone marrow guide continuous neutrophil lineage determination

“…Also, 30% of terminally differentiated neutrophils become positive within 24hrs after start labeling. These data suggest extremely short generation times and post-mitotic transfer times in mice in vivo that are not recapitulated in vitro 88 .…”

“…These data suggest extremely short generation times and postmitotic transfer times in mice in vivo that are not recapitulated in vitro. 82 …”