Cell cycle status of stromal cells in long-term haematopoietic cultures

This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.     

Cell kinetic status of haematopoietic stem cells

The haematopoietic stem cell (HSC) population supports a tremendous cellular production over the course of an animal's lifetime, e.g. adult humans produce their body weight in red cells, white cells and platelets every 7 years, while the mouse produces about 60% of its body weight in the course of a 2 year lifespan. Understanding how the HSC population carries this out is of interest and importance, and a first step in that understanding involves the characterization of HSC kinetics. Using previously published continuous labelling data (of Bradford et al. 1997 and Cheshier et al. 1999 ) from mouse HSC and a standard G₀ model for the cell cycle, the steady state parameters characterizing these HSC populations are derived. It is calculated that in the mouse the differentiation rate ranges between about 0.01 and 0.02, the rate of cell re-entry from G₀ back into the proliferative phase is between 0.02 and 0.05, the rate of apoptosis from the proliferative phase is between 0.07 and 0.23 (all units are days⁻¹), and the duration of the proliferative phase is between 1.4 and 4.3 days. These values are compared with previously obtained values derived from the modelling by Abkowitz and colleagues of long-term haematopoietic reconstitution in the cat ( Abkowitz et al. 1996 ) and the mouse ( Abkowitz et al. 2000 ). It is further calculated using the estimates derived in this paper and other data on mice that between the HSC and the circulating blood cells there are between 17 and 19.5 effective cell divisions giving a net amplification of between ~170 000 and ~720 000.     

Cell cycle of neural crest cells in the early migratory stage in vivo

Abstract. The fact that directional migration of neural crest cells (NCC) in vivo occurs in narrow pathways at high cell density, together with our preliminary results showing their high proliferative behaviour, supports the view that a 'population pressure' could be an important factor in the mechanism of early dispersion of NCC.
The purpose of this work was to establish the cranial proliferative pattern of chick embryo NCC during their early migratory stage in vivo . Growth rates and parameters of cell cycle were obtained from cell populations at several cephalic levels by means of autoradiography after labelling with [³H]dT. The labelled cell index of NCC (Forebrain, 0.288; Midbrain, 0.206; and Hindbrain, 0.134) was significantly greater than in other cells populations (e.g. for the neural tube cells: 0.085, 0.030, and 0.031, respectively). The cell generation time was the shortest in NCC (16 h), compared to ectoderm (33 h), mesoderm (58 h) and endoderm (72 h). The duration of the cell cycle phases for NCC were: M, 0.29 h; G₁, 11.23 h; S, 3.40 h; and G₂, 1.02 h.
These quantitative results show that NCC have the greatest proliferative rate in young chick embryos. In relation to cranial regions, the data are consistent with the idea that, in the early migratory phase of this cell population in vivo , migration is in part driven by population pressure.     

Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.     

Cell cycle distribution in-vivo of mononuclear blood cells of renal allograft recipients

In 47 healthy blood donors (controls) and 29 renal allograft recipients (patients) the relative contents of RNA and DNA of peripheral blood mononuclear (PBM) cell populations were estimated from the intensities of green and red fluorescences emitted by complexes that form with DNA and RNA, respectively, after staining the cells with the metachromatic dye, acridine orange. Based on the correlated DNA and RNA estimates for large numbers of cells, the percentages and the relative RNA contents of cells in particular compartments of the cell cycle were determined. PBM cell populations of controls contained less than 0.5% proliferating (SG2M) cells with highly variable relative RNA contents. Among controls, neither percentages nor relative RNA contents of SG2M-cells were correlated with percentages or relative RNA contents of G1-cells with an RNA content 2 (2SD) or 3 (3SD) standard deviations above the mean of the entire G0/1-cell population. Unlike controls, PBM cell populations of patients contained significantly higher percentages of SG2M-cells which were significantly correlated with the relative coefficients of variation (rCV) of the F530 histograms of G0/1-cells; the rCV represents the ratio of a patient's CV to a control's CV. Moreover, significant statistical correlations existed between percentages and relative RNA contents of 2SD-, 3SD-, SG2M- and G0/1-cells, suggesting a well-orchestrated progression of cells through the cell cycle. Different pairs of correlated parameters characterized clinically stable, acutely rejecting, and infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.     

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in Pig spleens

Summary Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a ³H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar.The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G₂, about 1.2 to 1.3 hours for M; about 17 to 16.5 hours for S and about 1.5 to 1.7 hours for G₁. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.The expert technical assistance of Mrs. A. Fischer is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 112.     

Effect of ribonuclease on cell cycle dynamics in tumor cells in vitro and in vivo]

The effects of RNAase from Bacteroides intermedius and pancreatic RNAase on the dynamics of cellular cycle phases were studied in the cells of ascitic lympholeukemia NK/Ly in vivo and in vitro, in the cells of the human ovary carcinoma CaOv and in the cells of ascitic leukemia P388 in vivo. It was shown that both the RNAases induced cell accumulation (blocking) in the G2/M phase of a cellular cycle with simultaneous certain depletion of the proportion of DNA-synthesizing cells (S-phase). In in vitro experiments, there was a higher efficiency of pancreatic RNAase. In animal experiments, RNAase from B. intermedius was more efficient. The likely target of the in vitro and in vivo effects of the enzymes on the cellular cycle dynamics and biological activity are discussed.     

Cell cycle dynamics and complement expression distinguishes mature haematopoietic subsets arising from hemogenic endothelium

The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.     

Density distribution analysis of in vivo and in vitro colony forming cells in bone marrow

The technique of buoyant density separation in gradients of Bovine Serum Albumin has been used to separate hemopoietic cell populations in mouse bone marrow that form in vivo spleen colonies and in vitro colonies of granulocytes and macrophages in an agar culture system. The density distribution profiles showed a number of reproducible density subpopulations of both in vivo and in vitro colony forming cells (C.F.C.'s). The mean density of in vitro C.F.C.'s exceeded that of the in vivo but overlap of the density profiles of the two populations was evident. Density-related differences in seeding efficiency of in vivo C.F.C.'s were observed. Freund's adjuvant treatment increased marrow and spleen in vitro C.F.C. populations. Marrow density profiles obtained three and seven days after adjuvant showed a progressive increase in in vitro C.F.C.'s in a restricted density region with no associated elevation of in vivo activity. The antimitotic agent, vinblastine, revealed differences in mitotic activity between the two cell populations, reducing the in vitro C.F.C. population to .07% and the in vivo to 5% of normal in 24 hours. Density separation of vinblastine-treated marrow produced density regions devoid of in vitro activity but containing in vivo in vivo C.F.C.'s which, upon transfer to irradiated recipients, regenerated both in vivo and in vitro density distribution profiles.     

Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice

The relative proliferative capacity of haematopoietic cell populations derived from 22-week-old adult bone marrow and 14–18 day foetal liver has been studied in lethally irradiated syngeneic recipients by means of chromosome markers. Although starting at a disadvantage in terms of the number of colony-forming units (stem cells) injected, the foetal liver-derived populations steadily increased their relative numbers in the myeloid and lymphoid tissues over a period of several weeks until a plateau was reached. It is suggested that stem cells in foetal liver have, on average, a higher intrinsic capacity for self-renewal than do those in bone marrow, and that this capacity falls to the adult level within about ten weeks of transfer.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

Cell cycle regulation in hematopoietic stem cells

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy.     

In vivo haematopoietic activity is induced in neurosphere cells by chromatin-modifying agents

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2'-deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurosphere-derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells.     

Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo

Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination. Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl-. EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53. The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries. Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase. The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells. Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries. No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications. Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest. Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.