Stem Cells, Vol. 16, No. 2, 107-111,
March 1998
© 1998 AlphaMed Press
Estimation of Extent of Cell Death in Different Stages of Normal Murine Hematopoiesis
Emanuel Ne
asa,
Lud
k 
efca,
Karel ulca,
Edda Barthelb,
Hans-Joachim Seidelb
a Institute of Pathophysiology, First Medical Faculty, Charles University, Prague, Czech Republic;
b Institute of Occupational and Social Medicine, University of Ulm, Federal Republic of Germany
Key Words. Apoptosis • Hematopoiesis • Cell cycle • Hydroxyurea • Mouse • Turnover of blood cells • Kinetics
Prof. Emanuel Neas, Institute of Pathophysiology, First Faculty of Medicine, Charles University, U nemocnice 5, 128 53 Praha 2, Czech Republic.
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Abstract
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Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative evaluation of this dynamic process. In this study, the capacity of several populations of the bone marrow clonogenic cells (progenitors) to produce blood cells was compared with their actual production. The cell cycle progression rate was directly measured in the following types of hematopoietic progenitors: day 8 colony-forming units-spleen, GM-colony-forming cells, BFU-E, and CFU-E in normal mice. The cell cycle progression rates of the individual progenitors, together with their numbers in the whole hematopoietic tissue, were used to calculate the absolute numbers produced daily in each population. The data reviewed from literature were analyzed in parallel. The capacity of the progenitors to produce mature blood cells was derived from the daily production of progenitors multiplied by their clonogenic potential. This theoretical capacity to produce blood cells was compared to the actual blood cell production determined from the turnover of circulating blood elements. The comparison strongly suggested an intensive cell death rate occurring at the early stages of differentiation and its decline as the hematopoietic cells become more differentiated and mature.
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Introduction
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There is a general consensus that most of the hematopoietic cells die through apoptosis; however, the actual extent and distribution of this death is not known [1, 2]. We try to estimate these parameters in normal murine hematopoiesis using new experimental and previously published data.
The majority of hematopoietic cells can be classified under several differentiation pathways where the cells are amplified and mature into functional blood elements. In this way, hematopoiesis replaces senescent blood cells. These differentiation pathways are fed by progenitor cells. The intensity of cell fluxes in differentiation pathways of murine hematopoiesis has been obtained previously by integrating available data [3]. However, this analysis has not accounted for cell losses due to apoptosis. The comparison between expected production of blood cells from the early progenitor day 8 colony-forming units-spleen (CFU-Sday 8) and their actual production showed that the actual production was less than 0.1% of the expected one [4]. This suggested that a high rate of physiological apoptosis occurs in normal hematopoiesis. The experiments of Drize et al. [5] using labeled progenitors provided support for such a conclusion since they demonstrated the existence of CFU-S which did not contribute to steady-state hematopoiesis.
Our knowledge of the quantitative aspects of hematopoiesis relies heavily on our knowledge of its various developmental stages. While sufficient data are available for consideration of the late developmental stages [3], corresponding data are relatively sparse for the early developmental stages of clonogenic progenitors. Therefore, we have tried to supplement the existing data with direct determination of the cell cycle progression rate in several populations of the hematopoietic progenitor cells examined in vivo. Results of these direct experimental measurements were compared with those derived from previously published data regarding normal hematopoiesis of the mouse. Both sets of kinetics data were used to test and quantify the hypothesis of a significant physiological apoptosis.
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Materials and Methods
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Mice
Female CBF1 (CBA/Ca x C57Bl/10) mice and BDF1 (C57Bl/6 x DBA/2) hybrid mice of age 8 to 12 weeks were used. The mice were fed commercial pellets and water ad libitum.
Hydroxyurea Administration
Hydroxyurea (Sigma; St. Louis, MO) was dissolved in distilled water, and a group of five mice was injected intraperitoneally (i.p.) with a single dose of 1g/kg b.w.
Bone Marrow Collection
Mice were killed by exsanguination under ether anesthesia, and femurs from three to five mice (treated in the same way) were collected into petri dishes with ice-cold phosphate-buffered saline (PBS) or tissue culture medium. Both ends of the femurs were opened and their cavities were flushed with 1 ml of either PBS with 1% bovine serum albumin (CFU-S experiments) or Iscove's medium (colony-forming cells; CFC-GM, BFU-E, and CFU-E experiments). The nucleated cells were counted and aliquots were subjected to 3H-thymidine suicide.
3H-thymidine Suicide
107 bone marrow cells in 1 ml were incubated for 20 min with or without 200 mCi or 400 mCi of 3H-thymidine of specific activity 0.925 TBq(25 Ci)/mmol [6]. A medium with cold thymidine (to achieve a 100-fold concentration over radioactive thymidine) was then added, and incubation continued for another 10 min. Cells were then washed three times with an excess of medium without cold thymidine, since its presence interfered with the growth of CFU-E colonies. Cells were counted and used for a particular type of clonogenic assay. Before and after the incubation, the cells were maintained and manipulated at 4°C.
CFU-S Assay
Recipient mice were irradiated with 9 Gy from a 60Co source. Groups of 10 irradiated mice were transplanted with diluted samples of bone marrow cells to obtain approximately 15 to 20 spleen colonies. Spleens were collected eight days after transplantation, and mean colony numbers were corrected for the effect of colony overlap, as has been described previously [7].
CFC-GM, BFU-E, and CFU-E Assays
Clonogenic cell assays were previously described in detail [8]. CFC-GM agar cultures were supplemented with heat-inactivated serum from NMRI mice three h after endotoxin (50 µg) treatment, and were scored after seven days. CFU-E were cultivated in methylcellulose with 0.4 IU erythropoietin (EPO)/ml and counted after two days. BFU-E were grown in methylcellulose supplemented with postendotoxin serum and 2 IU EPO/ml and were counted after nine days.
Statistics
In Figures 1 through 4
, the linear part of the curve (closed symbols) was used for regression analysis, and 95% confidence intervals were calculated. The slope represented the increase in the DNA-synthesizing cell population (S-phase filling rate, 
% per hour) after initial depletion by hydroxyurea. The average cell cycle time (Tc) was calculated as the time necessary for all the cells to enter S-phase (100/slope).
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Figure 2. GM-CFC from normal BDF1 mice: cells in S-phase after hydroxyurea administration.  %/h: % of cells entering S-phase per h. ( Fig. 1 legend.)
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Figure 3. BFU-E from normal BDF1 mice: cells in S-phase after hydroxyurea administration. %/h: % of cells entering S-phase per h. ( Fig. 1 legend.)
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Figure 4. CFU-E from normal BDF1 mice: cells in S-phase after hydroxyurea administration. %/h: % of cells entering S-phase per h. ( Fig. 1 legend.)
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Calculation of the Expected Production of Blood Cells (
bc exp)
Average numbers of studied progenitors in the femoral bone marrow (n) of control mice were used. In the case of CFU-S, the number of cells was multiplied by 10, assuming a seeding efficiency of the assay of 10%. These numbers were multiplied by 16.7, (assuming that one femur represents 6% of the bone marrow [9]), in order to calculate the total number of progenitors (N) in the bone marrow (contribution of the spleen was neglected). N remained constant in the steady state. We have introduced the factor "f", calculated as the ratio of 24 h/average cell doubling time (Td), of the progenitor (f = 24/Td), in order to estimate the number of divisions corresponding to one progenitor per day. Thus, p = N x f describes the absolute number of divisions occurring inside the progenitor pool per day, i.e., the number of new progenitors made available in whole hematopoiesis during one day that must leave the progenitor cell pool in order to maintain a constant number of progenitors. This number was multiplied by the proliferation potential (p) of a particular progenitor to obtain bc exp. The p of different progenitors was set arbitrarily as 64 for CFU-E, 5,000 for CFC-GM and 10,000 for BFU-E. These arbitrary numbers were based upon publications of Metcalf [10], Heyworth and Spooncer [11] and Novak and Neas [3]. For CFU-Sday 8, the number of cells in the colony was calculated on the basis of the mean colony volume from Neas and Znojil [12]. The CFU-Sday 8 proliferation potential was then 860,000. This figure corresponded with similar calculations of Reincke et al. [13], leading to mode values exceeding 1,000,000 cells. For CFU-Sday 13, only large colonies (mean size 2.52 ± 0.92 mm) were taken into account. These large 13-day colonies represented 41% ± 5% of all colonies visible on day 8, when the same cell dose was injected into recipient mice [12]. The p of the average 13-day colony was 16,000,000 cells.
Calculation of the Actual Production of Blood Cells (bc act)
Actual blood cell production (bc act) was previously calculated from the published life spans or half-lives of different types of circulating blood elements. (For details see [4]).
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Results
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Figures 1 through 4
show the S-phase refilling rate after initial depletion of replicating cells caused by hydroxyurea. The results are highly scattered throughout the whole observation period. Due to measurement error, some values are presented as negative; however the scatter did not exceed the error that is inherent to measurement of 3H-thymidine kill [7]. Despite the scatter, a well-defined period of growth of the fraction sensitive to 3H-thymidine could be delineated. This is indicated by closed symbols (closed squares) as opposed to open symbols, which show either normal pretreatment values (circles), or post-hydroxyurea results not included in the regression analysis (squares) because they reached plateau or started to decrease. Results of the linear regression through the data depicted by closed symbols are presented, along with a 95% confidence interval. The calculated S-phase filling rate is expressed as the percentage of change in 3H-thymidine kill per hour.
CFU-S have exhibited the slowest recruitment to S-phase among tested progenitors. Figure 1 shows CFU-Sday 8 data from CBF1 mice. Almost 60% of CFU-S were in S-phase 15 h after hydroxyurea. Clonogenic in vitro assays were done on BDF1 mice. Figures 2 and 3 show results for GM-CFC and BFU-E which are similar. CFU-E ( Fig. 4) had a higher proportion of cycling cells under normal conditions, and 3H-thymidine kill reached more than 90% at five h after hydroxyurea.
Table 1 summarizes average cell cycle times of studied progenitors, calculated from the filling S-phase rate as: Tc (h) = 100/filling rate in % per hour. The cell cycle varied from 5.91 h to 30.3 h for CFU-E and CFU-Sday 8, respectively.
Absolute numbers of CFU-S, CFC-GM and BFU-E in the bone marrow before and after hydroxyurea administration did not change significantly. CFU-E decreased to approximately 30% after hydroxyurea (data not shown) and remained low during the 12-h observation period. Average normal values in femoral bone marrow (n) were: CFU-S: 5,049 (CBF1 mice); CFC-GM: 39,989; BFU-E: 9,616 and CFU-E: 53,033 (all BDF1 mice). These values along with cell cycle times were used to calculate the daily efflux from progenitor cell compartments ( Table 2).
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Table 2. Effluxes from compartments of progenitor cells derived from current experiments and from published data summarized by Novak and Neas [3]a and from Neas and Znojil [12, 14]b
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Table 3 compares the expected production of blood cells from CFU-Sday 13, CFU-Sday 8, CFC-GM, BFU-E and CFU-E to the actual production. The efficiency of blood cell formation is then calculated as a percentage of cells actually produced from those expected. The results suggest that the efficiency increases with the advanced maturation from CFU-Sday 13 to CFU-E from 0.02% to almost 100% in normal mice.
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Table 3. The efficiency of hematopoiesis from different categories of progenitors in normal mice, derived from current experiments and from published data ( Table 2 legend)
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Discussion
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The aim of this study was to provide a quantitative analysis of normal murine hematopoiesis. This analysis has compared the capacity of the bone marrow to produce blood cells with the actual production. We have used our experimental determination of CFU-S, CFC-GM, BFU-E and CFU-E effluxes from the progenitor cell compartments and, separately, the effluxes derived from published papers in order to estimate the capacity of the bone marrow to produce blood cells.
Our experiments, aimed at determination of progenitor production rate in vivo, used hydroxyurea to specifically destroy cells in the S-phase and monitor the rate of their replacement. The replacement kinetics indicated the intensity of cell passage through the cell cycle. Thus the measured kinetics were used to calculate the average cell cycle time in examined populations of CFU-Sday 8, CFC-GM, BFU-E and CFU-E hematopoietic progenitors ( Table 1).
The cell kinetics data obtained from animals are necessarily always limited regarding special strains, number of animals that can be examined, and experimental techniques. Therefore, we have also used reviewed published data on murine hematopoiesis [3] and CFU-Sday 8 and CFU-Sday 13 in parallel [12, 14], to arrive at results regarding functional reserve of different populations of murine hematopoietic progenitors. These two approaches thus checked each other. The fact that they led to compatible results increased the validity of our final conclusions.
The capacity of various progenitors to multiply was derived from the determination of cell numbers in hematopoietic colonies in clonogenic assays [3, 10-13]. In fact, these capacities are actually underestimated because part of the colonies would still continue to grow after their enumeration time. Therefore, the estimated capacity of various progenitors to produce blood cells is at the lower end of their normal range.
The actual production of circulating blood elements in the mouse, except the lymphocytes, has been determined previously from published values of their numbers in the blood and from data regarding their normal life span or half-life [4].
The comparison of the expected and actual production rates of blood cells, expressed as the efficiency of hematopoiesis in Table 3, is of interest because it suggests that the actual production is only a very small fraction of the bone marrow capacity. This fraction, however, increases in the progenitor differentiation hierarchy from less than 0.1% for early progenitor CFU-S, to almost 100% for CFU-E. The presented results therefore suggest existence of a steep gradient of the physiological apoptosis, declining with advancing differentiation and maturation of hematopoietic progenitors. This conclusion is in concert with the present concept of the hematopoietic tissue functional organization with "stem cells" endowed with considerable functional reserve. However, this might be the first attempt to really quantify this reserve and to compare it for different populations of progenitor cells. The results suggest that this reserve operates by means of an extensive physiological apoptosis occurring among early progenitors and diminishing with advancing progenitor differentiation. The difference between the apoptotic rate of CFU-S and CFU-E by more than three orders of magnitude makes it possible that small changes in these characteristics could significantly influence the hematopoiesis.
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Conclusions
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The present analysis of normal murine hematopoiesis suggests a steep gradient of apoptosis occurring among proliferating hematopoietic progenitors. The apoptotic rate appears to be the highest among early progenitors, affecting more than 99.9% of produced cells at this stage. However, the apoptotic rate declines to almost zero as the progenitors become more differentiated and mature.
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Acknowledgments
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This work was supported by research grants Nos. 306/94/0876 and 106/95/1440 from the Grant Agency of the Czech Republic.
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References
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accepted for publication December 12, 1997.
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Top
Abstract
Introduction
: untreated mice; 
: hydroxyurea- treated mice—values used in linear regression analysis; 
: hydroxyurea- treated mice—values not included in the analysis;