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Stem Cells 2004;22:1111-1120 www.StemCells.com
© 2004 AlphaMed Press

Cell Division Rates of Primary Human Precursor B Cells in Culture Reflect In Vivo Rates

Jonathan Coopermana, Robert Neelya, David T. Teachey, Stephen Gruppc, John Kim Choia,b

a Pathology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA;
b Pathology and Laboratory Medicine, University of Pennsylvania, Pennsylvania, USA;
c Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Key Words. Cell division • Precursor B cell • Lymphoblastic leukemia • Stroma-based culture

Correspondence: John Kim Choi, M.D., Ph.D., Children’s Hospital of Philadelphia, 802F ARC, 3516 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-590-7194; Fax: 215-573-0342; e-mail: jkchoi{at}mail.med.upenn.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone marrow stroma-based cultures provide a powerful model for studying cell division and apoptosis of primary human precursor B cells. Studies using this model are elucidating the mechanisms by which stromal cells inhibit apoptosis of cultured normal precursor B cells and have demonstrated that the apoptotic rate of cultured leukemic precursor B cells can predict clinical outcome in acute lymphoblastic leukemia. In contrast to apoptosis, cell division in this model has not been well characterized. In this study, we quantified the rates of cell division in cultured primary human normal and leukemic precursor B cells by labeling precursor B cells with the fluorescent dye carboxyfluorescein diacetate, succinimyl ester. Based on the rate of decreasing fluorescent signal over 3 weeks, normal CD19+, CD10+ precursor B cells divided once every 90.5 hours, a number that correlates well with the known in vivo rate of 65.5 hours. The division rates were similar among different cultures and constant throughout the 3 weeks of culture, suggesting that the variable expansions of precursor B cells seen among different samples and culture durations are not secondary to different cell division rates. Unlike normal cells, cultured leukemic B cells had a heterogeneous division rate that ranged from once every 26–240 hours. These rates correlated well with their respective in vivo proliferation index. These findings indicate that the stroma-based cultures faithfully replicate in vivo cell division rates and can be used to elucidate the pathways that regulate cell division of primary human precursor B cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Primary normal and leukemic human precursor B cells can be cultured on bone marrow stromal cell layer for at least 3–4 weeks [1–5]. Studies using this model have provided important insights into the cell pathways that promote the expansion of human precursor B cells. For example, expansion of normal or leukemic human B cells does not require interleukin-7 (IL-7) [2,6], a cytokine necessary for the expansion of murine precursor B cells. The rate of expansion is dictated, in part, by a balance of apoptosis and cell division. Apoptosis in both normal and leukemic precursor B cells is inhibited by direct contact with stromal cells [7,8] and is mediated by vascular cellular adhesion molecule-1, {alpha}4ß1 integrins, and multiple antiapoptotic proteins [9,10]. Although the rate of apoptosis is similar among different samples of normal human precursor B cells, primary leukemic precursor B cells have variable rates of apoptosis; decreased apoptotic rate in culture is an independent predictor of poor clinical outcome [11].

Cell division is also an important contributor to precursor B cell expansion and may occur at a high rate even without expansion if there is offsetting apoptosis and differentiation. Studies of freshly isolated bone marrow cells demonstrate that the rates of cell division are similar among normal human precursor B cells but variable among leukemic precursor B cells. Furthermore, low rates of cell division in leukemic cells may correlate with poor response to chemotherapy [12] and poor clinical outcome [13]. As with apoptosis, stroma-based cultures provide a potentially powerful model for studying cell pathways that regulate cell division of primary normal and leukemic human precursor B cells. However, cell division has not been well characterized in the stroma-based culture model, and it is unknown how well precursor B cells divide in culture. In this study, we followed cell division using the fluorescent dye carboxyfluorescein diacetate, succinimyl ester (CFSE). This approach has not been previously used to study human lymphocytes but has been used to quantify cell division in murine lymphocytes [14–16]. We report that different patient samples of normal precursor B cells have a relatively constant rate of cell division that is similar to reported in vivo rates. Cultured primary human leukemic cells have more heterogeneous rates of cell division that correlate with their in vivo proliferation index. These findings indicate that stroma-based cultures and CFSE can be used to study cell division of cultured primary human precursor B cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Under institutional review board approval, human precursor B cells were procured from excess clinical bone marrow aspirates. Normal precursor B cells were isolated from aspirates from patients without precursor B lymphoblastic leukemia (ages 5–18 years). CD19+ cells were isolated by Ficoll gradient followed by immunomagnetic selection using the magnetic cell sorting (MACS)-positive selection system for CD19 (Miltenyi Biotec, Auburn, CA). Pediatric leukemic precursor B cells were isolated from patients with precursor B lymphoblastic leukemia and purified by Ficoll gradient. Adult leukemic B cells purified in the same manner were obtained from the Stem Cell and Leukemia Core Facility of the Abramson Family Cancer Center of the University of Pennsylvania. Human primary bone marrow stromal cells were obtained by culturing CD19-depleted bone marrow cells in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. The stromal cells were incubated at 37°C, 5% CO2, and 90% humidity, passaged at least three times, trypsinized, irradiated with 2000 rads, and plated onto 24-well plates at 105 cells per well in RPMI 1640 medium with 10% FBS. After overnight incubation, the wells were washed with X-vivo 10 media.

Coculture
Using a modification of previously described protocol [4], 105 CD19+ cells per well were cultured. Irradiated primary human stromal cells were used rather than the AFT024 murine stromal cell line. CD19+ and stromal cells were cocultured in X-vivo 10 media supplemented with 5% FBS and the growth factors IL-7 (20 ng/ml), stem cell factor (SCF) (20 ng/ml), insulin-like growth factor (IGF)-1 (20 ng/ml), and FMS-like tyrosine kinase 3 (FLT-3) (10 ng/ml). Every week, half of the culture media was replaced with fresh media, FBS, and growth factors.

BrdU Incorporation
Cocultures were incubated with BrdU for 16 hours, and the incorporated BrdU was detected using the In Situ Cell Proliferation Kit (Roche, Indianapolis) following the manufacturer’s protocol. Fluorescent signal was detected by inverted UV microscopy.

Terminal Deoxynucleotidyl Transferase–Mediated Rhodamine-dUTP Nick-End Labeling Assay
Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP-X nick-end labeling was used to detect DNA fragmentation using the In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol. Fluorescent signal was detected by inverted UV microscopy.

CFSE Labeling
CD19+ cells were labeled with CFSE as previously described [17]. A total of 106 to 107 cells were washed twice with phosphate buffered saline (PBS) and then incubated with 5 nM CFSE in PBS for 3 minutes at room temperature. The cells were washed two times with RPMI media supplemented with 10% FBS.

Flow Cytometry
The cultures were trypsinized for 3 minutes at room temperature, washed with RPMI media supplemented with 10% FBS, and incubated with the antibodies following the manufacturers’ protocols. The antibodies used were allophycocyanin-conjugated anti-CD19, peridinin chlorophyll protein–conjugated anti-CD10, R-phycoerythrin (PE)–conjugated anti-CD10, and PE-conjugated annexin V (Pharmingen, San Diego). Intracellular staining using PE-conjugated anti-CD79a; Pharmingen) was performed using Fix and Perm reagents (Caltag Laboratories, Burlingame, CA) following the manufacturer’s protocol. 7-amino-actinomycin D (7-AAD) (Pharmingen, San Diego, CA) was used at a final concentration of 2.5 µg/ml, following the manufacturer’s protocol. Analyses were performed on FACSTAR and analyzed using Cell-Quest software (Becton, Dickinson, San Jose, CA).

Paraffin Immunohistochemistry
Four-µm sections of paraffin-imbedded tissue were stained with hematoxylin and eosin (H&E) for morphologic evaluation. Ki67 was detected using a modification of previously described protocol [13]. Slides were microwaved for antigen retrieval in citrate buffer, pH 6.0, for 10 minutes and then incubated with a monoclonal antibody (M7240; DAKO, Carpinteria, CA) for 30 minutes, biotin-conjugated horse anti-mouse antibody (Vector, Burlingame, CA) for 20 minutes, horseradish peroxidase–conjugated strepavidin (Vector) for 30 minutes, and diaminobenzidine (DAKO) for 5 minutes. Slides were counterstained with hematoxylin.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and Characterization of Primary Normal Human CD19+ B Cells
Progenitor B cells were isolated by Ficoll gradient followed by using antibodies against CD19. Flow cytometry analysis of the purified cells showed >95% of the cells were positive for the B cell–specific antigen CD79a; of these, 70%–100% were positive for the precursor B cell antigen CD10 (Fig. 1Go). The CD19+, CD10+ cells were negative for the more mature B cell antigen, surface immunoglobulin heavy chain (IgM), and surface immunoglobulin light chains. The remaining 0%–30% of the CD19-selected cells was more mature CD79a+, CD10–, IgM+ B cells. More than 98% of the CD19+ cells were viable by 7-AAD exclusion (data not shown).



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  		Figure 1. Characterization of isolated CD19+ B cells. Mononuclear bone marrow cells, before and after immunomagnetic selection with CD19 antibodies, were stained with phycoerythrin-conjugated anti-CD79a and allophycocyanin-conjugated anti-CD10 and analyzed by flow cytometry.

 
The CD19+ B cells were cocultured on stromal cells in X-vivo 10 media supplemented with 5% FBS and the growth factors IL-7, SCF, IGF-1, and FLT-3, as previously described [4]. The AFT024 murine stromal cell line was replaced by primary pediatric bone marrow stromal cells, because in our hands the irradiated AFT024 cells continued to proliferate and overgrew the culture after 2 weeks (data not shown). At 0, 1, 2, and 3 weeks, the cells were trypsinized, counted, and analyzed by flow cytometry for CD19 and CD10 expression. Studies using a leukemic precursor B cell line, Nalm-6, indicated that trypsinization up to 15 minutes had no effect on the detection of CD19 and CD10 (data not shown). The expansions of CD19+, CD10+ precursor B cells (n = 6) were variable among different samples, with the cell numbers being zero to four times the initial number of plated cells at the end of 3 weeks. Most samples increased in number during the first 2 weeks and then decreased in number during the third week (Fig. 2Go). The variable expansions among samples and the decreased numbers after 2 weeks in culture are similar to the findings reported by others [3,18].



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  		Figure 2. Expansion curves of CD19+, CD10+ precursor B cells from six patients. Isolated CD19+ B cells were cultured on bone marrow stroma. After 1, 2, and 3 weeks of culture, precursor B cells were enumerated by multiplying the total number of cells by the percentage of CD19+, CD10+ cells that was determined by flow cytometry analysis. The numbers were normalized to the numbers at initial plating.

 
Expansion of Cultured B Cells Is Determined by a Balance of Cell Division and Apoptosis
Two models can explain the variable, and in some cases lack of, expansion of precursor B cells among different samples with prolonged culture. In the first model, human B cells in some samples divide very slowly or not at all during prolonged culture. This predicts that cultures with no increase or net decrease in B cells have no cell division. In the other model, B cells are dividing but do not expand in number because of concomitant apoptosis. This predicts that all cultures contain dividing B cells. To determine which model is more accurate, we analyzed our cultures for active DNA synthesis by BrdU incorporation. Three-week-old cultures that had a net loss in B cells were incubated with BrdU, and the incorporated BrdU was detected using fluorescein-conjugated monoclonal antibodies to BrdU. UV microscopy showed many positive nuclei (Fig. 3Go), indicating that even in cultures without obvious B cell expansion, there was active cell division. The dividing cells were likely B cells, because the stromal cells were irradiated and thus unlikely to divide. In support, 3-week-old cultures of irradiated stromal cells without B cells demonstrated no BrdU incorporation (data not shown).



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  		Figure 3. DNA synthesis and apoptosis in culture. Three-week-old cultures were incubated without (top row) and with (middle row) BrdU for 16 hours, stained with anti-BrdU antibodies, and examined under phase and UV microscopy (magnification x200). Three-week-old cultures were analyzed by terminal deoxynucleotidyl transferase–mediated rhodamine-dUTP nick-end labeling (TUNEL) and examined under phase and UV microscopy (magnification x 200).

 
Because there was a net decrease in B-cell number in some cultures despite DNA synthesis, apoptosis was likely present in our culture. To confirm this, 3-week-old cultures were examined for DNA fragmentation by TdT-mediated rhodamine-dUTP nick-end labeling (TUNEL). UV microscopy showed many positive nuclei (Fig. 3Go), confirming the presence of apoptosis. Three-week-old cultures of irradiated stromal cells without B cells showed virtually no TUNEL activity, suggesting that the dying cells were of B-cell origin (data not shown). These studies indicate that B-cell expansion in culture is dictated by a balance of cell division and apoptosis.

Rates of Cell Division in Different Samples of Cultured Normal Human B Cells Are Similar
Next, we asked if the variable expansions of precursor B cells could be explained by different rates of cell division among different samples. To quantify the rate of cell division, isolated human CD19+ B cells were labeled with CFSE, cultured, and followed for decreasing CFSE signal. In this approach, CFSE is covalently bound to intracellular macromolecules and eventually produces a stable fluorescent signal for each cell. With each division, the resulting daughter cells have half of the CFSE signal [17,19]. At 0, 1, 2, and 3 weeks of culture, the cells were trypsinized, stained with antibodies against CD19 and CD10, and analyzed by three-color flow cytometry (Fig. 4AGo). Our CFSE analysis of human precursor B cells did not produce individual fluorescent peaks as seen with CFSE analysis of murine peripheral T lymphocytes [14]. The lack of individual peaks most likely represented the heterogeneous sizes of the precursor B-cell population leading to overlapping peaks. Backgating on CD19+, CD10+ B cells confirmed a wide size distribution as determined by forward scatter; in contrast, the more mature CD19+, CD10– B cells showed more uniform size (data not shown). Nevertheless, this assay permitted a good estimation of the number of cell divisions.



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  		Figure 4. CFSE analysis of normal human precursor B cells. (A): Isolated CD19+ B cells were labeled with CFSE and cultured on bone marrow stroma. After 4, 7, 14, and 21 days of culture, cells were stained with phycoerythrin-conjugated anti-CD19 and allophycocyanin-conjugated anti-CD10. Cells in the blast and lymphocyte region by forward and side scatter were subgated into CD19+, CD10+ and CD19+, CD10– B cells and then analyzed for CFSE expression. Figure is representative of six samples. (B): Same sample as in (A), except the growth factors were removed. Figure is representative of three samples. Abbreviations: CFSE, carboxyfluorescein diacetate, succinimyl ester; MFI, mean fluorescent intensity

 
Under our culture conditions, the more mature CD19+, CD10– B cells had a stable CFSE signal that decreased by only 25% over the 3 weeks of culture, suggesting little if any cell division within this population. The constant CFSE signal in this population confirmed the stable nature of the CFSE signal during prolonged culture. The constant CFSE signal also argued against significant differentiation of the dividing precursor cells, because this also results in CD19+, CD10– B cells with decreased CFSE signal. Hence, most of the CD19+, CD10– cells after 3 weeks in culture represented the initial plated cells.

Unlike the more mature CD10– B cells, cultured CD19+, CD10+ precursor B cells showed continuous decrease in CFSE signal throughout the 3 weeks of culture, indicating continuous cell division throughout the culture (Fig. 4Go). The rate of decreasing CFSE signal was examined for six different samples at weekly intervals (Fig. 5Go). The CFSE signals were normalized to the stable signal of the CD19+, CD10– B cells. This normalization compensated for the inherent variations in CFSE labeling between samples and flow cytometry settings between weeks. The samples had similar rates of cell division despite the variability in the expansion. During week 1 of culture, the average CFSE signal decreased by 3.14-fold, corresponding to approximately 1.65 divisions. During week 2 of culture, the CFSE signal decreased further by 3.88-fold, corresponding to approximately 1.95 divisions. During week 3 of culture, the CFSE signal decreased further by 3.90-fold, corresponding to approximately 1.89 divisions. Over the 3 weeks of culture, the CFSE signal decreased by 47.6-fold, corresponding to approximately 5.6 divisions. This predicts an average increase in cell number by more than 47-fold. Instead, the measured average increase was less than two-fold, suggesting a high level of cell death.



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  		Figure 5. Summary of CFSE analysis of six samples. The CFSE signals of CD19+, CD10+ B cells (see Fig. 4AGo) at 1, 2, and 3 weeks of culture were normalized to those of CD19+, CD10– B cells. The latter signal is set at 100%. Numbers represent mean normalized signal, with standard deviation in parenthesis. Abbreviation: CFSE, carboxyfluorescein diacetate, succinimyl ester.

 
Growth Factors Increase Cell Division of Precursor B Cells
Previous studies demonstrated that growth factors can increase precursor B-cell expansion by up to 20-fold [3,18]. In our cultures, the number of precursor B cells decreased 7-to 10-fold when growth factors were removed (n = 3). To determine how much of the decrease was the result of decreased cell division, CFSE-labeled CD19+ B cells were cultured with and without growth factors (Figs. 4A, 4BGo). Cultures were analyzed by flow cytometry at weeks 0, 1, 2, and 3. When cultured without growth factors, the CD19+, CD10+ precursor B cells showed continuous decrease in CFSE signal. However, the mean CFSE signal at 3 weeks was approximately two-fold more than that of B cells with growth factors, indicating that increased cell division contributed approximately only two-fold of the 7- to 10-fold increase in B cell number. The remainder of the increased number of B cells must be mediated by other processes. One possibility may involve apoptosis, because growth factors have been shown to promote survival in addition to cell division of precursor B cells [3].

Heterogeneous Rates of Cell Division among Cultured Leukemic Primary Precursor B Cells
Next, we asked if primary leukemic precursor B cells also had the same rate of cell division as the normal precursor B cells. We predicted that different leukemic cells would have different rates of cell division in culture, because previous studies on freshly isolated human precursor B cells indicated that leukemic B cells have variable rates of cell division [20,21]. We studied precursor B leukemic cells from 10 patients (Table 1Go). No normal CD19+, CD10– B cells were present for normalization, preventing direct comparison between different samples. Instead, the signal was externally normalized to unlabeled Nalm-6, permitting comparison within a sample at different times. CD19+ B cells were isolated from excess bone marrow aspirates within 24 hours, labeled with CFSE, and cultured. At 0, 1, 2, and 3 weeks of culture, the cultures were trypsinized, stained with CD19 and CD10, and analyzed by three-color flow cytometry.


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  		Table 1. Patient information and characteristic of the precursor B lymphoblastic leukemia
 
As predicted, the leukemic cells had variable rates of cell division (Fig. 6Go). Leukemic cells from patient A had a rapid decrease in CFSE signal of approximately 50-fold over 6 days (day 1 through day 7), corresponding to approximately 6.5 divisions per week. This division rate is more than three times that of normal precursor B cells. During the second and third weeks, the signal decreased by another 3.5- and 1.5-fold, respectively. However, these signals were near or at background and likely underestimate the number of cell divisions. Leukemic cells from patient B had a decreased CFSE signal of approximately 25-fold over 10 days (day 1 through day 11), corresponding to approximately 3.2 divisions per week. These leukemic cells also died rapidly in culture such that no viable cells were detected at 14 days of culture. Leukemic cells from patient C had a decreased CFSE signal of approximately 3.9-fold over 20 days (day 1 through day 21), corresponding to approximately 0.69 division per week, a rate approximately one third of that in normal precursor B cells. Leukemic cells from the other seven patients had cell division rates between the extreme rates of patient A and patient C.



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  		Figure 6. CFSE analysis of three cases of precursor B lymphoblastic leukemia. Isolated CD19+ B cells from patients A, B, and C were labeled with CFSE, cultured, and analyzed as described in Figure 4Go. Abbreviations: CFSE, carboxyfluorescein diacetate, succinimyl ester; MFI, mean fluorescent intensity.

 
The slow rate of cell division of the leukemic cells from patient C was unexpected in a leukemia that is typically associated with an aggressive clinical behavior. Possible explanations for this unexpected finding included slowly dividing leukemic precursor B cells or culture conditions lacking an important proliferation signal present in vivo. To distinguish between these possibilities, we measured the proliferation index of the leukemic cells in the bone marrow biopsies from patients C and B. No biopsy was performed for patient A. The biopsies were placed in B5 fixative within 5 minutes of the procedure, ensuring rapid preservation of the cells near their original state. Examination of the H&E-stained sections of the biopsies revealed complete replacement of the marrow by leukemic cells (Figs. 7A, 7CGo). Proliferation cells were identified using paraffin immunohistochemical staining using antibodies against Ki67, a proliferation antigen with strong correlation to BrdU incorporation and mitotic rate [22]. Biopsy of patient C showed that <5% of the leukemic cells were positive for Ki67 (Fig. 7BGo). In contrast, biopsy from patient B showed that >70% of the leukemic cells were positive for Ki67 (Fig. 7DGo). The biopsies from the other patients were analyzed similarly. The percentages of Ki67-positive cells were plotted against rate of CFSE-decreasing intensity for the nine leukemic samples with biopsies (Fig. 8Go). There was a general trend in which leukemic cells with high percentage of Ki67+ cells tended to have higher rates of cell division. These findings suggest that the cell division rates in culture for many primary leukemic cells correlate well with the in vivo rates.



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  		Figure 7. Examination of bone marrow biopsy. (A, B): Patient C. H&E-stained section (A) and paraffin immunohistochemical-stained section with antibodies against Ki67 (B), magnification x400. Note the single strongly Ki67-positive cell in the lower left corner of (B). (C, D): Patient B. H&E-stained section (C) and paraffin immunohistochemical-stained section with antibodies against Ki67 (D), magnification x400. Abbreviation: H&E, hematoxylin and eosin.

 


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  		Figure 8. Scatter plot of nine leukemic samples by percentage Ki67-positive versus in vitro cell division rate (decrease in CFSE signal per week). These represent patients 2 through 10 of Table 1Go. No biopsy was available for analysis for the first patient (patient A). Abbreviation: CFSE, carboxyfluorescein diacetate, succinimyl ester.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Precursor B lymphoblastic leukemia, the most common cancer of childhood [23], is characterized by abnormal clonal expansion of precursor B cells. Because expansion is dictated, in part, by cell division, regulation of this cell function is likely perturbed in many precursor B lymphoblastic leukemias. In support, mutations or aberrant expression of the cell-cycle regulatory genes INK4A, INK4B, retinoblastoma, and possibly p21(CIP1/WAF1/SDI1) are frequently seen in both pediatric and adult precursor B lymphoblastic leukemias [24–29]. In pediatric leukemias, low rate of cell division and high expression of INK4A may be associated with in vitro drug resistance or poor clinical outcome [12, 13, 30]. Hence, additional understanding of the regulation of cell division in precursor B cells may provide insight into therapy-resistant precursor B lymphoblastic leukemias.

One powerful model for studying cell division of human B cells is the in vitro stroma-based culture that provides long-term expansion of primary human precursor B cells. However, this model has not been well characterized for cell division, in part because of the technical difficulties. Previous studies of freshly isolated primary human precursor B cells used simultaneous measurements of 3H-thymidine/BrdU incorporation and Ki67 expression. Application of this technically challenging approach to cultured B cells is additionally complicated by the significant numbers of stromal cells that are present in the coculture. Finally, significant levels of apoptosis present in these cultures can complicate these measurements, because increased apoptosis can decrease the number of cells leading to decreased incorporation of 3H-thymidine or BrdU even with increased division rate. Alternatively, apoptosis during or shortly after tritium/BrdU incorporation may cause increased incorporation without productive cell division.

In this study, we quantified the rate of cell division of cultured primary human precursor B cells using CFSE labeling. The rates of decreasing CFSE signal and thus the rates of cell division in six different normal human samples were similar. Furthermore, the rates of cell division during the first week of culture were similar to those during the third week of culture, indicating that the decreased expansion of precursor B cells during the third week is unlikely to be the result of decreased cell division. These findings suggest that the variable expansions among samples and different culture duration seen in this and other studies [2, 3, 18] are most likely the consequence of variability in apoptosis.

Cultured primary human precursor B cells underwent approximately 5.57 divisions over 3 weeks of culture. This change translates to approximately one division every 90.5 hours, which is similar to the mean cell-cycle transit time of 65.5 ± 3.5 hours that was determined by the more technically challenging approach of simultaneously measuring 3H-thymidine incorporation, 14C-thymidine incorporation, and Ki67 expression in freshly isolated precursor B cells [20]. In contrast to CD19+, CD10+ precursor B cells, the more mature CD19+, CD10– B cells had little to no cell division under our culture conditions, a finding that is consistent with the requirement for different factors for expansion of more mature B cells [4,5]. Despite little to no cell division, the numbers of CD19+, CD10– B cells remained constant (data not shown), suggesting that the apoptosis rate of these cells is significantly lower than precursor B cells in this culture system.

Unlike normal precursor B cells, leukemic B cells had variable rates of cell division. In one sample, the leukemic cells divided three times faster than normal precursor B cells. In another sample, the leukemic cells divided at an unexpected slow rate, on average nearly one third of the rate of normal B cells. The variable rates of cell division of leukemic B cells in culture (once every 26–240 hours) are similar to the variable rates reported for freshly isolated leukemia cells (once every 25–112 hours) [20,31]. To confirm that the cell division rates in culture reflected in vivo rates, we examined the expression levels of the proliferation marker Ki67 in rapidly fixed bone marrow biopsy containing the leukemic cells. Paraffin immunohistochemistry demonstrated the expression of Ki67 correlated with cell division rates in culture. Leukemic cells with low division rate had <5% Ki67-positive cells. Although unusual, other cases of precursor B lymphoblastic leukemias with <5% Ki67-positive cells have been reported and correlate with high frequency of treatment failure; in contrast, biopsies of most other precursor B lymphoblastic leukemias had >30% Ki67-positive cells [13]. These findings suggest that the division rates of precursor B lymphoblastic leukemia cells in culture reflect their rates in vivo.

Our results differ slightly from a previous measurement of cell division of primary precursor B leukemic cells [32]. In that study, seven primary leukemic cells were labeled and tracked using a lipophilic dye PKH-26 for 14 days. The leukemic cells divided 0.65 to 1.3 times per week compared with our 0.69 to 6.5 times per week. That study also noted a CD10 dim subpopulation that divided at a faster rate than most of the leukemic cells. No such subpopulation was detected in our study. The different findings may represent differences in the leukemic samples or differences in the culture conditions. They are unlikely to be secondary to the different tracking dyes, because in our hands, the PKH-26 and CFSE measured similar cell division rates for normal precursor B cells (data not shown).

In general, the rates of cell division in culture correlate well with in vivo rates, although additional samples are needed to confirm the validity of this trend. Even the exceptions to this trend might be important in understanding the signals for proliferation. For example, leukemic cells with rapid in vivo but slow culture rates of cell division suggest a proliferation signal that is present in vivo but lacking in culture. Our study demonstrates that CFSE can be used to quantify cell division in stroma-based cultures of normal and leukemic human precursor B cells and provide an approach to dissect the important pathways that regulate cell division in normal and leukemic human precursor B cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Sindhu Cherian for her help in obtaining pathology information.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 3, 2003; accepted for publication April 7, 2004.



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