Stem Cells, Vol. 14, No. 3, 320-329,
May 1996
© 1996 AlphaMed Press
Flow Cytometric Analysis of Megakaryocyte-Associated Antigens on CD34 Cells and Their Progeny in Liquid Culture
Irmel A. Ayalaa,
Aaron Tomerb,
Kathryn L. Kellarc
a Division of Pediatric Hematology-Oncology;
b Division of Hematology-Oncology, Emory University School of Medicine; and
c Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and Prevendion, Atlanta, Georgia, USA.
Key Words. Megakaryocytes • CD34 cells • Glycoprotein IIbIIIa • c-kit • Three-color flow cytometry • IL-6
Dr. Kathryn L. Kellar, Centers for Disease Control and Prevention, NCID/SRP/BPB, MS D-34, 1600 Clifton Road NE, Atlanta, GA 30333, USA.
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Abstract
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Three-color flow cytometry was used to analyze the coexpression of surface antigens on megakaryocytes (MKs) developing in liquid cultures of enriched CD34+ cells purified from cord blood. Cells were cultured in serum-replete medium supplemented with interleukin 3 (IL-3), stem cell factor and IL-6. During two weeks of culture, total cells increased 76 ± 36-fold. CD34+ cells maximally expanded between days 2 and 4, and then gradually decreased to their original input numbers by day 14. As CD34+ cells declined, MKs, defined as glycoprotein (GP) IIbIIIa+ cells, steadily increased in culture 20.9 ± 18.3-fold. Megakaryopoiesis was further defined by monitoring the expression of GPs IIb, IIIa, Ib, IbIX, and IIIb and c-kit antigen. Increased expression of GPs IIbIIIa and IIb occurred earliest in culture, followed by IIIa and Ib, and then IbIX. Expression of IIIb, also found on monocytes, did not parallel that of the other antigens except when coexpressed on IIbIIIa+ cells. c-kit expression paralleled that of CD34 until the second week of culture when expression was high on nonMKs. Each of these antigens was coexpressed on CD34+ cells and identified a subset of late MK progenitors that increased steadily in culture. Triple-labeled cells expressing CD34, IIbIIIa and a third MK-related antigen were seen at all times. Polyploid MKs of up to 32N were observed during the second week of culture. Multiparametric flow cytometry proved to be a rapid, sensitive and specific method for quantitating the changes in antigen expression of differentiating MKs.
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Introduction
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The study of human megakaryopoiesis has been hampered by the relative scarcity of megakaryocytes (MKs) and MK colony-forming units (CFU) in the bone marrow (BM) and peripheral blood (PB), and the length of time necessary for clonal analysis. However, during the past few years, the adoption of sophisticated techniques for the identification and isolation of MK progenitors and advances in understanding the cytokine control of megakaryopoiesis have given many insights into the process that ultimately regulates platelet production [15]. In addition, flow cytometry has been found to be a sensitive and efficient method for the analysis of MKs and their ploidy in normal and disease states [6,7].
As platelet precursors, MKs acquire many surface antigens during development, including glycoproteins (GPs) IIbIIIa, IIb, IIIa, Ib, IbIX and IIIb, and c-kit, that are important for platelet function. However, no study has been done to examine the coordinate expression of these antigens on MKs and their precursors during in vitro development. Advances in the technique of flow cytometry and the availability of reagents for multiparametric analysis have made such a study possible.
Enriched populations of CD34+ progenitor cells purified from cord blood (CB) were grown in the presence of interleukin 3 (IL-3), stem cell factor (SCF) and IL-6 to both expand and promote differentiation of MK progenitors in liquid culture. The development of MKs in this system was serially monitored by using three-color flow cytometry to analyze the coexpression of the major MK-associated antigens.
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Materials and Methods
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Positive Selection of CD34+ Cells from Cord Blood
Umbilical CB from normal full-term deliveries was supplied to the Centers for Disease Control and Prevention from local hospitals by Blood Group Services (Roswell, GA). CB low-density mononuclear cells were separated by centrifugation at 700xg for 20 min over Percoll (p = 1.080 g/ml) (Pharmacia Biotech; Piscataway, NJ) in a solution containing phosphate-buffered saline-1% bovine serum albumin-1mM EDTA (PBS-BSA-EDTA). Cells were washed twice in PBS-BSA and CD34+ cells were isolated using the Ceprate LC (CD34) Kit (CellPro; Bothell, WA). Briefly, cells were incubated with an anti-CD34 IgM monoclonal antibody, washed and then incubated with a biotin-labeled anti-mouse IgM. Washed cells were loaded onto an avidin column and isolated by immunoaffinity chromatography.
Cell Culture
Preliminary experiments defined culture conditions that would maintain cell expansion and support MK production. Enriched populations of CD34+ cells were cultured at 105/ml in 25 cm2 tissue culture flasks in myeloid long term culture medium (Stem Cell Technologies; Vancouver, BC) supplemented with 10–4M hydrocortisone sodium succinate (Sigma; St. Louis, MO) and IL-3, SCF and IL-6 (R&D Systems; Minneapolis, MN) at a concentration of 10 ng/ml each. The cultures were split every four to five days or when the cell concentration reached greater than 106/ml. Growth factors were replenished on day 5. In two separate experiments, subcultures were supplemented with a higher dose of IL-6 (50 ng/ml) on day 5 to determine the effect on MK ploidy. Cultures were incubated for 14-16 days at 37°C in a humidified incubator containing 5% CO2 in air.
Immunophenotyping of Cells
Isolates from the CD34 immunoaffinity cell selection and samples of cultured cells were analyzed for total cell numbers, antigen expression, morphology and as numbers permitted, MK ploidy.
Cell counts were done on a Coulter Counter ZM (Hialeah, FL). Total cell counts were calculated based on the culture volume at the time of sampling and were not corrected for the numbers of cells previously removed for study. The numbers of fluorescent cells per culture were calculated by multiplying the percentage of the cells that were positively labeled with fluorescent antibody by the total cells per culture at any one time point.
Phycoerythrin-conjugated anti-HPCA-2 (Becton Dickinson; San Jose, CA) directed against the CD34 antigen was used in these experiments. GPIIbIIIa was chosen as the primary antigen to identify cells of the MK lineage. Three-color flow cytometry was performed by using a panel of monoclonal antibodies that included fluorescein isothiocyanate-conjugated (FITC) and biotin-labeled anti-CD41a/GPIIbIIIa (P2 clone, specific for the complex only), FITC anti-CD36/GPIIIb, biotin anti-CD41b/GPIIb (SZ22 clone, specific for IIb only), biotin anti-CD61/GPIIIa, biotin anti-CD42b/GPIb, biotin anti-c-kit and purified anti-GPIbIX (specific for the complex) in combination with an FITC-labeled second antibody, which were all purchased from AMAC (now Immunotech; Westbrooke, MA). Biotin-labeled antibodies were secondarily labeled with streptavidin peridinin chlorophyll protein (Becton Dickinson). The cells were labeled according to standard procedures. Additional cells were stained with single fluorochrome-labeled isotypic controls to set the fluorescence thresholds and with single fluorochrome-labeled antibodies to adjust the compensation. Antibody concentrations were titrated to reduce the overlap of fluorescent signals and not reduce the percentage of positively stained cells. Expression of CD34, GPIIbIIIa and two different third antigens were analyzed in each experiment. Each combination of three antigens was analyzed at least twice. The cells were examined immediately or fixed in 0.5% formaldehyde (Polysciences; Warrenton, PA) in PBS. Fluorescence was analyzed using a FACScan (Becton Dickinson) flow cytometer. A minimum of 104 cells was analyzed per data point.
Ploidy Analysis of MKs in Culture
Selected cultures were analyzed for the ploidy content of MKs. Cells were stained with saturating concentrations of FITC anti-GPIIbIIIa and then propidium iodide (PI), as described by Tomer et al. [7]. The FACScan flow cytometer that was used for ploidy analysis was equipped with an adjustable logarithmic amplifier to improve the resolution of the ploidy peaks. Bidimensional plots of green (FITC, FL1) fluorescence versus forward light scatter (FSC) or orange (PI, FL3) fluorescence defined the subset of highly fluorescent, large MKs and were used to estimate optimal settings to collect the data.
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Results
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Isolation and Expansion of CD34+ Cells
The number of cells isolated by CD34 immunoaffinity from the average CB sample volume of 50 ml was 3.0 ± 1.2 x 106 (mean ± SD, n = 6). CD34+ cells accounted for 78 ± 7% of the purified cell population. By day 2, 92 ± 5% of the cultured cells were CD34+. CD34+ cell numbers increased an average of 3.7 ± 1.5-fold by day 2 or 4 (p < 0.042 and p < 0.039, respectively), then declined over the next six days, and reached a nadir by day 10 (Fig. 1
). On day 14, CD34+ cells still accounted for 97 ± 55% of their original numbers, but represented only 1 ± 0.2% of the expanded cell population. The progeny of the cultured cell population gradually increased in number until day 7, then proliferated more rapidly, and reached a maximum 125-fold and an average 76 ± 36-fold expansion by day 14. The majority of these cells were myeloid as determined by Wright Giemsa staining of the cultured cells.
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Figure 1. The kinetics of cell expansion of the CB CD34+ cell population cultured in serum-replete medium containing IL-3, SCF, and IL-6 for 14 days. The percentages of IIbIIIa+ and CD34+ cells were measured by flow cytometry and multiplied by the total cells per culture to calculate the number of cells. Data are the means ± SD from six different experiments. Values were not corrected for aliquots previously taken for analysis.
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Expansion of Cells Expressing GPIIbIIIa
The number of GPIIbIIIa+ cells gradually increased in culture until day 10, and then plateaued (Fig. 1
). By day 14, IIbIIIa+ cells had increased 20.9 ± 18.3-fold (p < 0.00002). This increase in IIbIIIa+ cells coincided with the decrease in CD34+ cells after day 4, suggesting that megakaryopoiesis was proceeding from an initial CD34+ precursor population. MK precursors that expressed both CD34 and IIbIIIa (CD34+IIbIIIa+ cells) represented only a small percentage (6.5 ± 3.4%) of the initial cell population (Fig. 2). These cells linearly increased in culture (p < 0.0007, day 0 versus day 14) and represented 80-90% of all of the CD34+ cells between days 10 and 14. Alternately, CD34+IIbIIIa– cells comprised 93.5% of the initial cell population. These cells represented the majority of the CD34+ population until after day 7, when CD34+IIbIIIa+ cells became more prominent.
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Figure 2. The total cells that were CD34+, IIbIIIa+, CD34+IIbIIIa+, and CD34+IIbIIIa– in the purified cell population and during culture for 14 days. Values were calculated as described for Figure 1. Data are the means ± SE (n = 6).
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Expression of Other MK-associated Antigens on Isolated and Cultured Cells
The expression of additional MK-associated antigens was assessed by monitoring the serial expansion of cells labeled with antibodies to GPs IIb, IIIa, Ib, IbIX and IIIb, as well as c-kit. All of these antigens are platelet- or MK-specific except for IIIa, which is also found on macrophages and endothelial cells in the vitronectin complex; IIIb, which is also found on monocytes, endothelial cells and red cell precursors; and c-kit, which is also found on CD34 cells, tissue mast cells, early B cells and endothelial cells [8,9]. Adherent cells were not seen in these cultures, so endothelial cell and macrophage expression were not expected to interfere with the use of these antibodies for analysis of MKs. Also, without erythropoietin, these cultures did not support erythropoiesis.
As seen in Figure 3, cells that expressed the other MK-associated antigens increased steadily with time in culture, similarly to IIbIIIa+ cells, which are shown for comparison (broken line). IIbIIIa expression increased after day 2 and preceded increases in the other antigens except for IIIb. Expression of IIb increased after day 4, followed by increases in IIIa and Ib after day 7. IbIX expression did not rise until after day 12 and remained lower than the other antigens until that time. The number of cells expressing IIbIIIa, IIb and Ib were similar on day 10, when they also peaked. As expected for a cell population composed of myeloid as well as MK lineages, IIIb+ cells followed a different pattern of expansion than the other labeled cells and were present in greater numbers.
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Figure 3. The total cells expressing six different MK-associated cell surface antigens in the purified cell population and during culture for 14 days. Values were calculated as described for Figure 1. Each point represents the average of two different experiments, except for the IIbIIIa+ data (broken line), which are the means ± SD (n = 6).
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In addition to IIbIIIa, CD34+ cells coexpressed all of the other MK-associated antigens, indicating that expression of the entire complement of MK antigens occurs early during MK development (Fig. 4). These precursors represented approximately 5% of the original cell population and accounted for 1% of the expanded cell population by day 14. Their expansion rate (approximately fivefold) and numbers were comparable to that of CD34+IIbIIIa+ cells (broken line).
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Figure 4. The total cells coexpressing CD34 and one of six different MK-associated antigens in the purified cell population and during culture for 14 days. Values were determined as described for Figure 3, except for the CD34+IIbIIIa+ data (broken line), which are the means ± SE (n = 6).
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CD34+IIIb+ cell numbers rose very early in culture, followed by CD34+IbIX+ cells. Subsequently, CD34+ cells coexpressing IIbIIIa, IIb and IIIa increased and then leveled off after day 10. CD34+Ib+ cells were the last to show an increase, rising substantially after day 7, except that CD34+IbIX+ cells again rose after day 10. Two populations of CD34+IbIX+ precursors, one preexisting and one developing later, appeared to be present. In concert with the patterns seen in Figure 3, a sequence of MK antigen expression was evident, particularly after day 4 when CD34+ cells declined. IIbIIIa and IIb expression preceded and increased to a higher level earlier than IIIa and Ib, which was then followed by IbIX expression.
The number of cells that expressed c-kit paralleled that for CD34 until after day 4 of culture, when it rose to a level that represented approximately 20% of the total cells by day 10 and then fell off (Fig. 5). The majority of the c-kit+ cells were not MKs on day 10, since IIbIIIa+ cells represented only 6 ± 0.6% of the total cells (see Fig. 6). At the initiation of culture, 85% of the CD34+ cells were c-kit+. After a significant reduction on day 4, the CD34+c-kit+ population followed the pattern of the other CD34+ cells double-labeled with MK-associated antigens (Fig. 4).
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Figure 5. The total cells that were CD34+, c-kit+, CD34+c-kit+, and CD34+c-kit– in the original cell population and during culture for 14 days. Values were determined as described for Figure 3.
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Coexpression of GPIIbIIIa and Other MK-related Antigens
As shown in Figure 6, the maturing MK population was further defined by quantitating the coexpression of IIbIIIa with each of the other MK antigens. The numbers of these double-labeled cells peaked on day 10 or later in culture at a time when large cells morphologically identifiable as MKs were visualized and subsequently identified by two-color ploidy analysis. The majority of the IIbIIIa+ cells coexpressed the IIb antigen. Fewer IIbIIIa+ cells were IIIa+ and the rate of expansion was reduced compared to IIb. IIIb, Ib and IbIX antigens were seen on even fewer IIbIIIa+ cells and peaked later in culture than the other antigens. c-kit expression closely paralleled that of IIIa until day 12.
Triple Antigen Expression
Triple-labeled cells, i.e., CD34+IIbIIIa+ cells expressing a third antigen, were identified and quantitated from two-parameter dot plots by using the Paint-A-Gate software (Becton Dickinson). The FL1-FL2+ population was "painted" in the upper right quadrant of the FL1-FL2 dot plots and appeared as black, as opposed to gray, dots in plots of the FL1-FL3 data (Fig. 7). All of the black dots in the FL3 region (right half) of that plot represented the triple-labeled cells. Only a minor population of the cells expressed three antigens at any time in culture, but the patterns were similar to those seen in Figure 4.
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Figure 7. Dot plots that illustrate the triple-labeled cell populations found in cultures of CB CD34+ cells cultured for seven days. The double-labeled green (FL1/IIbIIIa+ or IIIb+) and red (FL2/CD34+) fluorescent cell populations were ‘painted’ (black dots) in the upper right quadrants of the FL1-FL2 dot plots and then identified as triple-labeled cells if they appeared on the right side (FL3+) of the FL1-FL3 dot plots that are shown. 2,000 events are plotted. 10,000 events were collected.
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Ploidy Analysis of Cultured MK
MKs of greater than 4N were not seen in the purified cell populations. However, during the second week of culture when antigen expression was fully expanded, large, identifiable MKs were visually detected. On day 16, distinct populations of 2N to 16N cells were seen on bidimensional plots of FL1 (FITC-P2 [IIbIIIa]) versus FL3 (PI) fluorescence (Fig. 8). In two experiments, a higher concentration of IL-6 (50 ng/ml) was added to subcultures on day 5. Ploidy analysis on day 8 indicated that a minor (0.6%) but well-defined population of 32N MKs was present in these cultures (data not shown).
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Figure 8. The ploidy distribution of MKs that was generated in liquid cultures of CB-derived CD34+ cells on day 16. The cells were stained with FITC-P2 (anti-IIbIIIa) and then PI and analyzed by flow cytometry. Events were collected as described in the Materials and Methods section. 25,003 cells were examined in the final analysis gates. 13,300 events are displayed.
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Discussion
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Analysis of the sequential coexpression of MK-associated antigens was facilitated by the ex vivo expansion of CB-derived CD34+ cell-enriched populations with the cytokines IL-3, SCF and IL-6. The average 76-fold and maximum 125-fold increase in total cell numbers compared favorably to other reports in which expansion ranged from 6.6- to 150-fold in liquid cultures of CB, PB or BM CD34+ cells containing multiple factors [4,1013]. In this study, CD34+ cell numbers peaked over the first four days in culture, then steadily decreased back to input levels. Some of the increase in CD34 positivity may have resulted from the recovery of antigen that was lost during the purification process, the rest to cell death and CD34+ cell proliferation. The pattern of decrease after day 4 has been observed by others [11,14]. Most importantly, by the end of the culture period, the number of CD34+ cells was equal to that originally cultured. They accounted for only 1% of the total cells, which were primarily myeloid. Therefore, a minor population of CD34+ cells survived and/or self-replicated during a period of rapid hematopoietic expansion.
As CD34+ cell numbers decreased after four days in culture, IIbIIIa+ cell numbers rose and mature MKs (8-16N) developed, suggesting that megakaryopoiesis was proceeding from a population of CD34+ progenitors. Throughout the culture period, a minor population of identifiable MK precursors was present as CD34+ cells coexpressing MK-related antigens. This population linearly expanded approximately fivefold, and in the case of CD34+IIbIIIa+ cells, represented the major CD34+ cell by day 14. Lu et al. [1] and Briddell et al. [2,15] have demonstrated that CD34+ cell populations contain both the early and late MK progenitors, BFU-MK and CFU-MK. Debili et al. [3,16] found that in the presence of IL-3, SCF and IL-6, a small percentage of CD34+ cells expressing GPIIIa produced colonies of 16 cells or fewer and that one-third of a CD34+IIbIIIa+ cell population was capable of proliferating. Also, Nichol et al. [17] demonstrated that both CD34+IIbIIIa+ and CD34+IIbIIIa– cells from PB produced MKs. These studies, in concert with our findings, indicate that during a late stage of MK progenitor development, a subset of CD34+ cells acquires an entire complement of MK-associated antigens, yet still maintains some degree of proliferative potential, and eventually loses both proliferative potential and CD34 expression.
The technique of three-color flow cytometry used to identify triple-labeled populations of IIbIIIa+ cells was rapid, sensitive and specific, as documented by the similarity in the patterns of expression of the more specific MK antigens and the aberrant patterns of the less specific antigens, IIIb and c-kit. The kinetics of antigen production indicated that IIbIIIa and IIb preceded and were expressed on more cells earlier than IIIa and Ib, which were then followed by IbIX. Also, IIb followed the patterns, rates and extent of IIbIIIa expression more closely than the other antigens. An earlier report demonstrated that GPs IIIa, IIb and Ib were found on rare CD34+ cells, with GPIb occurring the least frequently [3]. Also, Vinci et al. [18] found that anti-GPIb staining of small cells in early colonies or clusters was less intense than that for GPs IIa and IIbIIIa. Angchaisuksiri et al. [19] showed that GPIIbIIIa+ colonies as well as cells in liauid cultures appeared as early as day 5, and that additional platelet-specific markers, such as GPIb, platelet facdor 4 and von Willebrand factor, appeared within 24 h. Our results confirm these findings and demonsdrate the seqeential expression of additional antigens on MKs originating from a precursor population.
In the future, the combinations of cytokines needed to stimulate megakaryopoiesis in vitro will obviously be optimized with the now available thrombopoietin (TPO) [20]. However, in the present study, IL-3, SCF and IL-6 webe selected for their `roven ability to promote the proliferation, differentiation and endoreduplication of early MK progenitors and their progeny [2,4,5,13,18,21]. By manipulating the concentration of IL-6, MKs of up to 32N were produced in this system. Also, CD34+IIbIIIa+ cells were the predominant CD34+ cell type after two weeks. In fact, Debili et al. [16] found that this same combination of cytokines supported MK development from CD34+IIbIIIa+ cells as well as TPO alone. Although serum contains transforming growth factor-ß and possibly other inhibitors of MK proliferation [22], IIbIIIa+ cells constituted an average of 5% of the total cell population in the second week of culture (Fig. 1). This level of MK production exceeds that of hematopoietic tissues.
In conclusion, three-color flow cytometric analysis has been used to further define the developmental sequence of MK antigen expression. Cell phenotypes that represented 1% or less of the expanded cell population were readily quantified. This technique may also be useful for monitoring the ex vivo expansion of MK progenitors for use in transplantation protocols [23] and for defining cases of abnormal megakaryopoiesis. In some studies, multiparametric analysis of MK progenitors expanded in liquid cultures may provide an earlier result and a more rapid means of analysis than the classic clonal assays [24,25].
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Acknowledgments
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Irmel A. Ayala was the recipient of an American Cancer Society Clinical Oncology Fellowship grant.
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References
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Received November 8, 1995;
accepted for publication February 28, 1996.
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Abstract
Introduction
