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From the Department of Medicine, the Division of Hematology and Oncology, University of Tübingen, Tübingen, Germany; U.119 INSERM, the Division of Molecular Oncology, Marseille, France; Matthew Roberts Laboratory, the Division of Haematology, Hanson Centre for Cancer Research, Adelaide, South Australia, Australia; SyStemix Inc, Palo Alto, CA; Childrens Hospital, University of Tübingen, Tübingen, Germany; The Institute of Immunology, University of Vienna, Vienna, Austria; The Molecular/Cancer Biology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; and Max-Planck-Insititute for Biochemistry, the Division of Molecular Biology, Martinsried, Germany.
The class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3. Clonogenic assays and morphological characterization of FACS-sorted BM CD34+ cells demonstrate that colony-forming unit-granulocyte-macrophage (CFU-GM) and immature myelo-monocytic precursor cells are enriched in the subpopulation staining most brightly with the FLT3 MoAb whereas the majority of the burst-forming units-erythroid (BTU-E) and small cells with lymphoid morphology are found in the FLT3- population. In contrast, statistically indistinguishable proportions of CFU-granulocyte-erythrocyte-megakaryocyte-macrophage (CFU-GEMM) and more primitive cobblestone area forming cells (CAFC) were detected in both fractions, albeit the FLT3+ fraction consistently showed more CAFC activity than the FLT3- fraction. Although in both, BM and CB the majority of CD34+CD117+ (KIT+), CD34+CD90+ (Thy-1+), and CD34+CD109+ cells coexpress FLT3, three-color phenotypic analyses are consistent with the functional findings and suggest that the most primitive cells defined as CD34+CD38-, CD34+CD71low, CD34+HLA-DR-, CD34+CD117low, CD34+CD90+, and CD34+CD109+ express low levels of cell-surface FLT3 and were therefore not enriched to a statistically significant extent with the bright versus negative sorting scheme. Thus, clear segregation of the most primitive progenitors from BM CD34+ cells was confounded by low apparent levels of FLT3 cell-surface expression on these cells, whereas myeloid progenitors unambiguously segregated with the FLT3 brightest cells and erythroid progenitors with the FLT3 dimmest. Additional phenotypic analyses using MoAbs against progenitor/stem cell markers including the mucinlike molecule MGC-24v (CD164), the receptor tyrosine kinases TIE, FMS (CD115), and KIT (CD117) further illustrate the differences in surface antigen expression profiles of BM and CB CD34+ cells. Notably, CD115 is rarely detected on CB CD34+ cells, whereas 20% to 25% of the BM CD34+FLT3+ cells are CD115+. Furthermore, 80% to 95% of the CB CD34+CD117+ but only 60% to 75% of the BM CD34+CD117+ cells coexpress FLT3. Only a negligible amount of CD34+CD19+ are detected in CB, while in BM 20% to 30% of CD34+CD19+ presumed pro/pre-B cells coexpress FLT3. In contrast, the majority of the CD34+CD164+ and CD34+TIE+ subsets in both CB and BM coexpress FLT3. Analysis of unseparated cells showed that FLT3 expression is not restricted to CD34+ subsets. About 65% to 70% of lymphocyte-gated BM CD34-FLT3+ cells are positive for the monocytic marker CD115 whereas 25% to 30% of these cells consist of CD10 expressing B-cell precursors. Finally, CD34- monocytes in BM, CB, and PB express FLT3 whereas granulocytes are FLT3-. Our data show that detectable FLT3 appears first at low levels on the surface of primitive multilineage progenitor cells and disappears during defined stages of B-cell development, but is upregulated and maintained during monocytic maturation.© 1997 by The American Society of Hematology.
THE ANTIGENIC profile of hematopoietic stem cells and/or their progeny has been extensively studied using monoclonal antibodies (MoAbs) against surface antigens selectively expressed on primitive cells. Antibodies against the heavily glycosylated transmembrane protein, CD34, are of particular interest because its expression is restricted to the stem/progenitor cell compartment.1-4 Thus, bone marrow (BM) cells that express CD34 contain multipotential cells with long-term engraftment capacity.5
In vitro functional activities within the CD34+ population are heterogeneous containing the spectrum from relatively mature colony-forming units (CFUs) to the most primitive cells detectable in vitro, cobblestone area forming cells (CAFC)/long-term culture initiating cells (LTC-IC). In the mouse, the latter activity has been highly correlated with cells contributing to both short-term and durable multilineage engraftment.6-8 To further define phenotypically the CD34+ population enriched for primitive stem cells, additional antibodies have been raised. Antibodies to several receptor tyrosine kinases (RTK) have proven to be suitable to discriminate functionally and phenotypically distinct progenitor/stem cell subsets.9-14 The class III RTK15 CD115/FMS/M-CSF receptor (CD115),9 CD117/KIT/stem cell factor receptor (CD117),10,11 and FLT3/FLK2 (FLT3),12-14 recently clustered as CD135,16 are known to play important roles in the regulation of hematopoiesis. Several studies have shown that both CD117 and FLT3 are expressed on the surface of CD34+ subsets.10-13 Additionally, CD117, but not FLT3 expression, is found on CD34-, immature erythroid cells,17,18 whereas normal and leukemic B-lymphoblastic precursor cells preferentially express FLT3.12-14 In contrast, CD115 expression is apparently restricted to cells showing monocyte-macrophage development potential.9
Recently, expression of the genes encoding the related RTK TIE/TIE-1 (TIE) and TEK/TIE-2 (TEK)19,20 has been described not only in endothelial cells but also in a subset of primitive hematopoietic cells. Moreover, TIE cell-surface expression has been preferentially detected on CD34+ cells with a primitive phenotype.21,22
CD90 (Thy-1) and CD109 belong to a group of glycophosphatidylinositol (GPI)-linked molecules and appear to be selectively expressed on the most immature subpopulation of CD34+ cells.23,24 Craig et al23 have shown that the CD34+CD90+ fraction is highly enriched in LTC-IC. Additionally, Baum et al25 have shown that these cells capable of multilineage repopulation of human fetal bones engrafted into severe combined immunodeficient (SCID) mice. Similarly, LTC-IC enrichment has also been reported in the CD34+CD109+ subset.24,26
Several reports have demonstrated that MoAbs with specificities for the recently clustered variant form of "multi-glycosylated core protein of 24 kD" (MGC-24v; CD164),27 a mucinlike molecule originally identified in a gastric carcinoma cell line,28 not only recognize solid tumors from various origins27 but also erythroid precursor cells and the majority of CD34+ BM cells.27,29-32 Further studies have shown that the highest levels of CD164 expression on CD34+ cells is found in the CD34+CD38- BM subset, suggesting that this molecule plays a crucial role in primitive progenitor cells.30,32
Murine flt3 mRNA expression has been detected in hematopoietic and nervous systems, the gonads, and the placenta.33 In addition, FLT3 cell-surface expression has been described on cycling murine progenitor cells from fetal liver and BM cells.34 Human FLT3 receptor expression has been detected on leukemic blasts,12,35 leukemic cell lines,12,35,36 and on BM cells from normal donors.12,37 Because of the low FLT3 receptor density on normal human hematopoietic cells,37 little information exists about the precise phenotype of the FLT3+ populations. In this study we analyze the coexpression of candidate stem/progenitor as well as lineage-restricted antigens on BM and cord blood (CB) CD34+FLT3± and CD34-FLT3+ cells, and describe the functional and morphological differences of FLT3+ and FLT3- BM CD34+ cells.
Cells
Selection of CD34+ Cells by MACS
Immunization and Hybridoma Production MoAb 4G8 (anti-FLT3) was raised by immunization of 4- to 8-week-old female Balb/c mice with Ba/F3 cells transfected with the complete coding sequence of the human FLT3/FLK2 cDNA.38 The mice were injected five times intraperitoneally with 107 cells in 2-week intervals, and 10 days after the last injection an intrasplenic boost of 2 × 105 cells was applied. The spleens were removed 4 days later for fusion with the SP2/0 myeloma cell line. The resulting hybridomas were grown in RPMI 1640 (GIBCO-BRL, Eggenstein, Germany) containing 10% fetal calf serum and hypoxanthine-aminopterine-thymidine (HAT; Sigma Chemicals, München, Germany). Culture supernatants were screened on the cells used for immunization, and hybridoma cells secreting antibodies selectively recognizing the transfectant cell line but not parental Ba/F3 cells were cloned twice by limiting dilution. The clone was cultured on a large scale in serum-free medium supplemented with 1% Nutridoma (Boehringer Mannheim, Mannheim, Germany), and antibody was purified from supernatant using T-Gel affinity columns (Bender & Hobein, Munich, Germany) as described by the manufacturer. The IgG1 isotype of the resulting MoAb 4G8 was determined by ELISA (Boehringer Mannheim). MoAb 4G8 was recently assigned to the CD135 (FLT3) cluster during the VIth International Workshop and Conference on Human Differentiation Antigens (HLDA) in Kobe, Japan.16Biotinylation of Anti-FLT3 MoAb SF1.340 Purified FLT3-specific antibody SF1.34012 was dialyzed against 0.1 mmol/L sodium borate buffer, pH 9.3, and concentrated to about 1 mg/mL by ultrafiltration (Ultrafree PF-filter, 10.000 NMWL; Millipore, Eschborn, Germany). Antibody concentration was estimated by Coomassie protein assay reagent (Bender & Hobein, Munich, Germany). A freshly prepared solution of 1 mg/mL -amino caproic acid N-hydroxy succinimide biotin (Biotin-X-NHS; Calbiochem, Heidelberg, Germany) was added to the antibody at a molar ratio of 30:1 and incubated for 2.5 hours at room temperature. The reaction was stopped by thorough dialysis against Hanks' balanced salt solution (HBSS). The optimal concentration of the biotinylated antibody for flow cytometric analysis was evaluated by incubating FLT3 transfected Ba/F3 cells with serial dilutions of the conjugate and staining with streptavidin-phycoerythrin (SA-PE).
Fluorescein Isothiocyanate (FITC)-Conjugation of FLT3-Specific MoAb 4G8 Purified antibody was dialyzed against 0.1 mol/L sodium carbonate, 0.1 mol/L sodium chloride, pH 9.2, and concentrated to 1 mg/mL. An FITC-celite solution (Sigma, Munich, Germany) of 100 µg/mL (10 µL) dimethylsulfoxide (DMSO) was prepared and slowly added to the antibody at a molar ratio of 40:1. After 30 minutes of incubation the reaction was stopped by pelleting the FITC-celite and conjugated antibody was separated by gelfiltration using Sephadex G-25 (Pharmacia, Freiburg, Germany). Activity, specificity, and fluorescence intensity of 4G8-FITC was estimated by comparative staining of FLT3 transfected and nontransfected Ba/F3 control cells with serial dilutions of the conjugate.Immunoprecipitation and Western Blot Analysis Proteins were extracted from Ba/F3 cells transfected with the complete coding sequence of the human FLT3 cDNA.38 Samples were solubilized by 1.5% NP40-TSE-buffer (50 mmol/L TrisHCl, 150 mmol/L NaCl, 1 mmol/L EDTA, pH 8) containing proteinase inhibitors (2 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 20 µg/mL leupeptin, 20 µg/mL aprotinin). Fifty microliters of coated goat-antimouse IgG-Sepharose (Sigma Chemicals) was preincubated for 9 hours on ice with 400 µL hybridoma culture supernatant of MoAb SF1.340, MoAb 4G8, or IgG1 control MoAb, respectively. Antibody-Sepharose complexes were washed twice with TSE and equal amounts of cleared cell lysate were added. After incubation on ice for 3 hours immunocomplexes were washed twice each with 1.5% NP40-TSE, 0.15% NP40-TSE and TSE. Bound proteins were eluted with 2× reducing Laemmli sample buffer, boiled for 3 minutes, and separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred to nitrocellulose, and the membranes were blocked with 3% nonfat milk-TBS-T (10 mmol/L TrisHCl pH 7.5, 100 mmol/L NaCl, 0.1% Tween). A rabbit polyclonal IgG raised against a peptide corresponding to the carboxy terminus of human FLT3 (STK1; Santa Cruz Biotech, IC, Ismaning, Germany) was used as primary antibody (1 µg/mL in blocking buffer), and after thorough washing with TBS-T the filters were incubated with horseradish peroxidase-conjugated secondary antibody (Amersham, Braunschweig, Germany) and detected by ECL (Amersham).Immunofluorescence Staining and Flow Cytometric Analysis Two-color staining. Unseparated or MACS-purified CD34+ BM, CB, or PB cells were labeled with HPCA-2-FITC (CD34; Becton Dickinson, Heidelberg, Germany) and SF1.340-biotin (FLT3) for 30 minutes on ice. After washing, cells were stained with streptavidin-PE (SA-PE) for 15 minutes and washed twice before FACS analysis or sorting.Cell Sorting Cell sorting was performed on a FACSVantage (Becton Dickinson) equipped with an air-cooled argon laser. FITC and PE fluorochromes were excited at 488 nm, and emission of FITC was detected through a 530-nm band pass filter, PE through a 570-nm band pass filter. Instrument alignment, and compensation of FITC versus PE signals was accomplished using a mixture of Calibrite beads (Becton Dickinson). For morphological analysis and colony assays, cells stained with anti-CD34 and anti-FLT3 were sorted into tubes containing 200 µL of phosphate-buffered saline (PBS)/40% fetal bovine serum (FBS). Sort windows were set as shown in Figs 5 and 6, respectively. For morphological analysis cells were cytocentrifuged onto glass slides and stained with May-Grünwald-Giemsa.
Colony Assays To determine the number of erythroid (BFU-E), myeloid (CFU-GM), and multipotent (CFU-GEMM) progenitors 2,000 sorted cells were plated in 35-mm tissue culture dishes (Greiner, Nürtingen, Germany) containing 1 mL of methylcellulose-based semisolid culture medium (MethoCult SFBIT H4436; Cell Systems, Remagen, Germany). Each test was performed in duplicate. After 14 days of incubation at 37°C, 5% CO2, the CFU-GM, BFU-E, and CFU-GEMM colonies were enumerated using an inverted microscope. Results are expressed as mean values ± SD from three experiments. Statistical significance was determined using the Student's t-test.CAFC Assays CAFC assays were performed as described by Breems et al42 using the murine FBMD-1 stromal cell line. Briefly, confluent stromal layers cultured in 96-well plates in long-term culture medium (MyeloCult H5100; Cell Systems, Remagen, Germany) with freshly added hydrocortisone sodium hemisuccinate (final concentration 10-6 mol/L; Sigma, Munich, Germany) were overlaid with sorted BM CD34+FLT3+, CD34+FLT3-, and unfractionated CD34+ cells, respectively. Serial dilutions of ten replicates ranging from 10 to 320 cells per well were used each. After 5 weeks of coculture at 33°C, 10% CO2 with weekly medium changes, the CAFC frequencies were estimated by limiting dilution analysis. All wells with at least one phase-dark cobblestone area localized under the stromal layer were scored. CAFC frequencies were determined by Poisson statistics and Probit analysis was performed for comparison of groups.43 The recovery was determined as follows: [(CAFC frequency of CD34+ subset × % of sorted CD34+ subset in gate): (CAFC frequency of total CD34+ cells × % of total sorted CD34+ cells)] × 100.Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis of flt-3 Expression on Sorted CB CD14+ and CD3+ Cells CB cells stained with anti-CD14-FITC and anti-CD3-PE (Becton Dickinson) were sorted into CD14+ and CD3+ fractions with a FACSVantage cell sorter (Becton Dickinson). RT-PCR analysis was performed essentially as described previously.44 Briefly, 500 CB CD14+ or CD3+ cells were sorted into 100 µL of lysis buffer (4 mol/L guanidine isothiocyanate, 25 mmol/L sodium citrate, pH 5.0, 0.5% sodium lauroyl sarconisate [wt/vol], 100 mmol/L -mercaptoethanol) containing 20 µg Escherichia coli rRNA (Boehringer, Mannheim, Germany) as carrier, and layered on top of a 100-µL 5.7 mol/L CsCl cushion in RNAse-free 0.3 mL polyallomer tubes. After centrifugation for 6 hours at 45,000 rpm in a TST 60.4 swinging bucket rotor (Du Pont, Bad Homburg, Germany) the total RNA pellet was processed first for DNAse I digestion45 and then for cDNA analysis. Total RNA was dissolved in 10 µL RT buffer (50 mmol/L Tris/HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2) containing 100 U Moloney murine leukemia virus (MMLV) reverse transcriptase (Superscript BRL/LTI; Eggenstein, Germany), 0.2 µg oligo(dT), 15 U RNasin, 10 mmol/L dithiothreitol (DTT), and 0.5 mmol/L of each dNTP and then reverse transcribed for 1 hour at 42°C. For RT-PCR analysis, cDNA from each sample was divided into two aliquots. One aliquot was amplified by an intron-spanning primer pair for the 2-microglobulin gene45 and the other for amplification of the flt-3 cDNA. flt-3 primers recognized positions 2598-2617 and 3003-2983 of the flt-3 cDNA. Each 50-µL PCR reaction contained 2 U AmpliTaq DNA polymerase (Perkin Elmer Cetus, Überlingen, Germany), 200 µmol/L of each dNTP, and 0.5 µmol/L MgCl2, 0.001% gelatin. Reactions were amplified in a DNA thermal cycler (PCR 9600 system; Perkin Elmer Cetus) for 40 cycles using predetermined optimal cycling parameters for each primer pair. The RT-PCR products were electrophoresed through a 2% SEAKEM (FMC BioProducts, BIOzym, Oldendorf, Germany) agarose gel and visualized using ethidium bromide staining.
MoAb 4G8 and SF1.340 Recognize FLT3 The specificity of MoAb SF1.340 and MoAb 4G8 against the extracellular domain of the FLT3 receptor tyrosine kinase was determined by their specific reactivity with Ba/F3 cells transfected with the complete coding sequence of the human FLT3 cDNA (4G1).38 Both MoAbs reacted strongly with the transfected cells but were negative on the parental Ba/F3 cells (not shown). In addition, immunoprecipitation of 4G1 lysates with MoAbs SF1.340 and 4G8 followed by Western blot analysis with a polyclonal anti-FLT3/STK-1 antiserum showed that both MoAbs are able to immunoprecipitate FLT3 from transfected Ba/F3 cells (Fig 1, lanes 2 and 3). Two bands of 155 kD (mature N-glycosylated form expressed on the cell surface) and 130 kD (immature high mannose glycosylated form) appeared which correspond to published molecular weights of FLT3.12 No bands appeared after blotting the sample obtained with the IgG1 control antibody (lane 1) whereas two faint bands appeared after immunoblotting of the whole lysate (lane 4). The specificity of both MoAbs was also confirmed during the VIth International Workshop and Conference on Human Leukocyte Differentiation Antigens in Kobe, Japan (November 1996) and assigned to the CD135 cluster.16
Differential Expression of FLT3 on BM, CB, and PB Cells In a first experiment Ficoll-separated BM, CB, and PB cells were double-stained with anti-CD34 and anti-FLT3 antibodies and analyzed by flow cytometry. Cells were either gated on low, intermediate, or high side scatter populations which correspond to lymphocytes, monocytes, or granulocytes, respectively (Fig 2, top row). Because Ficoll-separated PB cells are depleted of granulocytes, a 0.5:1 ratio of erythrocyte-lysed granulocytes were added before staining. Figure 2 (second row) shows that 63% of the BM FLT3+ lymphocytes (range, 45% to 71%; mean, 51%; n = 6) and 71% of the CB FLT3+ cells (range, 41% to 78%; mean, 55%) are positive for CD34. Vice versa, 63% to 82% of the BM CD34+ cells (mean, 74.2%) and 88% to 95% (mean, 92.3%) of the CB CD34+ cells coexpress FLT3. A small percentage of CD34-FLT3+ cells (2% to 3%) was also detected on lymphocyte-gated PB cells. Because FLT3 expression was detected on monocytes from all cell sources (Fig 2, third row) the small FLT3+ population on PB lymphocytes is most likely due to contaminating monocytes not excluded in the dual scatter gate defined in Fig 2 (top right). This is also substantiated by the fact that these cells were CD14+ and FLT3 expression was not found on cells with very low side scatter intensities (data not shown). In contrast to monocytes, granulocytes from all sources were negative for FLT3.
Flt3 mRNA is Expressed on CB CD14+ Monocytes but not on CD3+ T Cells Monocytes are known to express high levels of Fc receptors and suboptimal prevention of unspecific antibody binding easily results in an apparent unspecific detection of low levels of surface antigen expression. To address this question flt3 mRNA expression on purified monocytes was analyzed by RT-PCR. CB cells double-stained with anti-CD14 and anti-CD3 were separated by FACS and 500 cells of the resulting populations were used for RT-PCR analysis. Figure 3 (lane 2) shows a strong flt3 signal in the pre-B cell line NALM-1 which is known to express cell surface FLT3.12 A weaker, albeit significant, signal is seen in CD14+ monocytes (lane 3). In contrast, CD3+ T cells (lane 4) and erythroid K562 cells (lane 5) were negative for FLT3. These data are consistent with the detection of FLT3 cell-surface expression on monocytes and suggest that antibody binding to these cells was specific.
FLT3 Expression on CD34- BM and CB Cells Figure 2 shows that 1.4% of the lymphocyte-gated BM cells and 0.9% of the CB cells were CD34-FLT3+. To determine the precise phenotype of these populations, Ficoll-separated cells stained with anti-CD34, anti-FLT3, and antibodies against known cell-surface antigens were analyzed by FACS and sequentially gated on the low side scatter population and on FLT3+ cells (Fig 4, top left). Figure 4 shows that about 30% of the BM CD34-FLT3+ cells express CD10. A similar portion was also positive for CD20 (not shown), suggesting that about one third of BM CD34-FLT3+ cells consist of B-cell precursors. The majority of the CD34-FLT3+ cells (about 65%), however, expressed the monocyte-restricted antigen FMS (CD115), and about 50% of the non-B cells were also positive for CD33. In contrast, the granulocyte-specific antigen CD15 and the late-appearing monocyte antigen CD14 were found only in minor fractions (5% to 7% and 12% to 13.5%, respectively), suggesting that about two thirds of the CD34-FLT3+ cells in the lymphocyte gate consist mainly of not fully differentiated monocytic cells. Our data further show that almost none of these cells express KIT (CD117), suggesting that CD117 is concomitantly lost with CD34 during monocytic differentiation (Fig 4, bottom right). In contrast, the majority of the CD34-FLT3+ cells are CD71low and HLA-DR+.
FLT3 Expression on CD34-Selected BM Progenitor Cells In the next experiment, FLT3 expression on MACS-selected BM CD34+ cells was analyzed. The purity of the CD34+ cells was routinely 95% to 99.9% (mean, 97.5%; two purification cycles on separate MiniMACS columns were performed) and the ratio of MACS-selected versus unselected CD34+FLT3+ and CD34+FLT3- cells remained relatively unchanged. Two-color FACS analysis of anti-CD34 and anti-FLT3 stained column-purified CD34+ cells shows that the selected cells varied considerably in size (Fig 5A, top left). To estimate differential expression of FLT3 on CD34+ progenitor cells that differ in size, gates were set on small, intermediate, and large cells (Fig 5A, top left). About 40% of the small-sized CD34+ cells, which mainly consist of CD19+ pro/pre B cells (unpublished observation), expressed FLT3 at a comparably low level (Fig 5A, top right). In contrast, about 82% of the medium-sized cells (Fig 5A, middle left) and about 79% of the large-sized cells (Fig 5A, middle right) expressed FLT3 at high average levels. Thus, small BM CD34+ cells express lower FLT3 levels than the majority of the medium-sized and large cells.Clonogenic Capacity of BM CD34+FLT3+ and CD34+FLT3- Progenitor Cells The colony-forming capacity of column-purified, sorted CD34+FLT3+ and CD34+FLT3- BM cells was assayed in methylcellulose medium containing defined growth factors. After dual-scatter gating (Fig 5B, left) sort windows were set as indicated in Fig 5B (right). Results from three independent experiments performed in duplicate are summarized in Table 1. The CD34+FLT3+ subsets were highly enriched for CFU-GM but contained only a small population of BFU-E. Conversely, the BFU-E were highly enriched in the CD34+FLT3- subpopulation. In 2 of 3 experiments, the CFU-GEMM frequency was similar in both fractions, whereas in one experiment the multipotent progenitors were predominantly found in the FLT3+ fraction (Table 1). Thus, in contrast to KIT, FLT3 is only rarely found on erythroid progenitor cells.
CAFC Frequency in BM CD34+FLT3+ and CD34+FLT3- Progenitor Cell Subsets The frequency of more primitive progenitor cells was estimated using the murine stromal cell line FBMD-142 as a feeder layer. BM CD34+ cells were preselected as described for the colony assays, and defined cell numbers were FACS-sorted with the ACDU onto confluent FBMD-1 cells in 96-well plates. The CAFC frequency of sorted CD34+FLT3+, CD34+FLT3-, and CD34+ cells was determined. After 5 weeks of coculture, all wells with at least one cobblestone area were enumerated and the CAFC frequency determined by limiting dilution analysis as described.43 Table 2 shows that the highest mean CAFC frequency was found in the CD34+FLT3+ population. The estimated values derived from three independent experiments ranged from 1/40 to 1/110 CD34+FLT3+ cells compared with CD34+ control cells of 1/50 to 1/200. In contrast, the CAFC frequency in the CD34+FLT3- fractions ranged between 1/80 and 1/250. These data show that immature progenitor cells were present in both, the CD34+FLT3+ and CD34+FLT3- fractions. Furthermore, no significant difference in CAFC frequencies could be assigned to any of the fractions since by probit analysis the 90% confidence intervals from µ and were overlapping for each experimental group.43 However, in all experiments a higher frequency was determined in the CD34+FLT3+ subpopulation.
Morphological Characterization of BM CD34+FLT3+ and CD34+FLT3- Subsets MACS-selected CD34+ BM cells were stained with anti-FLT3 and anti-CD34, and FLT3+ and FLT3- populations size-fractionated by cell sorting (Fig 6, top left). Cytospin preparations of sorted cells were stained with May-Grünwald solution and microscopically examined. Morphological evaluation of 100 cells from the small-sized populations (gate R1, top left) resulted in 95% lymphocytes and 5% lymphoblastoid cells in the CD34+FLT3- (gate R4, top middle) and in 48% lymphocytes and 51% lymphoblastoid cells in the CD34+FLT3+ fraction (gate R3; top middle). Figure 6 (middle left) illustrates that the majority of CD34+FLT3+ cells are somewhat larger in size with a broader cytoplasmic seam than the CD34+FLT3- population (bottom left). Counting of cells with high forward scatter (gate R2, top left) resulted in 6% lymphocytes/lymphoblastoid cells and 94% blasts in the CD34+FLT3- (gate R4, top right) and in 11% promyelocytes and 88% blasts in the CD34+FLT3+ population. Figure 6 (middle right) shows some cells which are characterized by a promyelocyte-specific azurophile granulation (example indicated by arrow). The other cells in this gate represented agranulated myeloblasts or monoblasts. In contrast, the majority of the large-sized CD34+FLT3- cells appeared to be agranulated and contained nuclei with significant variations in shape (Fig 6, bottom right). Some of these cells were strikingly larger in size and showed a proerythroblast-like morphology (indicated by arrow).Phenotypic Characterization of BM and CB CD34+FLT3± Cells Coexpression of defined stem/progenitor cell antigens on BM and CB CD34+FLT3+ and CD34+FLT3- cells was analyzed by three-color analysis. Figures 7 and 8 illustrate contour plots of MACS-purified CD34+ cells (A1) which were gated on the CD34+ population (B1) after triple-staining with anti-CD34-PerCP, anti-FLT3-FITC, and each of the indicated PE-conjugated antibodies. About 65% of the BM CD34+ and about 85% of the CB CD34+ cells were FLT3+.
Characterization and purification of the most primitive hematopoietic progenitor cells is one of the major goals in experimental hematology. However, targeting of stem cells for transplantation or gene therapy is hampered by the very low frequency of such cells in BM, CB, or mobilized PB. Thus, additional markers are necessary to precisely define the phenotype of stem cells. Using MoAbs against the recently identified "stem cell" receptor tyrosine kinase FLT312 we FACS-separated purified BM CD34+ cells into FLT3+ and FLT3- fractions and analyzed the functional properties of the sorted subsets. Additionally, we characterized the phenotype of CD34+FLT3+, CD34+FLT3-, and CD34-FLT3+ cells in BM and CB using antibodies against defined cell-surface antigens.
Submitted November 6, 1996;
accepted February 18, 1997.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hearly marked ``advertisment'' in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract
haugnessy JA,
Doren S,
Carter C,
Berenson R,
Brown S,
Moen RC,
Greenblatt J,
Stewart FM,
Leitmann SF,
Wilson WH,
Cowan K,
Young NS,
Nienhuis AW:
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85:3048,
1995
