- Top of page
- MATERIALS AND METHODS
The transplantation of mobilized progenitor cells after high-dose chemotherapy shortens haemopoietic engraftment. CD34 cell subsets were examined in 20 consecutive mobilized progenitor cell collections obtained from patients with solid tumours that had not been previously treated. The analysis of CD34 cells was based on the expression of intracellular antigens, surface antigens including CD38, and cell size using multi-dimensional flow cytometry. We also correlated the numbers of stem cell subsets reinfused to haemopoietic recovery.
The majority of CD34+ cells expressed CD13 and CD33. A significant proportion was cytoplasmic myeloperoxidase (cMPO) positive. CD34+ MPO+ cells increased significantly in late collections. MPO expression was related to cell size. Cells expressing CD13 also increased in late collections in parallel to CFU-GM count. Small subpopulations of CD34+ CD38+ were committed to B cells, T cells and erythroid cell lineages. A small population expressing the megakaryocytic antigen had a small size and were predominantly CD38−. A minor subpopulation expressed stem cells antigens. These were significantly higher in late collections (CD34+ Thy-1+ and CD34+ CD33−). After mobilization, patients received three cycles of intensive chemotherapy followed by reinfusion of mobilized progenitors (5.45 × 106/kg CD34+ cells, range 3.4–11.88). The numbers of reinfused CD34 cells or the individual subsets did not influence recovery of leucocytes (9 d) or platelets (9 d).
In conclusion, the numbers of stem cells and their subsets differed between collections and, in unpretreated patients receiving intensive chemotherapy, there was no delayed engraftment when sufficient numbers of stem cells were reinfused. The recovery period was short and not correlated to any stem cell subsets.
The progenitor cells are phenotypically characterized by the expression of CD34 antigen and by their capacity for haemopoietic reconstitution. These cells are made up of two populations: (1) the primitive progenitors which have low rhodamine 123 uptake, high Thy-1 expression, lack the expression of maturation-linked surface antigens (Lin−) and CD38 ( Andrews et al, 1989 ; Papayannopoulou et al, 1991 ; Terstappen et al, 1991 ; Bernstein et al, 1991 ; Craig et al, 1993 ), and (2) the committed progenitors which express antigens associated with myeloid (CD13, CD33), erythroid (CD71), B lymphoid (CD19), T lymphoid (CD7) and megakaryocytic lineages (CD61 and CD41) ( Vainchenker et al, 1982 ; Kurtzberg et al, 1989 ; Loken et al, 1987a , b; Pierelliet al, 1993 ).
Despite detailed studies on the characterization of CD34+ cells using multi-colour flow cytometry, insights into haemopoietic cell development and differentiation have been hampered by the limitation of cell surface studies alone ( Knapp et al, 1994 ).
Little is known about the expression of intracellular antigens by haemopoietic stem cells, in particular those mobilized into peripheral blood. These antigens are lineage specific and are used to define the precise sequence of events during cellular differentiation ( Knapp et al, 1994 ). Their expression was first detected in the cytoplasm and later on the membrane ( Campana et al, 1987 ; Mason et al, 1987 ; Janossy et al, 1980 ; Strobl et al, 1993 ). The expression of, e.g., cytoplasmic CD3 (cCD3) in T lymphocytes, cCD22 in B lymphocytes and c-myeloperoxidase in myeloid cells have helped in establishing the differentiation features and the commitment of the early precursors in normal bone marrow and peripheral blood.
Combination analysis of cell surface and intracellular antigens have been used intensively in the diagnosis of acute leukaemia, particularly in those with aberrant expression of different antigens or with mixed lineage ( Mirro et al, 1985 ; Janossy et al, 1989 ; Knapp et al, 1994 ). This approach has led to better classification of leukaemia and helped in decisions regarding patient prognosis and therapy ( Knapp et al, 1994 ). In PBPC such analysis may provide a more direct approach and better understanding of the specificity of a particular subpopulation of committed or uncommitted CD34 cells to be reinfused. The classification of progenitor cells infused may assist in the more precise prediction of the engraftment period than indirect methods such as colony-forming assays. Traditionally, measurements are made using mononuclear cell count, CFU-GM assay and phenotypic evaluation of CD34+ cells ( To et al, 1986 ; Kessinger et al, 1988 ; Fruehauf et al, 1995 ; Haas et al, 1995b ; Millar et al, 1996 ). None of these tests is a direct measure of stem cell content. The CFU-GM are committed myelomonocytic precursor cells and stem cells are a small minority of the cell population expressing the CD34 antigen ( Gordon & Blackett, 1995). As yet the role of various CD34 subpopulations in achieving rapid haemopoietic recovery is not well documented. As suggested in a recent report, the numbers of CD34+/CD33− and CD34+/CD41+ subpopulations may help in predicting short-term repopulating capability of PBPC collections ( Dercksen et al, 1995 ). The present study analysed the CD34 subpopulations by using a wide range of monoclonal antibodies and evaluating by flow cytometry the expression of lineage-specific intracellular antigens as well as membrane antigens. We based the analysis on cell size in order to follow-up the maturation pathway of CD34+ cells. We then phenotypically characterized and compared the expression of CD34 cell subpopulations in successive collections of mobilized peripheral blood progenitor cells and obtained a more accurate assessment of haemopoietic recovery by correlation with the number of the different subpopulations of progenitor cells.
- Top of page
- MATERIALS AND METHODS
In this study we showed that mobilized peripheral progenitors form a heterogenous mixture of cells. Phenotypic analysis of CD34 subsets demonstrated the presence of several cell types belonging to different lineages.
Only a small percentage of CD34+ cells expressed the multipotent stem-cell-associated antigens Thy-1 and CD45RO. In addition, small subpopulations of lineage-negative cells (CD34+/CD38−, CD34+/CD33− and CD34+/HLA-DR−) were also identified. The majority of cells (>62%) expressed myeloid lineage-associated antigens. Small but distinct subpopulations of CD34+ cells expressed the megakaryocytic lineage-associated antigens and a limited number of cells expressed erythroid and T- and B-lymphoid antigens.
These results are consistent with the previously reported data ( Steen et al, 1994 ; Inaba et al, 1994 ; Bender et al, 1992 ; Fukuda et al, 1994 ). New subpopulations of CD34+ cells emerged in the present analysis based on CD34+ cell size, intracellular antigen expression and the acquisition of the differentiation antigen CD38 ( Terstappen et al, 1991 ). We have identified for the first time three new subpopulations of mobilized peripheral blood progenitors.
Small size CD34s+ cells (20–25%) lacked the expression of the CD38 antigen but expressed CD61 (the megakaryocytic lineage-associated antigen) suggesting an early commitment to the megakaryocytic lineage. This is in agreement with a previous report ( Buscemi et al, 1995 ) showing a strong correlation between time to platelet recovery and reinfusion of CD34+/CD38− subpopulation and a recent report demonstrating that the negative depletion of lineage-positive CD34 cells produced a significantly larger quantity of megakaryocytic progenitors ( Bertolini et al, 1997 ). Another support for this finding comes from our data on the ex-vivo expansion of megakaryocytic lineage. We obtained a substantial increase in CD61+ cells between days 12 and 14 despite extremely low numbers of colony-forming units (CFU-mega), suggesting that the majority of early megakaryocytic progenitors cannot be detected by means of colony assay (unpublished observations).
Intermediate size CD34s+ cells (20–30%) expressed the differentiation antigen CD38 as well as Thy-1 and CD45RO (the ‘stem-cell-associated’ antigens) but lacked the expression of lineage-associated antigens (CD7, CD10, CD33) and TdT, suggesting an early stage of committed haemopoietic progenitor cell differentiation. The expression of Thy-1 by CD34+ cells as previously shown is not homogenous and contains not only primitive progenitor cells but also progenitors producing colony-forming unit granulocyte macrophages ( Humeau et al, 1996 ).
Intermediate to large size CD34+ cells (5–60%) expressed intracellular myeloperoxidase (cMPO). These cells were CD33+ and CD13+. The expression of MPO increased amongst the large cells accompanied by a concordant decrease in CD34 antigen density demonstrating the commitment to myeloid-granulocytic lineage.
MPO was the most predominant intracellular antigen amongst the mobilized CD34 progenitors. This is in agreement with previous reports showing that G-CSF induces myeloid cell differentiation of stem cells ( Fukuda et al, 1994 ). Similarly, cMPO expression was reported in normal bone marrow mainly in large CD34 progenitors with low-density CD34 antigen ( Strobl et al, 1993 ). Other intracellular antigens, e.g. cCD22 and cCD3 (B- and T-lymphoid), TdT (immaturity) and cGly-A (erythroid), were also expressed but only in small subpopulations of CD34+ cells ranging in size from small to large accompanied by the expression of one or more surface lineage-associated antigens. The identification of new subpopulations at early stages of cellular differentiation or cells expressing lineage-associated molecules gives new insight into the process of progenitor cell differentiation and the generation of the cellular elements of the blood. This could further refine the process of progenitor cell transplantation. The expression of, e.g., CD61 by CD34s+/CD38− cells can be used in an experimental approach to select or increase the megakaryocytic precursors, most of which are likely to represent the progenitor cells responsible for early platelet rescue. Since we identified CD34 subsets in PBPC collections, we analysed these subpopulations in each of the leukapheresis collections (days 1, 2 and 3). There were no differences between the percentage of any of the subsets amongst the leukapheresis collections; this is in agreement with an earlier report ( Inaba et al, 1994 ). However, differences were found when the absolute numbers rather than percentages of these cells were compared. We obtained significantly higher yields of lineage-negative multipotent stem cells and more cells committed to the myeloid/granulocytic lineage in later collections (days 2 and 3) compared to day 1. We obtained median absolute numbers of 0.62 × 106/kg CD34+/CD33− cells and 1.6 × 106/kg CD34+/Thy-1+ when the leucocyte count reached 15 × 109/l (day 2) compared to 0.23 × 106/kg CD34+/CD33− and 1.26 × 106/kg CD34+/Thy-1+ when the leucocyte count was <6.5 × 109/l (day 1), P = 0.02 and P = 0.08, respectively.
Similarly, when the leucocyte count in peripheral blood reached 15 × 109/l we obtained a significant increase in CD34+/MPO+ cells (1.53 × 106/kg) in late collections compared to early collections (0.88 × 106/kg) (P = 0.02). We also observed a trend of increase in CD34+/CD13+ (6.74 × 106/kg) in late, compared to 4.87 × 106/kg in early, collections (P = 0.08). In line with the phenotypic results, we obtained a significant increase in CFU-GM colony numbers in late (29.32 × 104/kg) compared to early leukapheresis (21.82 × 104/kg).
Our results demonstrate that late leukaphereses contain sufficient numbers of primitive progenitor cells for engraftment as confirmed by CD34 subset analysis ( Haas et al, 1995a ). The presence of higher numbers of primitive haemopoietic cells in a PBPC transplant may contribute to a better quality long-term reconstitution ( To et al, 1992 ). The CFU-GM colony assay has been used to predict haemopoietic reconstitution by correlating it with numbers of mononuclear cells and CD34+ cells ( Kessinger et al, 1988 ; To et al, 1986 , 1997; Faucher et al, 1996 ). In some studies a strong correlation between CFU-GM colony count and CD34+ cells was reported ( Haas et al, 1995b ; Millar et al, 1996 ), but in others no correlation was found ( Serke et al, 1996 ). These differences may be related to the sensitivity of the assay and the phenotypic characterization of CD34 cells ( Fruehauf et al, 1995 ).
In the present study we combined the phenotypic analysis of CD34+ subsets and the clonogenic assays. The data shows that the increase in CFU-GM colony numbers in late collections paralleled the increase of those cells expressing the myeloid/granulocytic phenotype (CD34+/MPO+). No such increase was found in CFU-Mix colony count (data not shown) as compared to percentage of CD34+/Thy-1+ or CD34+/CD33− primitive progenitors. Probably the LTC-IC assay would have been the best candidate for such analysis. We believe that phenotypic determination of CD34+ subsets is reliable and facilitates a rapid quantitation of all the different subpopulations of CD34+ cells ( Dercksen et al, 1995 ). This type of analysis can be used routinely in clinical laboratories.
An important clinical issue is whether a relationship exists between CD34 cell number and the rate of haemopoietic recovery after PBPC transplantation. It is known that a correlation exists between reinfused CD34+ cell number and haemopoietic recovery with a minimal threshold of progenitor cells around 2–2.5 × 106/kg ( Haas et al, 1995b ; Millar et al, 1996 ). However, with less cells being reinfused, the time to platelet recovery was prolonged ( Millar et al, 1996 ; Ketterer et al, 1998 ). Moreover, the value of an optimal threshold is not well known, and recent data suggests a potential benefit of higher CD34+ cell doses for reinfusion ( Ketterer et al, 1998 ). In the present study the numbers of reinfused CD34+ cells (5.45 × 106/kg, range 3.4–11.88) did not influence recovery of leucocytes (9 d, range 7–10 d) or platelets (9 d, range 7–12 d). This was in contrast to the data reported by Dercksen et al (1995 ) where a correlation with neutrophil recovery was found when 6.0 × 106 CD34+ cells/kg (10 d, range 8–21 d) or <6.0 × 106/kg CD34+ cells were reinfused (11 d, range 9–8 d).
Studying individual subsets rather than the total CD34 population might shed more light on the mechanism of haemopoietic recovery. In one study a significant correlation was found between the number of CD34+/CD33− cells with neutrophil recovery ( Dercksen et al, 1995). Reinfusion of 2.79 × 106/kg CD34+/CD33− resulted in shorter recovery time (10 d, range 8–17 d) than when <2.79 × 106/kg cells were reinfused (12 d, range 10–28 d). In our patients the numbers of reinfused CD34+/CD33− cells (0.58 × 106/kg, range 0.25–4.37) did not influence leucocyte cell recovery. The time from reinfusion to recovery was 9 d (range 7–10 d). Reinfusion of cells committed to the myeloid lineage (CD34+/MPO+ or CD34+/CD13+) did not influence leucocyte recovery either.
The median megakaryocyte recovery was 9 d when 0.45 × 106/kg CD34+/CD41+ (range 0.25–0.69 × 106/kg) was reinfused. All the patients achieved platelet recovery (20 × 109/l) in less than 12 d, unlike the previous report ( Dercksen et al, 1995 ) where a longer recovery time (11 d, range 7–16 d) was achieved when 0.54 × 106/kg of CD34+/CD41+ cells/kg were reinfused. However, when <0.54 × 106/kg CD34+/CD41+ cells were reinfused, a delay in platelet recovery was seen (19 d, range 9–37 d). Only 16.1% of patients from the latter group recovered within 14 d.
The data presented in the present report did not reveal a significant correlation between the subpopulations of stem cells and haemopoietic recovery. This may be related to the small number of patients studied, the use of partially ablative chemotherapy, and the chemonaivity of the patients. These factors probably contributed to the rapid engraftment. Thus, the discrepancy between the results reported in the present study and previous reports may be related to differences in patient characteristics, such as the degree of pretreatment, different chemotherapy regimens, and different protocols for collections and transplantation. The analysis of CD34+ subsets might still provide an important tool in monitoring heavily pretreated patients undergoing stem cell transplantation, or in patients receiving ex-vivo processed or gene modified progenitor cells.