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Stem Cells, Vol. 15, No. 3, 207-213, May 1997
© 1997 AlphaMed Press

In Vitro Growth of Mobilized Peripheral Blood Progenitor Cells is Significantly Enhanced by Stem Cell Factor

Clara Cesana, Carmelo Carlo-Stella, Lina Mangoni, Ester Regazzi, Daniela Garau, Gabriella Sammarelli, Cecilia Caramatti, Camillo Almici, Vittorio Rizzoli

Department of Hematology - Bone Marrow Transplantation Center, University of Parma, Parma, Italy

Key Words. Stem cell factor • Blood cell transplantation • Long-term culture initiating cells • Hematopoietic growth factors • G-CSF • Cyclophosphamide

Dr. Carmelo Carlo-Stella, Cattedra di Ematologia, Centro Trapianti di Midollo Osseo, Università di Parma, via Gramsci 14, 43100 Parma, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self-renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC). Addition of SCF (50 ng/ml) to methylcellulose cultures stimulated with maximal concentrations of G-CSF, GM-CSF, interleukin 3 and erythropoietin significantly increased the growth (mean ± SE) of CFU-Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p <= 0.0001), BFU-E (47 ± 10 versus 32 ± 6, p <= 0.002) and CFU-GM (173 ± 31 versus 112 ± 20, p <= 0.0001). Mean (± SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. Mean (± SE) LTC-IC growth per 2 x 106 nucleated cells was 221 ± 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r = .87; p <= 0.0001) between LTC-IC and SCF-dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Hematopoietic reconstitution following autologous blood cell transplantation (BCT) is likely to rely on the self-renewal ability and multilineage differentiation potential of stem cells which were mobilized into peripheral blood, collected and reinfused after high-dose myeloablative therapy [1, 2]. The demonstration of functionally competent, "true" stem cells in mobilized peripheral blood is prevented by the lack of an adequate assay system allowing stem cell detection, and complicated by the theoretical possibility that marrow stem cells surviving conditioning regimens might reacquire functional activity and regenerate hematopoiesis following high-dose chemotherapy [3, 4]. Recently, the demonstration of the capability of mobilized progenitors to reconstitute hematopoietic function has been provided in the setting of allogeneic BCT [5, 6].

Quantification of primitive progenitors in mobilized blood represents a crucial issue for the qualitative and quantitative standardization of graft collection and manipulation. Both phenotypic and in vitro cell culture techniques are now available which allow investigation of the stem cell compartment [7-9]. Different types of progenitors can be measured directly by multiparameter phenotyping of CD34+ cells with the significant advantage that the results are essentially immediate and can therefore be used to guide clinical decisions. However, although empirically useful for some applications, correlations between progenitor cell phenotype and functional activity are not yet refined enough to become clinically applicable [9]. In addition, CD34 antigen is virtually expressed by all progenitor cells, but the percentage of CD34+ cells with a clonogenic activity assayable in vitro ranges from 10% to 50%, thus suggesting that the majority of CD34+ cells are apparently devoid of proliferative potential, at least in conventional semisolid culture systems. Evaluation of the engraftment potential of mobilized blood can be achieved by means of the long-term culture initiating cell (LTC-IC) assay which measures a primitive, self-renewing progenitor capable of initiating and sustaining hematopoiesis for weeks in culture [10, 11]. Indeed, the LTC-IC assay is time consuming, difficult to standardize and requires a high methodological expertise which prevents its use as a routine laboratory technique for reliably monitoring primitive cell content of autografts [12].

Stem cell purging studies have shown that mafosfamide treatment destroys cycling progenitors known to be responsive to several myeloid growth factors, including G-CSF, GM-CSF and interleukin 3 (IL-3), but spares a class of noncycling primitive progenitors which can be recruited into cycle by recombinant human stem cell factor (SCF) [13, 14]. SCF is the ligand of a protein tyrosine kinase receptor and has been shown to selectively control stem cell self-renewal, proliferation and differentiation by directly acting on primitive progenitors in synergy with other cytokines [15-18].

In order to quantify the content of primitive progenitors collected by leukapheresis in patients mobilized with cyclophosphamide (CY) followed by G-CSF or G-CSF alone, we investigated the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of SCF. In addition, primitive progenitors were quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients, Mobilization Regimens, Collection Criteria
Clinical characteristics of the patients (multiple myeloma [n = 7], non-Hodgkin's lymphoma [n = 7], Hodgkin's disease [n = 1]) included in this study are reported in Table 1. Patients were mobilized with either CY (4 or 7 g/m2 iv, day 0) followed by G-CSF (Filgrastim, 5 µg/kg/day s.c. from day 1 until completion of leukaphereses) (n = 8) or G-CSF alone (10 µg/kg/day s.c. until completion of leukaphereses) (n = 7). In patients mobilized with CY + G-CSF, leukaphereses were started when circulating CD34+ cells were >=20/µl, whereas patients mobilized with G-CSF alone were collected on day +5 following G-CSF administration. Leukaphereses were performed using a Cobe-Spectra (Cobe; Lakewood, CO) continuous flow cell separator and continued until at least 5 x 106 CD34+ cells/kg were collected.


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  		Table 1. Clinical characteristics of the patients at the time of the study
 
CFU-Mix, BFU-E, and CFU-GM Assay
The assay for CFU-Mix, BFU-E and CFU-GM was carried out as described elsewhere [19]. Briefly, 1 to 5 x 104 nucleated cells were plated in 35 mm Petri dishes in 1 ml aliquots of Iscove's modified Dulbecco's medium containing: 30% fetal bovine serum ([FBS], Stem Cell Technologies; Vancouver, Canada); 10–4 M 2-mercaptoethanol (GIBCO; Grand Island, NY); and 1.1% (w/v) methylcellulose. Cultures were stimulated with IL-3 (10 ng/ml, Sandoz; Basel, Switzerland), G-CSF (10 ng/ml, Amgen Inc.; Thousand Oaks, CA), GM-CSF (10 ng/ml, Sandoz) and erythropoietin ([EPO], 3 U/ml, Amgen, Inc.). SCF (Amgen, Inc.) was added at a concentration (50 ng/ml) which, in preliminary experiments, was shown to maximally stimulate colony formation. To avoid the effects of the variable stimulating activity of FBS, the same pretested batch of FBS was used throughout our study. Progenitor cell growth was evaluated after 14-18 days of incubation (37°C, 5% CO2) in a humidified atmosphere. Four dishes were set up for each individual data point per experiment. CFU-Mix defined as containing at least erythroid and granulocytic cells, BFU-E with >=500 cells and CFU-GM with >=40 cells were scored from the same dish.

LTC-IC Assay
The LTC-IC assay was performed according to Sutherland et al. [20]. Briefly, test cell (5 x 106 nucleated cells) suspension was seeded into cultures containing a feeder layer of irradiated (8,000 cGy) murine M2-10B4 cells (3 x 104/cm2, kindly provided by Dr. C. Eaves) engineered by retroviral gene transfer to produce human IL-3 and human G-CSF [21]. Test cells were resuspended in complete medium consisting of alpha-medium (GIBCO) supplemented with FBS (12.5%), horse serum (12.5%), L-glutamine (2 mM), 2-mercaptoethanol (10–4 M), inositol (0.2 mM), folic acid (20 µM) and freshly dissolved hydrocortisone (10–6 M). Cultures were fed weekly by replacement of half of the growth medium containing half of the nonadherent cells with fresh complete medium. After five weeks in culture, nonadherent cells and adherent cells harvested by trypsinization were pooled, washed and assayed together for clonogenic cells in standard methylcellulose cultures at an appropriate concentration. The total number of clonogenic cells (i.e., CFU-Mix plus BFU-E plus CFU-GM) present in five-week-old LTC provides a relative measure of the number of LTC-IC originally present in the test suspension [22]. Absolute LTC-IC values were calculated by dividing the total number of clonogenic cells by four, which is the average output of clonogenic cells per LTC-IC, according to limiting dilution analysis studies reported by others [23].

Statistical Analysis
Four plates were scored for each data point per experiment, and the results were expressed as the mean ± 1 standard error (± SE). Statistical analysis was performed with the statistical package Statview (BrainPower Inc.; Calabasas, CA) run on a Macintosh Performa 6300 personal computer (Apple Computer Inc.; Cupertino, CA). The Student's t-test for paired or unpaired data was used to test for significance of changes in the incidence of progenitor cells. Correlations were determined using the least square linear regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
In vitro growth of mobilized hematopoietic progenitors was studied in 15 patients with lymphoid malignancies undergoing a total of 32 leukaphereses. As shown in Table 2, addition of SCF (50 ng/ml) to methylcellulose cultures containing 5 x 104 nucleated cells and stimulated with maximal concentrations of a standard growth factor mixture, including G-CSF, GM-CSF, IL-3 and EPO, significantly increased the mean (± SE) growth of CFU-Mix (7.7 ± 1.7 versus 2.4 ± 0.6, p <= 0.0001), BFU-E (47 ± 10 versus 32 ± 6, p <= 0.002) and CFU-GM (173 ± 31 versus 112 ± 20, p <= 0.0001). Mean (± SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 ± 5%, 19 ± 5%, and 33 ± 4%, respectively. When progenitor cell growth was calculated on the basis of the absolute number of nucleated cells collected per leukapheresis, SCF addition resulted in a significant increase of the mean numbers of CFU-Mix (27 ± 8 x 105 versus 7 ± 2.4 x 105, p <= 0.0025), BFU-E (170 ± 52 x 105 versus 116 ± 34 x 105, p <= 0.009), and CFU-GM (563 ± 151 x 105 versus 352 ± 104 x 105, p <= 0.0003) (Table 3).


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  		Table 2. In vitro colony growth from leukapheresis stimulated with and without SCF
 

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  		Table 3. Hemopoietic progenitor yield on 32 leukaphereses
 
With the exception of patient numbers 6, 9, and 11, who had been heavily pretreated before being mobilized and showed a limited increase in progenitor cell growth upon SCF stimulation, all the remaining patients displayed a consistent, although variable, increase in colony number and size when cultures where supplemented with SCF (Tables 2 and 3). Due to a shift from erythroid to multilineage progenitors, SCF-induced percentage increase for CFU-Mix (347 ± 88%; range 0 to 2,650%) was higher than for BFU-E (39 ± 8%; range –38 to 138%) and CFU-GM (60 ± 8%; range -5 to 200%).

Figure 1 shows the mean (± SE) percentages of SCF-dependent progenitors in the first (31 ± 3%; range –3 to 49%), second (31 ± 4%; range -14 to 56%) and third (28 ± 4%; range -1 to 48%) leukapheresis. In patients undergoing multiple leukaphereses, the percentage of SCF-dependent progenitors was substantially unchanged over time, thus showing that mobilization of SCF-dependent progenitors occurs during the entire mobilization period.



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  		Figure 1. Daily percentages of SCF-dependent progenitors (CFU-Mix + BFU-E + CFU-GM). Mean (± SE) SCF-dependent percentages were 31 ± 3% on day 1 (n = 10), 31 ± 4% on day 2 (n = 10) and 28 ± 4% (n = 6) on day 3. These percentages were not significantly different.

 
In 24 out of 32 leukaphereses, the growth of the more primitive LTC-IC progenitors could be investigated. Mean (± SE) LTC-IC growth per 2 x 106 nucleated cells inoculated on day 0 over a murine stroma was 221 ± 53 (range 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r = 0.87, p <= 0.0001) between LTC-IC and SCF-dependent progenitors (CFU-Mix + BFU-E + CFU-GM) (Fig. 2).



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  		Figure 2. Correlation between LTC-IC concentration per 106 NC in individual leukapheresis and the correspondent concentration of SCF-dependent progenitors (CFU-Mix + BFU-E + CFU-GM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
As compared to marrow, reinfusion of autologous blood results in a significant acceleration of hematopoietic recovery following myeloablative therapy [24]. Due to the lack of in vitro assays allowing to quantify self-renewing hematopoietic stem cells, the long-term reconstituting potential of mobilized circulating progenitors cannot be easily evaluated and the search for methods allowing an adequate quantitative detection of primitive progenitor cells in leukapheresis is an important issue [9]. In addition, based on the increasing use of mobilized blood for both autografting and cell manipulation procedures, quantitative and qualitative graft characterization represents a clinically relevant issue.

Several studies have demonstrated that mafosfamide-purged autografts, although deprived of cycling progenitors, are capable of reconstituting hematopoietic function due to the property of mafosfamide to spare noncycling stem cells [25, 26]. The addition of SCF to a growth factor combination including G-CSF, GM-CSF, IL-3 and EPO has been found to be critical for in vitro proliferation of quiescent primitive progenitors surviving marrow purging with mafosfamide [13, 14]. Recently, primitive blood progenitors have been detected by stimulating mafosfamide- or 5-FU-resistant cell populations with SCF-based cytokine combinations [27, 28]. A number of in vitro studies suggests that SCF acts on immature stem cells in synergy with other growth factors [15-18], whereas it has no proliferative effect on committed progenitors [29-31]. Therefore, it may be postulated that primitive and committed progenitor cell populations represent distinct and largely nonoverlapping cell classes with distinct hierarchical response to different cytokines. While "intermediate-acting" and "late-acting" growth factors stimulate committed progenitor cell proliferation, in vitro growth of primitive progenitors requires the presence of "early-acting" factors, such as SCF.

In the present study, we demonstrate that SCF addition to cultures stimulated with maximal concentrations of "intermediate-acting" and "late-acting" growth factors induces a statistically significant enhancement of in vitro colony growth in the majority of leukaphereses performed in patients with hematological malignancies. These data are confirmatory of a number of previously published investigations analyzing the effects of SCF on normal hematopoiesis [15, 16, 29-31], and demonstrate that SCF is able to increase blood colony formation from patients which were pretreated with cytotoxic drugs and mobilized with different regimens.

The type of mobilizing regimen as well as pretreatment patient characteristics have been shown to be the most important factors affecting progenitor cell mobilization and collection [27]. Stem cell damaging drugs are known to affect yields and performance of blood cell autografts by damaging both marrow progenitor cell compartments and microenvironment [32, 33]. We found significant but heterogeneous amounts of SCF-dependent progenitors in individual leukapheresis carried out on consecutive days from patients mobilized either with CY + G-CSF or G-CSF alone. As suggested by some patients (numbers 6, 9, 11) who had been previously treated with repeated cycles of an alkylating agent-containing regimen and mobilized negligible amounts of both SCF-dependent progenitors and LTC-IC (for example, patient number 6 mobilized 1, 7 and 2.5 x 106 LTC-IC in three consecutive leukaphereses), the heterogeneous SCF effect is likely to be due to quantitative and qualitative differences in premobilization treatment.

The significant enhancement of CFU-Mix, BFU-E and CFU-GM which was observed after SCF addition to IL-3, G-CSF, GM-CSF and EPO is consistent with the synergistic action of SCF with several growth factors and the capability of SCF to induce progression of primitive progenitors into committed compartments [15, 29-31]. The marked SCF-dependent increase of CFU-Mix confirms previous evidence suggesting that primitive progenitors with mixed differentiation potential represent the primary hematopoietic target cells of SCF [30]. The mobilization of SCF-dependent, primitive progenitors in the majority of our patients clearly suggests that SCF supplementation of methylcellulose cultures is mandatory in order to adequately quantify the incidence of multilineage, erythroid and granulocyte-macrophage progenitors collected by leukapheresis.

The LTC-IC assay detects a class of primitive stem cells with phenotypic characteristics of transplantable murine in vivo repopulating cells [34] and self-renewal potential [11]. Both steady-state and mobilized blood have been shown to contain LTC-IC [23, 33]. In our study, a statistically significant correlation was observed between LTC-IC and SCF-dependent progenitors. Monitoring of SCF-dependent progenitors in patients undergoing multiple leukaphereses revealed that these progenitors were mobilized from the marrow into peripheral blood with a kinetic that is similar to that reported for LTC-IC [23]. As compared to LTC-IC, SCF-dependent progenitors had a higher concentration, indicating that they are not equivalent to LTC-IC and suggesting they might be considered a progeny of LTC-IC. While clarification of the ontogenetic relationship between LTC-IC and SCF-dependent progenitors will require further studies, detection of SCF-dependent progenitors by a straightforward methylcellulose assay might overcome the main drawbacks of the LTC-IC assay and might be a routine technique candidate for monitoring the primitive progenitor cell content of blood cell grafts.

Despite the fact that pluripotent stem cells surviving myeloablative therapies might be responsible for post-transplantation hematopoiesis, the absence of primitive progenitors in mobilized blood virtually makes patients ineligible for blood cell autografting procedures. This is especially true for heavily pre-treated graft recipients with significantly damaged hematopoietic progenitor cell compartments. Further studies correlating the amount of reinfused SCF-dependent progenitors with engraftment parameters are required to establish the limiting number of SCF-dependent progenitors which are necessary to achieve a successful long-term hematopoietic reconstitution following autologous BCT.

In conclusion, our data suggest that: A) the optimal quantification of progenitor cell incidence in leukapheresis requires SCF supplementation of methylcellulose cultures and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic cells might be clinically relevant for predicting the reconstituting potential of autografts.


   Acknowledgments
 
This work was supported in part by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST, 40%-60%), and Associazione Italiana per la Ricerca sul Cancro (A.I.R.C., Milano).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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accepted for publication February 18, 1997.




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