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Stem Cells, Vol. 16, No. 6, 404-412, November 1998
© 1998 AlphaMed Press

Mature Accessory Cells Influence Long-Term Growth of Human Hematopoietic Progenitors on a Murine Stromal Cell Feeder Layer

L. Mazinia, E. Wundera,b, H. Sovalata, D. Bourderontc, M. Baerenzunga, J. Bachorza, P. Hénona,b

a Institut de Recherche en Hématologie et Transfusion;
b Département d'Hématologie;
c Unité Biostatistique du Centre Hospitalier de Mulhouse, France

Key Words. CAFC assay • Progenitors • Stroma • Accessory cells

Dr. E. Wunder, Institut de Recherche en Hématologie et Transfusion, Hôpital du Hasenrain, 87 Ave. d'Altkirch, 68051 Mulhouse Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A recently described long-term culture system for early human progenitor cells was established with the murine preadipocyte stromal line FBMD-1 grown in 96-well plates; cobblestone areas formed by inoculated hematopoietic cells are determined in a limiting dilution setting after five weeks' culture. To compare the capacity of cobblestone-area-forming cell (CAFC) formation by bone marrow and leukapheresis products in this system, mononuclear cells (MNC) of both origins were cultured. As related to CD34+ cell content, CAFC yields after five weeks' culture were in the same range in bone marrow and leukapheresis stemming from patients with efficient mobilization of hematopoietic cells. In purified CD34+ cell fractions, the CAFC yield per inoculated cell number was considerably higher than in MNC; however, if the CAFC number was related to the inoculated CD34+ cell number in MNC and after purification, the yield was four to eight times decreased in purified fractions. Addition of the mature cells brought the CAFC yield back up to the numbers obtained in the unseparated MNC fraction. By contrast, slightly more advanced progenitors per CAFC were found in cultures of purified hematopoietic cells from both origins than in whole MNC. The results suggest that mature human accessory cells give noticeable support to recruitment of early progenitors on this feeder but lead to lower yield of GM progenitors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cobblestone-area-forming cell (CAFC) assay on FBMD-1 layers is a new alternative method for evaluating early progenitors. Confluent FBMD-1 feeders, although of murine origin, support growth of human progenitors efficiently; they have the advantage of growing more homogeneously than complete stromal layers, which may avoid variation in CAFC yields due to inconstant growth conditions. In this system, interleukin 3 (IL-3) and G-CSF are added to the medium so that cobblestone areas of larger size can be generated, facilitating their microscopic evaluation [1].

Long-term culture (LTC) of bone marrow (BM) cells has been used as an in vitro model of hematopoiesis to study interactions between early progenitors, stroma, and regulating factors. Dexter and colleagues first described murine marrow cultures [2, 3]. Early progenitors proliferating beneath stromal layers form cobblestone areas which release committed myeloid clonogenic progenitors into the medium for several months [4-12]. Primary BM cultures produce stromal layers [5, 7-9, 11, 12], which after irradiation are able to maintain long-term culture initiating cells (LTC-IC) being inoculated as mononuclear cell (MNC) fractions and enriched CD34+ populations from mice [4, 10, 13, 14] and humans [9, 11, 12]. Murine and human cell lines such as M2-10B4, MS5, and 14F1.1, mostly of embryonal origin, are also able to support growth of hematopoietic cells in vitro [1, 7, 15-19].

Production of advanced myeloid progenitors (CFU-GM) is used as a classical parameter for quantifying early progenitors in LTC of BM-MNC on preformed and irradiated human or murine stroma, or engineered murine feeder cell lines being transfected with human genes secreting supplementary growth factors [5-7, 16-18]. Also, MNC fractions stemming from leukapheresis products (LP) or the respective purified CD34+ cells show in vitro hematopoiesis when seeded onto these cell lines [1, 16, 18, 19].

Since early progenitors are thought to play a key role in regenerating hematopoiesis after grafting, there is considerable interest in analyzing their frequency in grafts of BM or the LP of patients after mobilization. Proliferating CAFC found after five to six weeks of culture (CAFC weeks 5-6) represent early progenitors [1, 4, 11], and can be quantified in 96-well plates by applying the limiting dilution method; in mice, they confer long-term repopulating ability. Transient CAFC (CAFC weeks 2-3) are produced by advanced progenitors corresponding to CFU in the spleen appearing 12 days after transplantation, and protect animals from acute death after irradiation damage [20, 21].

It has been noted that human feeder layers are more efficient in supporting growth of early human progenitors than mouse-derived feeders [16], implying that in a given cell sample different numbers of CAFC would be measured if seeded on different feeders. It is also conceivable that hematopoietic cells from different origins, after culture on the same feeder, would grow in different quantities, and that enriched CD34+ cell fractions, as recently used in autologous grafting in order to reduce contamination with residual tumor cells, would proliferate with different efficiency than would MNC fractions. Since FBMD-1 does not contain any accessory cells by itself, we used this system also for evaluating possible effects of mature cells by comparing CAFC formation from MNC and purified CD34+ target cells. Production of CFU-GM was determined in parallel to test whether this system would be universally applicable for determination of operationally defined early progenitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Collection
Sternal BM cells were obtained after informed consent from three donors (average age = 44 years) undergoing cardiac surgery, and collected in RPMI 1640 (Euro-Bio; Les Ulis, France) containing heparin (5,000 IU/ml, Roche; Neuilly-Seine, France). LP were obtained from six lymphoma, three multiple myeloma, and two solid tumor patients (average age = 47 years) after progenitor cell mobilization by G-CSF (Filgrastim; Amgen/Roche; Lucerne, Switzerland) following high-dose chemotherapy. The cells were collected on Ficoll (1.077 g/cm2, Life Technologies; Cergy Pontoise, France), centrifuged 30 min at 400 g, and washed twice with phosphate buffered saline (PBS) (Life Technologies) supplemented with 5 mM EDTA (Life Technologies).

Purification and Immunofluorescence Assays
Enriched CD34+ cell fractions were obtained by immunomagnetic separation using the Mini-MACS separation system (Miltenyi; Gladbach, Germany). MNC fractions from BM and LP were blocked with unspecific immunoglobulin, incubated with CD34 monoclonal antibodies (mAb) (QBEND-10), and, after one wash, with paramagnetic microspheres coupled to antimouse mAb. The marked CD34+ cells were retained in a strong magnetic field containing iron particles, and recovered as the positive fraction after removal from the magnet [22]. One hundred µl aliquots of the positive fraction and of unseparated cells (106 cells) were stained with 20 µl of anti-CD34-phycoerythrin or Ig G1-isotype control (Becton Dickinson; Le Pont de Claix, France) in 100 µl PBS for 15 min at 4°C and washed twice. CD34+ cell purity was subsequently measured with a FACStar flow cytometer (Becton Dickinson) as described [23].

Stromal Feeders
FBMD-1 cells, an untransformed preadipocyte line generated from mouse BM by S. Neben (Genetics Institute; Cambridge, MA), were grown in 25 cm2 flasks up to one-third confluency in Iscove's medium supplemented with 1% L-glutamine (Life Technologies), 1% peni-streptomycin (Sigma; St. Quentin Fallavier, France), 1 x 10–5M hydrocortisone (Roussel; Paris, France), 10% fetal calf serum (FCS) and 5% horse serum (HS), both from Life Technologies. Sera were pretested and the same batch used for all experiments. Stromal cells with fewer than 22 passages were utilized throughout. Ninety-six-well plates were precoated overnight with 1% gelatin in PBS; FBMD-1 cells were plated at a density of 1 x 103 cells per well and cultured in medium containing 12.5% FCS and 2.5% HS at 33°C, 10% O2 and 10% CO2 until confluent. Half of the medium was changed weekly.

Hematopoietic Growth Factors
G-CSF (Amgen/Roche) at 20 ng/ml and IL-3 (Genzyme; Paris, France) at 10 ng/ml were added to the culture medium at weekly half-medium change.

Long-Term Culture Assay
BM and LP-MNC fractions were seeded at 12 serial 1:1 dilutions (50,000 to 24 cells/well) into 15 wells for each dilution. The percentage of negative wells at each dilution of target cells was determined weekly under an inverted microscope. CAFC (Fig. 1Go) frequencies per inoculated cell number were calculated using Poisson statistics [1, 19, 21]. The cell dilution containing one proliferating progenitor per well on average is found in the row where 37% of wells contain no CAFC, while the others show one or more. CAFC frequencies were also calculated per CD34+ cell content in the inoculum, based on the percentage content previously determined by flow cytometry. Purified CD34+ cells from each sample were seeded likewise at twelve 1:1 dilutions (5,000-2 cells) and evaluated in the same manner. One-half of the medium was removed weekly and replaced by fresh medium containing IL-3 and G-CSF.



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  		Figure 1. Phase contrast micrographs of a week 5 LTC of human LP-MNC. Dark cells are cobblestone areas covered with the FBMD-1 stromal cells, while hematopoietic mature cells are light refringent on the surface of stromal cells and in the supernatant (Gx 400).

 
In order to further investigate the effect of accessory cells, and to exclude loss or damage of CD34+ cells during purification, original MNC fractions from LP were reconstituted after separation; the CD34– cells, which did not attach to the magnet and constitute the flow-through fraction, were collected and precisely counted. CD34+ cells from the positive fraction, corrected for 100% purity, were added. Reconstituted MNC (R-MNC) were cultured in parallel to untreated MNC, CD34+, and CD34– cell fractions alone.

Short-Term Colony Assay
Colony-forming (late) progenitors being produced during LTC were determined in two different ways: in bulk cultures in flasks containing confluent feeders the progeny of the inoculum (1 x 105 MNC or 1 x 103 CD34+ cells/25 cm2 flask) were assayed. Nonadherent cells were collected weekly during the half-medium change, washed once in RPMI, and counted; 5 x 104 cells were inoculated per Petri dish in duplicate in a standardized assay for CFU-C formation (Methocult H4431, Terry Fox Laboratories; Vancouver, Canada). After incubation for 14 days at 37°C in 5% CO2, myeloid colonies comprising more than 50 cells were counted under an inverted microscope. Total CFU-GM and CFU-GM per CAFC yields (as calculated from the limiting dilution cultures performed in parallel) were determined. Adherent layers were washed with RPMI 1640, and 5 ml of 1% trypsin (Life Technologies) were added per flask. After three min incubation at 37°C, trypsin was deactivated with 10 ml cold RPMI containing 10% FCS. Cells were then washed and counted, and 5 x 104 cells were cultured in Methocult as above.

In wells inoculated with a cell number prospectively inducing one CAFC at week 5 (dilution D), the cellular supernatants were harvested and the adherent layers trypsinized. Each week, 10 wells were washed and 20 µl of trypsin were added per well. After three min, 100 µl cold RPMI containing 10% FCS were added, and all cells from each well were transferred into one Petri dish containing Methocult for short-term culture as above.

Statistical Analysis
The yield of CAFC in the inoculum was determined in the limiting dilution assay according to the Poisson distribution as described above. Differences in the yields of CAFC in BM versus LP, in MNC versus CD34+ enriched fractions, and in CFU-GM yields of both were evaluated by the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cobblestone-Area Production During LTC

CAFC Yield and Kinetics in BM- and LP-MNC   BM samples contained 1.52 ± 0.62% CD34+ cells on average, and produced 21.5 ± 3.8 week 5 CAFC/105 MNC (n = 3, Table 1Go). 48.6 ± 8.01 CAFC/105 MNC were found in LP samples with good mobilization (LP-high, 2.8 ± 1.43% CD34+ cells, n = 5). In samples with modest mobilization (LP-low, 0.48 ± 0.24% CD34+ cells, n = 6) 27.8 ± 5.49 CAFC/105 MNC were observed. A marked difference of CAFC yield per CD34+ cell content was found between BM and LP-low (11,608 ± 819 and 7,687.6 ± 4,696 CAFC/105 CD34+ cells; p = 0.02) and between LP-high and LP-low (2,376.8 ± 1,838 and 7,687.6 ± 4,696 CAFC/105 CD34+ cells, respectively; p = 0.02), while it was similar for BM and LP-high (p = 0.45). The kinetics of CAFC formation showed differences in the initial phase: LP-CAFC presented a peak value after two weeks' culture with an average of 130 ± 10 CAFC/105 MNC (n = 6), whereas a similar peak occurred in BM at week 3 with 111.6 ± 15 CAFC/105 MNC on average (n = 3, Fig. 2AGo). These peaks disappeared rapidly in both instances. After week 4, a slow steady decrease was observed in both samples.


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  		Table 1. Week 5 CAFC yield in BM and LP
 


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  		Figure 2. CAFC formation in BM cells from donors 1-3 and LP cells from mobilized patients 1-3 and 6-8. Extrapolated CAFC numbers per 1 x 105 MNC (A) and per 1 x 105 initial cells of the CD34+ fraction (B) are indicated; bars indicate one standard deviation. CAFC yield in the same BM- and LP-MNC cultures was also calculated per CD34+ cells contained in the MNC fraction and compared with equivalent quantities of enriched CD34+ cells seeded alone (C and D).

 

CAFC Yield and Kinetics in Enriched CD34+ cells from BM and LP   Whereas purity of CD34+ fractions was in general high in LP samples (average = 87.29 ± 11.28%, n = 11), it varied in BM samples between 62% and 94% (average = 73.77 ± 17.6%, n = 3, Table 1Go). For appropriate comparison of CAFC yields in both positive cell fractions, they were corrected for the content of CD34+ cells. Enriched BM CD34+ cells produced an average of 362.7 ± 72 CAFC/105 CD34+ cells, while enriched LP-high and LP-low produced 384.8 ± 94.2 (n = 5) and 412 ± 216 CAFC/105 CD34+ cells (n = 6), respectively, after five weeks' culture (Table 1Go). Three weeks after the start of LP and BM culture, CAFC numbers decreased in both with a shallow slope and were of similar order throughout (Fig. 2BGo). CAFC frequency per inoculated cells in both materials remained higher in enriched CD34+ cell fractions than in MNC for the entire culture.

Comparison of CAFC Frequency Calculated per CD34+ Cell Number in Unfractionated and Enriched Cells   If the yield of week 5 CAFC is calculated per inoculated CD34+ cells, a significant difference between MNC and CD34+ cell fractions was observed in BM (1,608 ± 819 versus 362.7 ± 72 CAFC/105 CD34+ cells, p = 0.04), in LP-high (2,376.8 ± 1,838 versus 384.8 ± 94.2 CAFC/105 CD34+ cells, p = 0.009), and in LP-low (7,687.6 ± 4,696 versus 412 ± 216 CAFC/105 CD34+ cells, p = 0.004) (Table 1Go). If "accessory" mature cells are eliminated, the enriched CD34+ cells produced about one log fewer CAFC/CD34+ cells throughout in both BM and LP than in the native MNC fractions (Figs. 2C and 2DGo). In addition, in LP-low week 5 CAFC yield per CD34+ cells was in average up to three times higher than in LP-high (p = 0.03); this yield was similar in LP-high and BM MNC (p = 0.45) and significantly different between LP-low and BM MNC (p = 0.02).

CAFC Frequencies in R-MNC   In order to test whether the observed differences between total MNC and enriched CD34+ cells in culture are due to the quantity of accessory cells present or to a damage or loss of subsets of CD34+ cells during purification, additional experiments were performed in four patients. Positive and negative fractions from each LP sample were tested separately and after reconstitution of the original cell population. Results were compared with those obtained in the respective unseparated MNC population. CAFC kinetics for all fractions, as calculated per inoculated CD34+ cells, are shown in Figure 3Go. In separated CD34+ fractions, CAFC yields per CD34+ cell were clearly lower than in unseparated MNC fractions during the whole culture period. After reconstitution, similar CAFC yields were obtained as prior to separation, including the peak value at week 2. This suggests that the purification procedure had no negative effect on functioning of both hematopoietic and accessory cells, and that accessory cells in fact support CAFC formation.



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  		Figure 3. CAFC formation in MNC, CD34+ enriched and depleted CD34– LP cell fractions, and in the R-MNC. R-MNC was prepared by mixing purified CD34+ cells and the CD34– fraction in proportions matching the initial MNC samples (patients 1, 2, 7, 8). Results are the mean of four experiments; bars indicate one standard deviation.

 
CFU-GM Progenitor Production During LTC
CFU-GM production of the initial cell sample was taken as reference value for the cells harvested every week (100%).

Nonadherent Cell Fraction   MNC-LTC
In flasks seeded with BM-MNC, CFU-GM production remained stable over the 12 weeks of culture on the level of the input value (Fig. 4AGo). In LP-MNC, CFU-GM increased during the first three weeks of culture. One week later, this production decreased below the input value, and at week 8 equalized production in BM-MNC cultures; for the rest of the culture, CFU-GM production decreased slowly. At week 5, CFU-GM per CAFC in BM- and LP-MNC were not significantly different (Table 2Go).



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  		Figure 4. CFU-GM production in the adherent and non-adherent fractions of BM and LP cells after seeding 1 x 105 MNC or 1 x 103 enriched CD34+ cells per flask. Nonadherent cells from MNC-LTC (A) and CD34+ cell-LTC (B) performed in flasks were collected every week and seeded in Methocult for day 14 CFU-GM colonies. The adherent cells of these flasks being inoculated with MNC (C) and CD34+ cells (D) from both origins were washed, trypsinized, and stimulated for growth of short-term colonies. CFU-GM yields were calculated per CFU-GM numbers in the inoculum, relative to 1 x 103 CD34+ cells contained in MNC and enriched CD34+ fractions (= 1 arbitrary unit). BM donors 1-3 and LP donors 1-3 and 6-8 were tested.

 

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  		Table 2. CFU-GM/CAFC production at week 5 in LTC
 
CD34+ Cell-LTC
Enriched CD34+ cells from BM released more clonogenic progenitors into the culture supernatant during the first three weeks than MNC (Fig. 4BGo) and the numbers of CFU-GM increased progressively up to 25-fold the input value at week 6 in BM. In LP-LTC an enhanced production was observed in the first two weeks with 12-fold the initial value. During the following culture period, CFU-GM production was similar in enriched CD34+ cells from both origins and remained higher than the input values. At week 5, CFU-GM/CAFC for BM and LP were not significantly different (Table 2Go). The average yield was higher in purified CD34+ than in the respective MNC fractions in both materials, although significance could not be obtained (p = 0.25 in BM, and p = 0.91 in LP).

Adherent Cell Fraction   In a control experiment with fresh cells exposed to trypsin, it turned out that this treatment had no effect on CFU-GM yield.

MNC-LTC
In the adherent layer of both BM and LP-MNC, 10- to 20-fold more CFU-GM colonies were produced than in their culture supernatant. In BM cultures, CFU-GM production increased progressively until week 4 (Fig. 4CGo), and after five weeks of culture, it was 30-fold the initial value. During LP-LTC, up to an average of 60-fold input value was observed in the first two weeks. At week 5, CFU-GM per CAFC in BM and LP were not significantly different (Table 2Go). For the rest of culture, CFU-GM production was stable in BM, while in LP it decreased slowly after eight weeks but remained higher than the input value.

CD34+ Cell-LTC
During the entire culture period, the adherent fractions of BM and LP-LTC seeded with enriched CD34+ cells produced better than one log more CFU-GM than the respective MNC fractions (Fig. 4DGo). Clonogenic progenitor production increased rapidly in the two first weeks of LP and BM cultures to over 100-fold the initial yield; at week 5, BM reached a 300-fold increase, while a 200-fold increase was observed in LP culture. Both rates were maintained for the rest of culture. CFU-GM per CAFC produced at week 5 in BM and LP were not significantly different (Table 2Go).

All CFU-GM numbers in LTC of early progenitors from BM and LP CD34+ cell fractions were found amazingly similar. Production of clonogenic progenitors in both materials, related to the number of CAFC, was 13-20 times higher in the adherent than in the nonadherent fraction in both MNC and CD34+ LTC. CAFC frequencies, in contrast, always showed higher growth efficiency in MNC than in enriched CD34+ cell fractions if related to underlying CD34+ cell numbers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While the total number of hematopoietic cells can be counted readily by cytofluorimetry due to their common surface marker, CD34, the ability of early progenitors to produce progeny over long time spans can only be determined by long-term culture on feeder cells. In our study, it turns out that in this culture system hematopoietic cells in the MNC of BM and LP after good mobilization produce week 5 CAFC at similar efficiency. This is remarkable since other features show differences. First, CAFC numbers early after start of the culture show differences between both materials. While BM cells produce stromal layers in culture, LP-MNC are unable to do so. Also, grafts with LP cells in vivo lead to faster regeneration of hematopoiesis; this was ascribed on one side to a higher number of total hematopoietic cells transplanted and on the other to a higher content in advanced progenitors [24-26]. Our results indicate a similar proportion between early progenitors and total CD34+ cells in both materials. These early progenitors are thought to be responsible for the long-term recovery of hematopoiesis in the body [19] as is demonstrated in mice [27]. The observation of patients who mobilized lower numbers of CD34+ cells but nevertheless produced fair amounts of week 5 CAFC is of interest for graft quality evaluation, and more extended investigations may be appropriate to consolidate this finding.

The onset of CAFC over time is slightly different in both materials: early appearing CAFC reach a peak value after two weeks in LP and after three weeks in BM. They represent advanced progenitors which are known to be less resistant to 5-fluorouracil [1], and in mice enact short-term recovery from acute irradiation damage [20, 21]; their faster production of CAFC in LP may relate to the shorter aplasia seen after grafting. Longer maintained CAFC, which correspond to early progenitors [1, 6, 11, 16], show a steady slow decrease in both materials.

In enriched CD34+ fractions from BM and LP, week 5 CAFC yields were significantly lower when calculated per inoculated CD34+ cells. Evidently, this effect is due to the absence of additional stimulation by accessory cells as demonstrated; up to one log more CAFC is formed by CD34+ cells in the presence of CD34– cells, as anticipated [28]. Also, no initial CAFC peak is found after two to three weeks for both materials; so obviously, the development of advanced progenitors is impeded. In another study applying the same enrichment procedure, likewise they appeared not to be damaged by purification [29].

It was claimed recently that the CAFC generated by MNC are not exclusively committed to the myeloid line but may also contain some lymphoid precursors [30]. Short-term culture of cells being formed in CAFC+ wells after inoculation with cells at the limit dilution D always generated myeloid colonies after stimulation; selected growth of a subset of progenitors with different commitment in the absence of accessory cells can therefore be excluded. The results suggested that advanced as well as early progenitors develop in suboptimal conditions if CD34+ cells deprived of accessory cells are cultivated in this culture system.

Our previous investigations showed that accessory cells have a bystander effect in terminal differentiation of advanced progenitors: BM and LP CD34+ cells produced more myeloid cell colonies after stimulation in the presence of the CD34– cell fraction than in their absence [31]. Also, the modulating effect of IL-3 on myeloid colony formation from unfractionated MNC and semi-purified CD34+ cells from BM was ascribed to accessory cell interactions [32]. Similarly, LTC-IC numbers from human cord blood CD34+ Lin– cells decreased with increasing target cell purity [33].

Using CFU-GM production after five weeks as a parameter, Eaves' group found one LTC-IC per 2 x 104 MNC in human BM on average [5]; this corresponds (by extrapolating on the basis of an average of 1%-2% CD34+ cell content in the MNC fraction), to one LTC-IC per 100-200 CD34+ cells. In enriched CD34+ fractions, one LTC-IC per 50-100 CD34+ cells was found, implying that clonogenic progenitor production on a preformed and irradiated human BM stroma showed few differences in the presence or absence of added mature cells. However, CFU-GM generation in our MNC-LTC series was higher than in Eaves' system.

In BM and LP cells cultivated in the FBMD-1 system, more clonogenic progenitors were found per CAFC when accessory cells were absent, in both the adherent and nonadherent fractions. This may have two reasons—either the committed progenitors differentiated more rapidly to mature cells, or a subset of early progenitors might be recruited for forming cobblestone areas that produce higher numbers of committed progenitors. An eventual influence of trypsinization on these results could be excluded, which confirms previous observations [34].

Human stroma contains monocytes and adipocytes besides endothelial cells, and monocytes in particular, are known to produce cytokines. Evidently, complete stroma provides perfect conditions for development of early progenitors, obviating the need to add growth factors to the medium. FBMD-1 feeders show better efficiency of CAFC growth in the presence of mature cells, even though growth factors are added. After about the eighth week, some degradation is seen if MNC are cultivated, but not in cultures of purified CD34+ cells; thus, accessory cells and more specifically, monocytes, appear to be destructive for this feeder in the long run.

In conclusion, our study shows that human accessory cells play a supporting role in CAFC formation from early human hematopoietic progenitors on the murine feeder FBMD-1 and influence the clonogenic progenitor cell yield in this system. In view of the ablative treatment with cytostatics in high doses and ionizing irradiation on the body prior to grafting, which may also compromise functions of the marrow microenvironment and accessory cells, it is conceivable that enriched CD34+ cells after grafting may face suboptimal conditions in vivo; this would especially concern grafts of very highly purified CD34+ cells, and attentive observation and profound analysis of such clinical situations may be indicated. As a practical consequence for applying the CAFC assay on murine FBMD-1 feeder for testing graft material of different origin, it should be retained that in MNC from BM and LP grafts after good mobilization, week 5 CAFC yields may be compared directly. Also purified CD34+ fractions from both origins give equivalent yields, but no direct comparison should be made between MNC and purified CD34+ cell fractions, since the assay underestimates the latter due to the lack of bystander cells.


   Acknowledgments
 
We thank Mrs. H. Levandowsky, V. Chabouté, and P. Fuchs for help with cytofluorometric evaluation, Dr. R.E. Ploemacher for contributing FBMD-1 stromal cells and samples of pretested horse serum, Dr. E. de Wynter for discussion, and Drs. Jung and Brillard for statistical help.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication October 5, 1998.



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