Flt3 Ligand Enhances the Yield of Primitive Cells After Ex Vivo Cultivation of CD34⁺ CD38^dim Cells and CD34⁺ CD38^dim CD33^dim HLA-DR⁺ Cells

EX VIVO EXPANSION of hematopoietic precursors for transplantation is an area of intense investigation. The potential benefits of such studies include accelerated engraftment, reduced risk of infection, smaller stem cell harvests, and improved effectiveness of genetically modified stem cells.1,2 In murine models, the effects of expansion on subsequent engraftment are controversial.3-8 Human culture systems using interleukin-3 (IL-3) and c-kit ligand (KL) generally maintain at least some primitive cells.9-16 However, achieving a net expansion of early cells has proven elusive, although some laboratories have reported encouraging results.17-19 With the recent discovery of FLT3 and its ligand (FL),20-26 investigators have re-examined the possibility of expanding stem cells in vitro.

Studies of murine cells,27-29 human cord blood,16,30 fetal liver,31 and cadaveric marrow32,33 have shown that FL does indeed stimulate early hematopoietic cells. However, the response of early cells from adult donors has not been characterized as well. In addition, the extent to which late precursors respond to FL is not clear, because recent data suggest that FLT3 is present on both CD34^bright and CD34^dim cells.34 Finally, it is not clear how growth in FL might affect the number of primitive cells in culture. The response of the earliest precursors is a major concern, because stimulation by FL might promote their loss through activation, growth, and differentiation. Elegant single-cell and small-scale experiments have suggested that FL can support renewal of long-term culture-initiating cells (LTCIC) from cadaveric marrow.32,33 However, it is important to extend those investigations to cells collected from living donors.

Given that background, we initiated a study to determine how FL modulated the proliferative and maturational responses of cells collected from adult donors. This study focused on three issues. First, using four CD34⁺ subsets, we defined the populations most responsive to FL stimulation. Special emphasis was placed on determining cellular responses to combined exposure to FL and KL, about which little is known. Second, we determined the extent to which FL could enhance cell and progenitor expansion. Third, we defined the capacity of FL to modulate the number of primitive cells during ex vivo expansion. The results presented here show that FL-responsive cells are primarily found in CD34⁺ CD38^dim and CD34⁺ CD38^dim CD33^dim HLA-DR⁺ populations. Inclusion of FL stimulates the production of committed progenitors and significantly enhances the number of primitive cells as measured by immunophenotype and long-term culture assay.

This study was approved by the American Red Cross Committee for the Protection of Human Subjects. Aliquots of mobilized peripheral blood stem cells were obtained after granulocyte colony-stimulating factor mobilization with or without chemotherapy. Bone marrow cells (collected from healthy allogeneic donors or autologous donors under treatment for lymphoma or breast cancer) were retrieved from Fenwal bone marrow collection kits after filtration of the harvested product.

Bone marrow or mobilized blood cells were separated on Ficoll-Paque (Pharmacia, Piscataway, NJ) and the resulting mononuclear cells (MNC) were enriched for CD34⁺ cells using magnetic microbeads (Miltenyi Biotec, Auburn, CA). The enriched population was stored overnight at 37°C in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) with 20% fetal bovine serum (FBS; Hyclone, Logan, UT), penicillin/streptomycin, IL-3 (12.5 ng/mL), IL-6 (10 ng/mL), and KL (50 ng/mL; provided courtesy of Immunex, Seattle, WA). The next morning, cells were washed and labeled with anti-CD34-fluorescein isothiocyanate (FITC) (HPCA-2), anti-CD38-phycoerythrin (PE) (HB-7), and where specified, anti-CD33-PE (P67.6) and anti–HLA-DR peridinin chlorophyll protein (PerCP) (L243) or isotypic control antibodies (all from Becton Dickinson, Mountain View, CA). In some cases, biotinylated anti–HLA-DR/streptavidin-allophycocyanin (APC) was used (Becton Dickinson). Cell separation was performed with a Coulter ELITE ESP flow cytometer (Coulter, Miami, FL). When biotinylated anti–HLA-DR/streptavidin-APC was used in 4-color sorts, the fluorochrome was excited with a 10-mW helium neon laser (633 nm) and the fluorescent signal was processed through a gated amplifier. Propidium iodide (PI) was included as a viable stain. Viable cells (gated according to forward and right angle light scatter and PI negativity) were sorted for CD34 and CD38. The CD34⁺ CD38^dim sort gate was positioned around CD34⁺ cells with the lowest 10% to 15% CD38 fluorescence. Alternatively, CD34⁺ CD38^dim CD33^dim gated events were sent to a CD34 versus HLA-DR histogram for sorting (see Fig 4). Reanalysis of sorted cells indicated ≥90% purity (or ≥95% purity if allowance is made for photo-bleaching of the fluorochromes). After suspension culture, washed cells were labeled as described above for analysis. Alternatively, early CD34⁺ CD45RA^dim CD71^dim CD64^dim were identified with anti-CD34⁺-PerCP (HPCA-2), anti-CD45RA-FITC (L48), anti-CD71-FITC (L01.1) (all Becton Dickinson), and anti-CD64-FITC (22, Immunotec). At least 10,000 total events were collected in the light scatter gate.

Cells were cultured in IMDM with 20% FBS, penicillin/streptomycin, and 50 ng/mL recombinant KL, 12.5 ng/mL recombinant human IL-3 (rhIL-3), 10 ng/mL rhIL-6, and (where indicated) 50 ng/mL FL (all generously supplied by Immunex). Cultures were initiated with 1 to 10 × 10³ cells (dictated by sort yields) and cultured in 1 mL of medium in 24-well trays (Corning, Cambridge, MA) maintained at 37°C in a humidified atmosphere with 5% CO₂ . Fresh cytokines were added 3 times a week and media were completely changed once a week with no more than 1 to 2 × 10⁵ cells returned to the wells. Calculated yields of cells and colony-forming unit–granulocyte-macrophage (CFU-GM) were corrected for adjustments to cell numbers at the time of medium change.

CFU-GM were assessed in agarose essentially as described35-39 using rhIL-3 (12.5 ng/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF; 4 ng/mL), KL (50 ng/mL), 0.1 mmol/L hemin (Sigma, St Louis, MO), and an optimal concentration (2% vol/vol) of 5X 5637 CM. CFU-GM were also assayed in methylcellulose (Fig 1) supplemented with cytokines (Stem Cell Technologies, Vancouver, British Columbia, Canada; catalog no. 4230), as indicated. Colonies were counted after 14 days of incubation in a 5% CO₂ humidified atmosphere at 37°C.

When the number of cells available for analysis was high, limiting dilution analysis on irradiated allogeneic stroma was used (method 1). More often, when the initial number of cells available for analysis was quite small, LTCIC were quantitated in triplicate stromal cultures at 1 or 2 concentrations (method 2, described below). For any single experiment, the same quantitative method was used throughout to ensure the validity of the analysis. Primary stromal cultures were established as previously described.38,40,41 First or second passage stromal cells were irradiated (10 Gy) and seeded at 50% to 75% confluence and were used within 3 days. Cultures were fed immediately before the addition of test cells. For method I, LTCIC were quantitated on microstromal cultures in 96-well trays. Six blocks of 24 wells were recharged with varying numbers of cells. Wells were fed by weekly demi-depopulation. After 5 weeks, nonadherent cells and trypsinized adherent cells from each well were plated for CFU-GM. In the absence of added test cells, no CFU-GM were recovered from the wells. The frequency of LTCIC was determined by plotting the logarithm of the percentage of negative wells against the number of cells added per well.42,43 A curve was fitted using logistic regression and the method of maximum likelihood, with (LTCIC frequency)⁻¹ found at 37% negative wells.44 For method 2, test cells were plated at 1 or 2 concentrations in triplicate wells (24-well trays) containing stroma and maintained as described above. After 35 days, all cells were harvested as above and plated at two concentrations in triplicate in agarose. From these data, the total number of day-35 CFU-GM in the liquid culture was calculated and converted to LTCIC by multiplying by 0.25.39,43 For both methods, 95% confidence intervals are given.

Tests of significance were conducting using the Student's t-test.

Comparison of CD34⁺ CD38^dim and CD34⁺ CD38⁺ cells. The first goal of this study was to determine how early and late hematopoietic cells from adult donors responded to stimulation by FL. Towards that end, CD34⁺ CD38^dim and CD34⁺ CD38⁺ fractions were isolated as representative early and late cells. Before use, they were characterized to confirm their developmental status. When plated in agarose containing IL-3 and GM-CSF, the cloning efficiency of CD34⁺ CD38^dim cells was lower than CD34⁺ CD38⁺ cells (data not shown), the results expected for the primitive CD34⁺ CD38^dim fraction.11,32,45 Additional experiments showed that LTCIC frequencies were higher in the CD34⁺ CD38^dim fraction than in the CD34⁺ CD38⁺ fraction (7.4- ± 3.2-fold greater with blood [n = 5] and 3.7- ± 1.1-fold greater with marrow [n = 3]). These data confirmed the early and late nature of these fractions. We therefore analyzed their responses to FL in both short- and long-term culture.

When CD34⁺ CD38^dim marrow cells were cultured in methylcellulose, little growth was observed with single factors FL or GM-CSF (Fig 1). Cloning efficiency improved with multifactor combinations and, in each case, the addition of FL significantly enhanced the number of colonies in the CD34⁺ CD38^dim fraction. In contrast, FL provided no significant stimulation to the CD34⁺ CD38⁺ fraction. To better understand the effect of FL on cell proliferation, marrow fractions were grown in liquid culture (Fig 2). The addition of FL to [IL-3 + IL-6] and to [IL-3 + IL-6 + KL] was nearly 10 times more stimulatory for the CD34⁺ CD38^dim fraction than for the CD34⁺ CD38⁺ fraction (Table 1). Of the 4 cytokine combinations examined, [IL-3 + IL-6 + KL + FL] was the most supportive for CD34⁺ CD38^dim cell expansion (P < .05 in each case, see legend to Table 1). Interestingly, cultivation in [IL-3 + IL-6] confirmed the greater stimulatory requirements of the early CD34⁺ CD38^dim population, ie, CD34⁺ CD38^dim cells expanded only fourfold, whereas CD34⁺ CD38⁺ cells expanded 267-fold (Table 1).

Cultures were also examined for expansion of myeloid progenitors. Unlike CFU-GM in the CD34⁺ CD38⁺ fraction, progenitors in the CD34⁺ CD38^dim fraction did not survive cultivation in [IL-3 + IL-6] (Fig 3). CD34⁺ CD38^dim progenitors did grow when FL was added to [IL-3 + IL-6], although the response was highly varied (see legend to Fig 3). The greatest progenitor output was achieved by adding FL to [IL-3 + IL-6 + KL], a combination that significantly enhanced production of CFU-GM from CD34⁺ CD38^dim cells (P = .01) but not CD34⁺ CD38⁺ cells (P = .18).

Substantiating the FL-responsiveness of CD34⁺ CD38^dim cells, 9 additional trials showed that inclusion of FL with [IL-3 + IL-6 + KL] increased CFU-GM output 14.5- ± 5.6-fold (total CFU-GM expansion, 426- ± 154-fold). FL enhancement was significant for 7 normal marrows (all P values <.02) and 1 mobilized peripheral blood sample (P = 3 × 10⁻⁴), but not for one autologous marrow donor sample (P = .12).

FL-responsive cells within the CD34⁺ CD38^dim fraction. From the preceding data, it was not possible to discern whether all cells within the CD34⁺ CD38^dim fraction were uniformly responsive to FL. To answer that question, CD34⁺ CD38^dim cells were further subdivided according to their expression of HLA-DR antigen, an approach previously used with fetal cells.46,47 To ensure that early cells were depleted of committed progenitors, we selected for cells that were also CD33^dim. Thus, CD34⁺ CD38^dim CD33^dim cells from mobilized blood were separated into the HLA-DR⁺ subset (denoted HLA-DR⁺) and the HLA-DR^dim subset (HLA-DR^dim; Fig 4). The fractions were cultured with [IL-3 + IL-6 + KL] ± FL.

After 6 to 9 days of incubation, HLA-DR^dim cultures showed no growth with [IL-3 + IL-6 + KL] and a modest increase (10-fold) with the addition of FL (Table 2). In contrast, the HLA-DR⁺culture increased 16-fold without FL and 93-fold with it (P = .035). Furthermore, with FL, the HLA-DR⁺ subset grew more than the HLA-DR^dim subset (P = .042). CFU-GM in HLA-DR⁺ cultures expanded 43-fold without FL and 326-fold with FL (day 14, n = 5, Table 2). Thus, the CD34⁺ CD38^dim fraction from mobilized blood was immunophenotypically and functionally heterogeneous, with the CD34⁺ CD38^dim CD33^dim HLA-DR⁺ fraction showing the greatest responsiveness to FL. We next asked how FL stimulation affected the number of primitive cells in culture.

Effect of FL on early immunophenotypes. The effects of FL on the maintenance of early cells was first assessed by flow cytometric analyses. CD34⁺ CD38^dim blood cells were cultured in [IL-3 + IL-6 + KL] ± FL for 5 to 12 days. FL-supplemented cultures contained 11.5- ± 3.1-fold more CD34⁺ cells than cultures lacking FL (n = 7). The number of CD34⁺ cells averaged 196% ± 108% of input with FL and only 30% ± 16% of input without FL, differences that did not reach significance (P = .15) due to the variability of donor responsiveness. To show the persistence of primitive cells, cultures were analyzed for early CD34⁺ CD33^dim cells.48,49 In 3 trials, the number of CD34⁺ CD33^dim cells in the FL-supplemented culture was greater than the number in the FL-deprived culture (Fig 5).

A similar approach was used to analyze the FL-responsiveness of HLA-DR^dim and HLA-DR⁺ subsets. After 6 to 8 days of culture of HLA-DR⁺ blood cells, suspensions with FL contained 5.8- ± 1.1-fold more CD34⁺ cells than FL-deprived cultures (n = 4; P = .024). FL enhanced the number of early CD34⁺ CD33^dim cells 6-, 15-, and 17-fold in 3 separate trials. The analysis was extended to quantitate cells with a primitive CD34⁺ HLA-DR^dim phenotype.15,43,50,51 When HLA-DR⁺ cells were cultured with FL, the number of CD34⁺ HLA-DR^dim cells was elevated 3.5-, 5.9-, and 45-fold compared with cultures lacking FL.

Figure 6 shows a representative flow cytometric pattern obtained from HLA-DR⁺ cells cultured with or without FL. In the culture supplemented with FL (Fig 6A), one observes that a higher proportion of gated CD34⁺ cells were CD33^dim or HLA-DR^dim. Data from Fig 6 are displayed quantitatively in Fig 7. Figure 7 also shows the comparatively poor yield of early cells from the HLA-DR^dim fraction cultured with FL. In 3 trials, CD34⁺ cells in the HLA-DR^dim fraction did not respond to FL (104% ± 8% of control). In a direct comparison of cell outputs from these fractions, FL stimulated 2.4, 3.2, and 7.8 times greater production of CD34⁺ CD33^dim cells from the HLA-DR⁺ fraction than from the HLA-DR^dim fraction. Furthermore, the enhancement of CD34⁺ HLA-DR^dim levels by FL was 8.4, 8.4, and 1.2 times greater in the HLA-DR⁺ subset than in the HLA-DR^dim subset.

Thus, the addition of FL enhanced the size of early populations with CD34⁺ CD33^dim and CD34⁺ HLA-DR^dim phenotypes. Greater enhancement of early populations was obtained in cultures of CD34⁺ CD38^dim cells and HLA-DR⁺ cells than in cultures of HLA-DR^dim cells. Further support for this conclusion was obtained in a single trial analyzing cultured HLA-DR⁺ and HLA-DR^dim blood cells for their contents of early CD34⁺ CD45RA^dim CD71^dim CD64^dim cells. FL enhanced this early fraction 15.4-fold in the HLA-DR⁺ culture, but only 1.8-fold in the HLA-DR^dim culture.

Effect of FL on maintenance/expansion of LTCIC. The preceding data suggested that FL might enhance the recovery of LTCIC after stroma-free cultivation. To test that hypothesis, LTCIC levels were assessed after stroma-free cultivation in [IL-3 + IL-6 + KL] ± FL. Initially, CD34⁺ CD38^dim cells from bone marrow were examined. After 2 weeks of incubation, the number of LTCIC in the liquid cultures was close to input levels, provided that FL was present (Fig 8, trials 1 and 2). In the absence of FL, LTCIC yields were significantly smaller (P < .05). Similar results were obtained using CD34⁺ CD38^dim cells from blood (trial 3). To better understand how blood LTCIC respond to FL, 5 additional experiments were conducted using HLA-DR⁺ and HLA-DR^dim fractions (Fig 8, trials 4 through 8). A comparison of HLA-DR^dim and HLA-DR⁺ populations was of interest, because Figs 6 and 7 suggested that LTCIC in the HLA-DR⁺ fraction might be more responsive to FL than those in the HLA-DR^dim fraction. In freshly isolated HLA-DR⁺ and HLA-DR^dim subsets, LTCIC frequencies were not significantly different (3.6% ± 1.4% v 2.6% ± 0.9%, respectively; n = 5; P > .05). The HLA-DR⁺ and HLA-DR^dim fractions were cultured in [IL-3 + IL-6 + KL] ± FL for 14 days. The mean yield of LTCIC from the HLA-DR⁺ fraction was 214% ± 87% of input with FL and only 24% ± 16% without FL. Direct comparisons of LTCIC yields with and without FL were possible in 3 trials and the differences were significant in 2. Overall, Fig 8 shows that LTCIC yields were enhanced by FL in 6 of 6 experiments and that the differences were significant in 5 of the 6 studies. In contrast to these results, HLA-DR^dim LTCIC responded poorly to FL. In 3 trials, HLA-DR^dim LTCIC decreased below the level of detection within 14 days of culture (data not shown). Finally, in Fig 8, CFU-GM expansion in the presence of FL averaged 481- ± 169-fold. Thus, FL-supplemented, stroma-free cultures of CD34⁺ CD38^dim cells and HLA-DR⁺ cells can generate large numbers of committed progenitors without losing significant numbers of primitive LTCIC.

The ability to generate hematopoietic precursors ex vivo will profoundly affect future approaches to stem cell collection, transplantation, and gene therapy. In the present study, we determined how hematopoietic cell expansion was modulated by FL, a recently discovered cytokine that acts early in blood cell development. Our goal was to precisely identify hematopoietic populations responsive to FL and to determine the impact of FL on primitive cell numbers. Towards that end, four CD34⁺ subsets were isolated for growth and analysis in stroma-free culture. To ensure clinical relevance, this study focused on hematopoietic cells collected from living, adult donors.

The stimulatory effects of FL were largely confined to primitive hematopoietic subsets. FL increased the proportion of CD34⁺ CD38^dim cells (but not CD34⁺ CD38⁺ cells) that grew in methylcellulose (Fig 1). In suspension culture, the addition of FL to [IL-3 + IL-6] and to [IL-3 + IL-6 + KL] stimulated proliferation of CD34⁺ CD38^dim cells far more than proliferation of CD34⁺ CD38⁺ cells (Table 1 and Fig 2). FL also enhanced the generation of CFU-GM from CD34⁺ CD38^dim cells (Fig 3). Interestingly, the proliferative response of CD34⁺ CD38^dim cells obtained from different donors was quite variable in [IL-3 + IL-6 + FL]. Such variability was greatly reduced when KL was added to [IL-3 + IL-6 + FL] (Fig 2). Finally, in some experiments, FL stimulated the growth of CFU-GM from late CD34⁺ CD38⁺ cells (Fig 3), but on average, it was less than that obtained with early populations.

To extend the analysis of early cells, CD34⁺ CD38^dim precursors from blood were separated into CD34⁺ CD38^dim CD33^dim HLA-DR⁺ (HLA-DR⁺) and CD34⁺ CD38^dim CD33^dim HLA-DR^dim (HLA-DR^dim) populations. Although the populations were similarly enriched for LTCIC, the HLA-DR⁺ fraction was far more responsive to FL stimulation than the HLA-DR^dim fraction (Table 2). The HLA-DR^dim population also responded poorly to KL. That is, in [IL-3 + IL-6 + KL], the HLA-DR^dim fraction failed to grow (Table 2), whereas CD34⁺ CD38^dim cells (Table 1) and the HLA-DR⁺ fraction (Table 2) grew significantly.

FL enhanced the number of CD34⁺ cells in 6 of 7 cultures initiated with CD34⁺ CD38^dim blood cells and all 4 cultures initiated with HLA-DR⁺ blood cells. Cultures were also examined for the presence of early immunophenotypes. After 5 to 12 days of growth of CD34⁺ CD38^dim cells, early CD34⁺ CD33^dim cells were present and their numbers were enhanced by the addition of FL (Fig 5). When the analysis was extended to the HLA-DR⁺ subset, it was found that FL stimulated the absolute number of CD34⁺ CD33^dim cells and CD34⁺HLA-DR^dim cells (Figs 6 and 7). To validate flow cytometric data, suspension cultures of CD34⁺ CD38^dim cells and HLA-DR⁺ cells were evaluated for LTCIC after 2 weeks of incubation. Cultures supplemented with FL had significantly (P < .05) greater numbers of LTCIC in 5 of 6 trials.

In summary, both functional and immunophenotypic assays show that early cells are considerably more responsive to FL than are late cells. Instead of accelerating the loss of early cells, FL enhances the number of primitive precursors in expansion cultures. It is notable that stroma-free growth with [IL-3 + IL-6 + KL + FL] yielded a 300- to 400-fold expansion of committed progenitor cells under conditions supporting the maintenance or slight expansion of more primitive precursors. The enhanced generation of late cells (progenitors and mature cells) results from the stimulation of early HLA-DR⁺ cells, with weaker effects on CD34⁺ CD38⁺ cells.

Our finding that adult CD34⁺ CD38^dim cells are highly responsive to FL agrees with recent data obtained with human fetal liver31 and cadaveric marrow.32,33 Other investigators used stromal cultures to document the FL responsiveness of CD34⁺ CD38^dim cells.52 Our data are also consistent with a recent report that CD34⁺ lin⁻ high proliferative potential colony-forming cells (HPP-CFC) and CFU-blast respond to FL.53 To our knowledge, this is the first study to show that early blood cells are heterogeneous in their response to FL, ie, CD34⁺ CD38^dim CD33^dim subfractions differing in HLA-DR antigen density vary in their capacities to respond to FL. This report also demonstrates that late cells, which express FLT3,22,34 respond weakly to FL. Low FL-responsiveness has also been observed with CD34⁺ CD38⁺ cells in a stroma-based system52 and with late FLT3⁺ murine cells.29 In flow analysis, the proportion of cells coexpressing FLT3 and c-kit increases with the intensity of CD34 staining.34 Our results appear to support that finding, because the most primitive cell fractions responded to the simultaneous presence of FL and KL (Figs 1-3 and Table 1). In that regard, adult CD34⁺ CD38^dim cells and fetal liver CD34⁺ CD38^dim cells differ, because the latter are not further stimulated by combining FL with KL.31 When FL and KL were examined separately, ie, [IL-3 + IL-6 + KL] versus [IL-3 + IL-6 + FL], no clear distinction could be made in terms of total cell expansion. However, KL differed from FL in that KL stimulated both early and late populations (Figs 1 and 2).

The effect of FL on the number of precommitted cells in liquid culture has been examined previously, although generally with different cell sources and less highly fractionated cell populations. A role for FL in the maintenance of primitive cells was proposed based on studies of long-term cultures,23,53,54 but neither the target population nor the identity of synergizing factors nor the change in LTCIC number could be defined. In stroma-free culture, a 4.5-fold increase in cord blood LTCIC was reported, which was somewhat greater with FL than without.16 In studies of cadaveric CD34⁺ CD38^dim cells, Petzer et al32,33 reported that FL, KL, and IL-3 were key to stimulating a greater than 10-fold expansion of LTCIC. Although it is not clear why LTCIC expansion was greater in the latter studies than in the present work, methodologic differences are apparent. For example, Petzer et al32,33 used serum-free media for expansion. Furthermore, LTCIC and CFU-GM detection systems used by Petzer et al32,33 differed from those used in the present study. It is also possible that marrow harvested from cadaveric bone possesses an enhanced growth potential. For example, mechanical disruption of the bone could release cells adjacent to the endosteum, a region enriched for early precursors, including marrow repopulating cells.55-57 In other studies conducted without FL, recovery of LTCIC from nonperfused, stroma-free cultures generally decreases.12-15 Enhanced maintenance or expansion in the presence of stromal layers,14 stromal CM,15,58,59 or low-density marrow spinner cultures19 has been reported. Elegant experiments showed a fivefold expansion of LTCIC in stroma-noncontact cultures.48 

In this study, the HLA-DR⁺ population gave rise to CD34⁺ HLA-DR^dim cells, a fraction generally accepted as primitive in nature and enriched for LTCIC.15,43,51 The possibility that early cells may be either HLA-DR⁺ or HLA-DR^dim was addressed previously.60 Because the CD34⁺ HLA-DR^dim fraction and the HLA-DR⁺ fraction each constitute less than 10% of total CD34⁺ cells, the enrichment of primitive cells in both populations is not contradictory. The precise relationship between HLA-DR antigen expression and the nature and properties of primitive cells remains to be clarified. In the case of human fetal liver, HLA-DR⁺ and HLA-DR^dim subsets of the CD34⁺ CD38^dim lin^lo population contained similar numbers of HPP-CFC and had similar proliferative capacities.47 In the present study, the HLA-DR⁺ and HLA-DR^dim subsets contained similar numbers of LTCIC when freshly isolated, yet only HLA-DR⁺ LTCIC survived in stroma-free culture containing [IL-3 + IL-6 + KL + FL]. The superior proliferative potential of adult HLA-DR⁺ cells is similar to that observed in fetal marrow with CD34⁺ CD38^dim HLA-DR⁺ cells.60 Although not directly tested in this study, these results suggest that maintenance or expansion of LTCIC in the HLA-DR⁺ fraction should provide superior results to that obtained with CD34⁺ CD38^dim cells, because the latter include unresponsive HLA-DR^dim cells. The nature of the HLA-DR^dim fraction is under investigation.

In conclusion, functional and flow cytometric studies show that FL enhances the productivity of stroma-free expansion cultures. FL had its greatest stimulatory effects on a subfraction of the precommitted CD34⁺ CD38^dim population (HLA-DR⁺), which constitutes just a few percent of the total CD34⁺ cell pool. FL stimulated the HLA-DR⁺ fraction to generate increased numbers of cells, CFU-GM, and CD34⁺ cells as well as greater numbers of primitive CD34⁺ CD33^dim cells, CD34⁺ HLA-DR^dim cells, and LTCIC. The strong synergism between FL and KL suggests that these cytokines are not functionally redundant61 and that FL may make a distinct contribution to early hematopoietic development. Finally, these data show that a simple, stroma-free culture system can be used with cells from adult donors to expand progenitors several hundred fold while maintaining primitive precursors. These results may have clinical significance. Maintenance of LTCIC numbers during a time of extensive progenitor expansion raises important questions regarding the effect of FL on the dynamics of self-renewal and maturation in the primitive cell pool.