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Keywords:

  • AC133+ cells ;
  • flow cytometry;
  • in vitro expansion ;
  • cord blood

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte–macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62·3%, and median AC133+ population purity was 97·9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

Expression of the CD34 cell surface antigen has allowed the identification of early haemopoietic progenitor cells in bone marrow, peripheral blood and umbilical cord blood (UCB). CD34 expression has been widely used for the selection of primitive haemopoietic progenitor cells by means of sophisticated strategies. The availability of these techniques has also fostered research on the ex vivo expansion of CD34+ haemopoietic progenitor cells, a procedure that could find clinical application in the setting of transplantation.

Information obtained from these studies has been essential to the design of stem cell expansion procedures for clinical use (Henschler et al, 1994; Bertolini et al, 1995 ; Brugger et al, 1995 ) , experimental programmes involving gene transfer ( Bertolini et al, 1996; Corrias et al, 1998 ) and purging protocols aimed at reducing tumour cell contamination in reinfused autologous leukapheresis ( Handgretinger et al, 1997 ; Paulus et al, 1997 ).

The CD34 surface antigen is expressed on a phenotypically and functionally heterogeneous cell fraction that contains early and more mature haemopoietic progenitors. In the last decade, several studies have been carried out to define a subset more enriched for early progenitor cells compared with the total CD34+ population ( Peault et al, 1993; Mayani & Lansdorp, 1994 ; Humeau et al, 1996 ; Rappold et al, 1997 ; Gotze et al, 1998 ) . Recently a novel stem/progenitor glycoprotein antigen, AC133, has been identified and cloned ( Miraglia et al, 1997 ).

The function of AC133 is so far unknown. The expression of AC133 has been evaluated within the CD34bright haemopoietic stem and progenitor cells derived from human fetal liver, bone marrow and blood ( Yin et al, 1997 ), in normal donors undergoing granulocyte colony-stimulating factor (G-CSF) mobilization ( Durett et al, 1998 ), in chronic myeloid leukaemia ( Hömer et al, 1998 ), in acute myeloid ( Horn et al, 1998 ) and lymphoblastic leukaemia ( Snell et al, 1998 ) and in myelodysplastic syndromes ( Snell et al, 1998 ).

Because of the important role of new immunophenotypic markers in the characterization of stem/progenitor cells and the preliminary evidence that AC133 antibody could constitute an alternative approach to CD34 for the selection of cells capable of both short- and long-term engraftment, we have addressed in this study the characterization of AC133+ isolated from UCB cells and compared the functional features of these latter cells with those of CD34+ cells purified from the same source.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Collection and isolation of mononuclear cells UCB samples were collected in preservative-free sodium heparin during normal full-term deliveries. Informed consent was deemed unnecessary, as the UCB samples were obtained from placentas to be discarded after delivery. UCB samples were immediately diluted 1:4 in phosphate-buffered saline (PBS), and low-density mononuclear cells (MNCs) were isolated on Ficoll density gradients (Lymphoprep; Nicomed Pharma, Oslo, Norway). MNCs were next washed twice in PBS containing 0·5% bovine serum albumin (Fraction V, Sigma-Aldrich, Germany) and 5 mmol/l phosphate buffer ethylendiaminetetraacetate (PBE). An aliquot of UCB MNCs was collected from the suspensions to determine the initial percentage and absolute number of AC133+ cells.

Magnetic separation of AC133+ cells MNCs were resuspended in a final volume of 300 μl of buffer per 108 total cells, incubated with human IgG (blocking reagent) and, subsequently, with colloidal superparamagnetic microbeads conjugated to mouse anti-human AC133 monoclonal antibody (mAb) (MiniMacs; Miltenyi Biotec, Germany) for 30 min at 6–12°C. Cells were washed, applied to a prefilled separation column, placed in a magnetic field and allowed to pass through it. AC133+ retained cells were eluted by removing the magnetic field and flushing the column with PBE. To increase the purification rate, this fraction containing AC133+ cells was loaded onto a second freshly prepared column, and AC133+ cells were isolated as described above. A sample of the AC133+ purified cell fraction was processed for the determination of viability, cell number, morphology and immunophenotype.

Magnetic separation of CD34+-positive cells MNCs, resuspended at a concentration up to 108 cells/300 μl, were incubated with human IgG and subsequently with colloidal superparamagnetic beads coated with mouse anti-human CD34 antibody (MiniMacs) for 15 min at + 4°C. Cells were then washed and CD34+ cells isolated through a separation column as above. Magnetic separation was repeated once to increase the purification rate.

Flow cytometric analyses Immunophenotypic markers of MNCs were estimated by flow cytometry, acquiring 10 000 events for each sample, with a FACS analyser (FACScalibur; Becton Dickinson, Mountain View, CA, USA). Samples were labelled with either fluorescein isiothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAbs. Briefly, cells were incubated for 20 min at 4°C with the following mAbs: CD34-FITC (anti-HPCA-2); CD3-PE; CD19-PE; CD33-PE; CD38-PE; CD61-FITC; CD71-FITC; HLA-DR-PE (all from Becton Dickinson); AC133-1PE or AC133-2PE (Miltenyi Biotec). AC133-1PE and AC133-2PE recognize two different epitopes of the AC133 molecule. In our hands, the two antibodies differed slightly in fluorescence intensity. Most of the experiments reported below have been carried out using the AC133-1PE mAb.

Controls for flow cytometric analyses were untreated cells and cells treated with irrelevant isotype-matched FITC- or PE-conjugated mAbs.

Haemopoietic growth factors The human recombinant haemopoietic growth factors (HGFs) tested were erythropoietin (EPO), interleukin-6 (IL-6), interleukin-3 (IL-3), granulocyte–monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Flt-3 ligand (FL) and stem cell factor (SCF). All cytokines were purchased from Genzyme (Cambridge, MA, USA), with the exception of SCF, which was kindly provided by Amgen (Thousand Oaks, CA, USA). HGFs were used at the concentrations that induced the optimal responses in semi-solid and liquid cultures.

CFU-Bl progenitor assay The blast cell colony-forming unit assay was performed according to the method reported by Leary & Ogawa (1987). Briefly, 5–10 × 103 freshly purified AC133+ cells, CD34+ cells or 7-day cultured cells derived from AC133+ or CD34+ cells, respectively, were seeded in methylcellulose. IL-3 (10 ng/ml) and SCF (100 ng/ml) were added on day 14. Colonies were scored on day 28 of primary culture: single, well-isolated blast cell colonies containing 25 or more cells were picked up in a volume not greater than 100 μl of Iscove's modified Dulbecco's medium (IMDM) and dispersed into a single-cell suspension by gentle pipetting. The ability of individual cells from each blast colony to form secondary colonies was assayed subsequently by plating cell suspensions in 0·6% agar medium enriched with pooled human AB serum, 100 μmol/l 2-mercaptoethanol, 2 mmol/l glutamine and antibiotics (100 U of penicillin and 100 μg of streptomycin). These secondary cultures were stimulated with 20 ng/ml GM-CSF, 20 U/ml G-CSF, 20 ng/ml IL-3, 50 ng/ml SCF and 3 U EPO, incubated for 14 days and scored for colonies (> 20 cells by definition) with an inverted microscope. The ability of a single 28-day colony to give origin to secondary colonies was calculated as a percentage from the number of colonies seeded and the number of colonies formed.

Clonogenic assays for committed haemopoietic progenitors The assay for the growth of the CFU-E, BFU-E (colony- and burst-forming units erythroid respectively) and CFU-GM (granulocyte–macrophage) colonies was performed as described in detail elsewhere ( Pasino et al, 1998 ). Briefly, 5 × 103/ml freshly isolated AC133+ or CD34+ cells, as well as AC133+ or CD34+ cells that had been expanded in liquid culture, were tested in triplicate in semi-solid cultures containing 0·9% methylcellulose (CFU-E and BFU-E) or 0·6% agar (CFU-GM). Colony growth was stimulated with GM-CSF (100 ng/ml) for myeloid progenitors and EPO (2 U/ml) for erythroid colonies. Cultures were incubated for 7 days (CFU-E, early CFU-GM) and 14 days (BFU-E, late CFU-GM) under humidified conditions in a 5% CO2 atmosphere at 37°C. The absolute number of CFU-GM, CFU-E and BFU-E was calculated by multiplying the numbers of colonies (per cells seeded) by the number of living nucleated cells present after expansion in liquid culture.

Haemopoietic progenitor liquid cultures AC133+ and CD34+ cell cultures were performed in IMDM supplemented with 30% pooled human AB serum in the presence of 100 ng/ml SCF + 50 ng/ml IL-6 (HGF2); 100 ng/ml SCF + 50 ng/ml IL-6 + 50 ng/ml FL (HGF3); 50 ng/ml SCF + 10 ng/ml IL-6 + 40 ng/ml IL-3 + 20 ng/ml GM-CSF + 20 U/ml G-CSF + 3 U EPO (HGF6). Twenty-five thousands cells were cultured in 24-well plates in 1 ml of medium. Culture medium and HGFs were never replaced during the 7-day incubation at 37°C. After this period, cells were counted, immunophenotyped and the number of colony-forming cells (CFCs) evaluated as described previously. Morphology of cells was evaluated on cytocentrifuged preparations stained by May–Grünwald Giemsa. At least 100 cells were examined at 800× magnification under oil immersion.

Statistical evaluation The Wilcoxon test for pair differences was performed to estimate the significance of difference in magnitude between the initial value of cell input and the final value of HGF-generated total cells, CD34+ cells and AC133+ cells respectively.

Student's t-test for paired data was used for statistical analyses of HGF-dependent changes in CFU-GM in pre-expansion and post-expansion cultures. Student's t-test for unpaired data was performed to estimate the significance of difference between the input and the post-expansion CFU-Bls obtained from purified progenitor cell pools. P-values lower than 0·05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Flow cytometric analysis of AC133+ and CD34+ UCB cells

A proportion of CD34bright cells isolated from UCB expressed AC133 ( Fig 1). The percentage of AC133+ cells observed in MNCs from 16 UCB samples is reported in Table I. One colour fluorescence analysis of UCB MNC showed that AC133+ cells ranged from 0·3% to 2·4%, with a median value of 0·71%. The co-expression of CD34 and AC133 was assessed using two-colour immunofluorescence. Within the CD34+ purified cell population, a high percentage (78·7% median value, range 56·3–88·1%) of cells expressed both CD34 and AC133 antigens, whereas 21·3% (median value, range 11·8–43·7%) of cells expressed the CD34, but not the AC133, antigen. All cells that stained for AC133 also stained for CD34.

image

Figure 1. Flow cytometric analysis of MNCs from a UCB sample stained with CD34 FITC- and AC133 PE-conjugated monoclonal antibodies, showing (A) AC133+ population (R1 gate) and (B) the bright expression of AC133 within the CD34+ population (R2 gate).

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Purified AC133+ cells were also stained for CD3, CD19, CD33, CD38, CD61, CD71 and HLA-DR. Most AC133+ cells were CD33+ (> 85%), CD38+ (> 95%) and HLA-DR+ (> 91%). Extremely low percentages of AC133+ cells expressing CD3 (T lymphocytes) or CD19 (B lymphocytes) were detected (1–2% and 1% respectively). CD38 cells ranged from 0·2% to 4·5% and CD33 from 1·7% to 14·2%. HLA-DR cells were within the range 3·9–8·4% ( Fig 2). Small percentages (1–2%) of CD61 (platelets and megakaryocytes including very early forms)- and CD71 (transferrin receptor)-positive cells were present.

image

Figure 2. Flow cytometric analysis of purified AC133+ cells showing CD33 and HLA-DR expression.

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Isolation of AC133+ and CD34+ cells from UCB

Five samples of UCB were assessed for their content of AC133+ cells. MNCs isolated from 30–32 ml of UCB ranged from 66 to 90 × 106 (median 80 × 106) cells. The total number of AC133+ cells present within MNC-enriched UCB samples ranged from 309 to 1872 × 103 cells (median 1202 × 103), as determined by flow cytometry. The total number of AC133+ cells separated by MiniMacs ranged from 198 to 1410 × 103 (median 411 × 103), with a calculated median recovery range of 62·3% (range 31·8–75·3%). A yield lower than 50% was obtained in one out of five samples, without any apparent relationship to any of the following parameters: initial volume, hours from birth, time lapse from the separation of MNCs to AC133 mAb labelling.

The median purity of AC133+ cells isolated by MiniMacs was 97·9% (range 91·6–99·6%).

In all samples, percentage viability of AC133+ isolated cells was greater than 98%. The morphology of cytospin preparations showed that > 98% purified AC133+ cells were small to medium- sized MNCs with lymphoid features. Less than 1% of cells displayed morphological features typical of the late myeloid differentiation stages.

The purity of CD34+ isolated cells presented a median of 96% (range 63–99%). In all samples, the percentage viability of CD34+ isolated cells was greater than 98%.

Proliferative responses of purified UCB AC133+ and CD34+ cells to HGFs

Purified AC133+ cells were cultured for 7 days in the presence of different combinations of HGFs (HGF2, HGF3 and HGF6). There is general agreement ( Traycoff et al, 1994; Almici et al, 1997 ) that SCF, present in HGF2, is a crucial component of the culture medium. Additional factors were added (HGF3 and HGF6) in an attempt to identify the conditions whereby the optimal growth of progenitors could be achieved without affecting the pool of stem/progenitor cells.

Absolute numbers of initially seeded and HGF-generated total cells and AC133+ cells are listed in Table II. Fold increase expansion of total cells over initial cell input was demonstrated to be statistically significant (P ≤ 0·05) in all the tested conditions. In detail, the expansion ranged from a 1·9- to 12·9-fold increase (median value 3·7) in the presence of HGF2, from 4·9- to 39·9-fold in the presence of HGF3 (median value 10·9) and from 20- to 58·8-fold (median value 31·8) when the HGF6 combination was added.

Proliferation of the AC133+ cell population resulted in a median fold increase of 1·9 (range 0·9–5·2) in the presence of HGF2, 2·5 (range 0·5–5·9) in the presence of HGF3 and 1·8 (range 0·3–6·4) when the HGF6 combination was added. In parallel experiments, the number of CD34+ cells generated in liquid cultures supplemented with HGF was significantly (P ≤ 0·05) higher than the initial CD34+ cell input in all tested conditions. Median fold increases were 2·4 (range 1·1–9·1) in the presence of HGF2, 3·8 (range 1·5–12·6) in the presence of HGF3 and 8·1 (range 2–19) in the presence of HGF6.

Double staining showed that the total number of AC133+CD34+ cells cultured in the presence of HGF2 increased or remained unchanged in five out of six samples; a slight reduction was recorded in the remaining sample. In the presence of HGF3, the absolute number of AC133+ cells increased or was unchanged in five out of six samples, whereas AC133+ cells decreased in three out of six samples cultured with HGF6. Statistical evaluation demonstrated that the AC133+ cells increased significantly (P ≤ 0·05) only in the presence of HGF2, i.e. SCF + IL-6.

Examination of cell morphology (data not shown) demonstrated that the proportion of blast cells decreased from 100% at the start of the culture to a median value of 84%, 72% and 58% in the presence of HGF2, HGF3 and HGF6 respectively. Myeloblasts, predominantly type I, were present in HGF2- and HGF3-driven cultures, while both myeloblasts and early promyelocytes appeared in cultures supplemented with HGF6. Rare monocytes (< 1%) were observed and polymorphonuclear granulocytes were virtually absent in any of the culture conditions tested.

CFU-GM, CFU-E, BFU-E and CFU-Bl content in UCB AC133+ and CD34+ cells

The committed progenitor cell content of AC133+ cell fractions that expanded after 7-day cultures in the presence of different HGF combinations was investigated by performing CFU-GM, CFU-E and BFU-E assays. Figure 3 shows the number of CFU-GM obtained. In pre-expansion colony assays, AC133+ cells produced a median value of 553 (range 90–1100) early CFU-GM and 2047 (range 1050–2720) late CFU-GM. Early CFU-GM numbers generated in the presence of HGF2, HGF3 and HGF6 were eight (median value; range 6–22)-, 25 (range 9–65)- and 112 (range 40–824)-fold the input value respectively. Similarly, late CFU-GM progenitor cells increased over the initial number by three-, eight- and 23-fold for HGF2-, HGF3- and HGF6-supplemented cultures respectively. Statistical analysis of the absolute number of CFU-GM progenitors generated after expansion of AC133+ cells in different HGF-supplemented cultures showed that increments in colony numbers were significant in all the conditions tested (P ≤ 0·02), after both 7 and 14 days of culture in semi-solid medium.

image

Figure 3. Generation of CFU-GM from freshly isolated AC133+ cells and AC133+ cells expanded in 1-week liquid cultures in the presence of different combinations of HGF: HGF2, HGF3 and HGF6 (see text for their composition) respectively. Five × 103 cells were cultured in semi-solid medium containing GM-CSF (100 ng/ml) for 14 days. The number of early (7-day score) and late (14-day score) CFU-GMs was determined. The data from each sample are expressed as total CFU-GM content. The means ± SE of six experiments are shown.

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Concerning CFU-GM progenitors derived from CD34+ cells, early CFU-GM numbers obtained after HGF2-, HGF3- and HGF6-driven cultures were 11-, 20- and 18 (median value)-fold the input value respectively. Colony ranges were 391–45 600 in the presence of HGF2, 518–89 434 in the presence of HGF3 and 433–70 437 in the presence of HGF6. Late CFU-GM progenitor cells augmented over the pre-expansion input by seven-, nine- and eight-fold for HGF2-, HGF3- and HGF6-supplemented cultures respectively. Colony ranges were 2432–16 522, 3729–39 239 and 3153–30 518 for HGF2-, HGF3- and HGF6-driven cultures respectively. Increments in both early and late CFU-GM were statistically significant (P ≤ 0·05) in all the conditions reported.

CFU-E progenitor cells, undetectable in freshly isolated AC133+ cells, were detected in small numbers after culture of the latter cells in the presence of HGF2 and HGF3 (median value of the total expanded population 61 and 25 respectively); the CFU-E progenitor cell median value increased significantly when the HGF6 combination was added (P ≤ 0·01) in comparison with HGF2 and HGF3 (median value 3388). On the other hand, BFU-E increased over the initial input with all the HGF combinations tested (HGF2, HGF3 and HGF6), with median fold increases of seven, 9·5 and 20 respectively (data not shown).

CD34+-derived CFU-E progenitors were already present in the pre-expansion cultures (median value 55, range 1–210): their number increased over the initial value by nine-, four- and 227-fold in cultures performed in the presence of HGF2, HGF3 and HGF6 respectively. Colonies ranged from 0 to 1140 in HGF2-containing cultures, from 0 to 627 in HGF3-containing cultures and from 13 to 1571 in HGF6-containing cultures. BFU-E displayed a huge increment in comparison with the baseline values, i.e. 22-, 19- and 16-fold increase in HGF2-, HGF3- and HGF6-supplemented cultures respectively. In the presence of the latter cytokine combinations, BFU-E ranges were 1524–104 310, 3686–85 193 and 2273–6530 respectively.

As a potential measure of self-renewal, we next tested the replating capability of uncultured and cultured AC133+ and CD34+ UCB cells.

The absolute numbers (mean ± SE) of AC133+-derived CFU-Bls capable of generating secondary colonies were 226·4 ± 84 for freshly isolated AC133+ cells; 455·6 ± 317, 1072 ± 558 and 1361·4 ± 650·8 for AC133+ cells expanded in the presence of HGF2, HGF3 and HGF6 respectively (Table III). The replating potential was determined as the percentage of primary colonies giving rise to at least one secondary colony. The potential for initiating secondary colonies augmented with a pattern similar to that of more committed progenitors. Before liquid cultures were started, 85% (median value) of the CFU-Bls were capable of generating a mean of two secondary colonies after replating (Table III). After 7 days in suspension cultures, CFU-Bls underwent a progressive expansion, reaching 1·6-, 3·7- and 4·8-fold the input number in the presence of HGF2, HGF3 and HGF6 respectively (Table IV). In identical conditions, CFU-Bls were able to generate a mean of 1·9, 1·1 and 0·6 secondary colonies respectively.

The absolute number (mean ± SE) of CFU-Bls derived from CD34+ cells was 48·9 ± 23·7 for freshly isolated cells, 265·9 ± 209, 90·2 ± 61·2 and 50·2 ± 34·2 for CD34+ cells cultured in the presence of HGF2, HGF3 and HGF6 respectively (Table IV). Pre-expanded cells displayed a CFU-Bl replating efficiency equal to 46% (median value). After a 7-day expansion with the cytokine combinations HGF2, HGF3 and HGF6, replating efficiencies were 52%, 31% and 10% respectively. The absolute number of CFU-Bls with replating efficiency obtained from 104 cells expanded in the presence of HGF2, HGF3 and HGF6 was 1·7-, 1·5- and 0·4-fold the input number respectively. In these experimental conditions, CFU-Bls generated a mean of 1·0, 0·6 and 0·09 secondary colonies respectively.

Statistical evaluation parameters of CFU-Bl colonies derived from CD34+ or AC133+ cells are shown in Table V.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The immunophenotypic and functional characterization of a subset of CD34+ cells, which includes early haemopoietic stem/progenitor cells, is relevant to the potential applications of such cells in both laboratory and clinical investigation. FACS analysis showed that, in our UCB samples, AC133+ cells not only were exclusively contained in the population of CD34+ cells, but were also strictly related to the CD34bright cells. The latter cells are known to include most of the CD34+CD38 cells and contain the majority of CFU-GM, a proportion of CFU-GEMM and a minor population of BFU-E ( Mayani et al, 1993 ). Co-expression of AC133 and CD34 antigens might suggest that their expression is similarly regulated in haemopoietic development, even though the relationships between the two antigens have to be clarified. Further information on AC133/CD34 antigen expression in haemopoietic differentiation might be inferred from the characterization of immature cell populations in malignancies. For example, it has been reported that AC133 may be found on myeloid leukaemia blasts in the absence of CD34 ( Kratz-Albers et al, 1998 ) .

In our samples, AC133+ cells ranged from 0·3% to 2·4% of total MNCs. This range appears to be higher than that reported by others ( de Wynter et al, 1998 ). The higher percentage of AC133+ cells we found could be the result of physiologic variability, the modalities of sample collection and time of harvesting, as well as differences in laboratory procedures (percentage defined on unseparated samples vs. Ficoll gradient fractionated MNCs). Reports published so far on the AC133+ cell population in bone marrow, UCB and mobilized peripheral blood are limited ( de Wynter et al, 1998 ), and comparison between reported data is made difficult by the expression of the frequency of AC133+ cells as a percentage within the CD34+ cell population ( Yin et al, 1997 ; Durett et al, 1998 ). However, the high percentage of AC133+ cells that we also detected in the CD34+ cell population (median value 78·7%) and the finding that CD34+/AC133+ cells are capable of repopulating NOD/SCID mice ( de Wynter et al, 1998 ), coupled with the demonstration that AC133+ cells can be isolated at a high purity (> 91%), prompted us to investigate further the in vitro response to growth factors of this subset of stem/progenitor haemopoietic cells.

In this study, AC133+ cells isolated from UCB were found to contain 0·2–4·5% CD38 cells, and similar proportions (0·5–5%) of the latter cells were detected in CD34+ cells from the same source in a previous study by our group ( Pasino et al, 1998 ). Whether or not CD38 cells found in AC133+ vs. CD34+ UCB cells are completely, or only partially, overlapping remains to be ascertained in further studies. Likewise, how other early markers of primitive haemopoietic progenitors, such as c-kit or Thy-1, are comparatively expressed in AC133+ vs. CD34+ cells deserves further investigation.

Conditio sine qua non for the use of UCB as an alternative source of transplantable cells is that the AC133+ cell subset contains both short- and long-term reconstituting cells. A major advantage could also derive from the demonstration that AC133+ cells are more enriched in early progenitor cells than CD34+ cells. Assays suitable for the detection of human putative stem cells have been developed, including CFU-Bl ( Leary & Ogawa, 1987). Both AC133+ and CD34+ cells isolated by Mini-Macs from UCB contained an aliquot of cells capable of giving rise to second-generation colonies. Comparing equal concentrations of cells seeded, we found that CFU-Bls derived from AC133+ cells were significantly more numerous than CFU-Bls derived, under identical conditions, from CD34+ cells (Table IV). Moreover, the replating efficiency, measured as the number of CFU-Bls capable of generating secondary colonies, was significantly higher for AC133+-derived CFU-Bls than for CD34+-derived CFU-Bls. During the short-term HGF-supplemented culture, the absolute number of cells capable of generating secondary colonies, normalized for the absolute number of counted cells and compared with the input value, was significantly higher for AC133+-derived CFU-Bls than for CD34+-originated CFU-Bls. Taken together, these data suggest that the early stem/progenitor cells are present and enriched in the pool of AC133+ isolated cells compared with CD34+ isolated cells.

Ex vivo expansion has been regarded as a potential tool for increasing the number of more immature haemopoietic progenitor cells. However, the experimental conditions employed have rarely been encouraging in obtaining an effective increase in stem and early progenitor cells ( Piacibello et al, 1997 ). This failure has been ascribed to the lack of accessory cells, to the predominant effect of induction of differentiation by the cytokines added ( Haylock et al, 1992 ) or, alternatively, to the presence of very immature, deeply quiescent cells, which proliferate after a more prolonged time and/or do not respond to known cytokines ( Berardi et al, 1995; Hao et al, 1996 ). In our stroma-free culture system, the simple combination of two early acting cytokines, i.e. SCF and IL-6, resulted in an output of CFU-Bls that was augmented when FL was associated. This finding represents an additional demonstration of the capability of FL to sustain the growth of primitive progenitors ( Piacibello et al, 1997 ). The presence of lineage-restricted growth factors in the 7-day expansion culture before the CFU-Bl assay did not further enhance the CFU-Bl output (P = 0·201, Student's t-test for paired data).

It would appear that AC133+ cells behave differently from the whole pool of CD34+ cells, in the expansion culture conditions used, in terms of the production of clonogenic precursors from primitive cells. Undoubtedly, the AC133+ cell fraction is a heterogeneous pool of immature and more mature cells, as we have demonstrated in clonogenic assays in which CFU-GM, BFU-E and CFU-Bl colonies were generated. However, the possibility of maintaining and expanding, although with small numbers, more immature elements adds new support to the hypothesis that the AC133+ cell subset could become increasingly important in all those clinical procedures that benefit from the availability of a pool of expanded and/or gene-manipulated immature haemopoietic cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors wish to thank Miss Barbara Caruzzo for her excellent assistance in preparing the manuscript, the staff of the Division of Obstetrics for providing normal human umbilical cord blood specimens, and Amgen for the gift of SCF.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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