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Stem Cells 2004;22:704-715 www.StemCells.com
© 2004 AlphaMed Press

AC133+ G0 Cells from Cord Blood Show a High Incidence of Long-Term Culture–Initiating Cells and a Capacity for More Than 100 Million-Fold Amplification of Colony-Forming Cells In Vitro

Yvonne J. Summers, Clare M. Heyworth, Erika A. de Wynter, Claire A. Hart, James Chang, Nydia G. Testa

Cancer Research UK Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, United Kingdom

Key Words. AC133+ cells • CD34+ cells • Cord blood • Ex vivo expansion • G0/G1 phase of cell cycle

Correspondence: Dr. Yvonne J. Summers, Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, M20 4BX United Kingdom. Telephone: 0161-446-3741; Fax: 0161-446-3299; e-mail: yvonnejsummers{at}aol.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AC133+ cells may provide an alternative to CD34+ cells as a target for cell expansion and gene therapy protocols. We examined the differences in proliferative potential between cord blood selected for AC133 or CD34 in serum-free, stroma cell–free culture for up to 30 weeks. Because most hemopoietic stem cells reside within the G0/G1 phase of the cell cycle, we combined enrichment according to AC133 or CD34 expression with G0 position in the cell cycle to identify populations enriched for putative stem cells. Our results show that AC133+ G0 cells demonstrated a long-term culture-initiating cell incidence of 1 in 4.2 cells, had a colony-forming cell incidence of 1 in 2.8 cells, were capable of producing 660 million-fold expansion of nucleated cells and 120 million-fold expansion of colony-forming units–granulocyte-macrophage over a period of 30 weeks, and were consistently superior to CD34+ G0 cells according to these parameters. Furthermore, we have shown that AC133+CD34– cells have the ability to generate CD34+ cells in culture, which suggests that at least some AC133+ cells are ancestral to CD34+ cells. We conclude that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34 and that, in combination with isolation according to G0 phase of the cell cycle, AC133 isolation identifies a highly enriched population of putative stem cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemopoietic stem cells (HSCs) are capable of self-renewal and generating committed progenitors of the different myeloid and lymphoid compartments to sustain lifelong hemopoiesis. They are present in bone marrow (BM), umbilical cord blood (CB), and normal and mobilized peripheral blood (MPB). Recent years have seen a decline in the use of BM as a source of HSCs, whereas MPB is increasingly used for autologous and allogeneic transplantation. CB is an alternative source of hemopoietic repopulating cells. There are several theoretical advantages of CB compared with adult cells; besides a reduced incidence and severity of acute and chronic graft-versus-host disease compared with unrelated BM transplant [1], CB cells produce larger hemopoietic colonies in vitro, are able to expand additionally in long-term culture [2], engraft nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and have longer telomeres [3]. These characteristics theoretically should go some way in compensating for the limited numbers of cells available. However, in clinical studies, when CB is compared with BM, delayed engraftment remains a problem [1]. Because the number of stem cells available may be limiting, a major goal of experimental and clinical hematology is the characterization of the HSC and identification of ex-vivo conditions that support its self-renewal and expansion.

Most transplant protocols rely on a threshold number of CD34+ cells to predict engraftment [4–6], and in many experimental investigations, CD34 is used as a tool to select cell populations enriched for HSCs. However, previous studies in the murine system indicate that some stem cells capable of long-term repopulation do not express detectable levels of cell-surface CD34 [7], and Osawa et al. [8] demonstrated long-term multilineage engraftment of sublethally irradiated mice from a single murine CD34– lineage–negative cell [8]. Furthermore, CD34– human cord blood cells have been shown to successfully repopulate SCID mice using an intra-BM injection technique, which may overcome homing difficulties [9].

More recently, the surface glycoprotein AC133 has been described, which is expressed mainly, but not exclusively, on CD34+ cells. Gallacher et al. [10] identified a population of CD34–lin–AC133+ CD7– human cord blood cells that were capable of engrafting NOD/SCID mice and showed that CD34–lin–AC133+ cells can, unlike their AC133– counterparts, generate CD34+ cells in culture. AC133+ cells show a higher content of colony-forming units–granulocyte-macrophage (CFU-GM), CFU–mixed lineage, and NOD/ SCID-repopulating cells when coexpressed with CD34 [11, 12]. These features suggest that AC133 may identify a population of primitive cells that are not simply a subset of CD34+ cells but a population with distinct biological characteristics.

In addition to cell-surface markers, cell-cycle status may be useful in the identification of HSCs. Stem cells are quiescent, residing in the G0/G1 phase of the cell cycle [13, 14]. In MPB, virtually all of the primitive cells are found in G0/G1, compared with only 85%–90% of the BM primitive cells [15, 16]. Therefore, cell-cycle status may be critical in defining strategies for HSC selection. It is now possible to distinguish and isolate viable cells in G0 or G1 phase using a combination of the DNA and RNA binding dyes, Hoechst 33342 (Molecular Probes, Eugene, OR, http://www.molecularprobes.com) and Pyronin Y (Sigma, Poole, U.K., http://sigmaaldrich.com)[17].

Reported methods for expansion of HSCs in vitro have used a variety of conditions, including stroma-free or stroma-containing protocols in the presence or absence of serum and different combinations of growth factors [18–24]. In most reports, the increase in stem cell numbers, if observed, was small. Extensive amplification of the progenitor cell population over 6 months has been described in a fetal calf serum (FCS)–containing system supplemented with the cytokines Flt-3 ligand (FL-3) and thrombopoietin (TPO) [25]. We have recently reported similar levels of amplification shown by CD34+ cells, but under serum-free conditions [26].

In this study, we used simultaneous DNA/RNA staining and flow cytometric cell sorting to isolate and characterize cord blood CD34+ or AC133+ cells in G0 or G1 phases of the cell cycle and document that AC133+ G0 cells possess a much higher capacity to generate progeny in a serum-free culture system than CD34+ G0 cells. We also demonstrate that cells isolated from G0 phase of the cell cycle exhibit much greater proliferative capacity than those isolated from G1 phase of the cell cycle and are capable of generating colony-forming cells (CFCs) for more than 6 months in serum-free culture. By the combination of these selection steps, we have shown thatAC133+ G0 cells are superior to the other populations and provide the best protocol to date for purification of putative stem cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Preparation
Human CB cells were obtained from full-term normal deliveries with informed consent. The mononuclear cell (MNC) fraction was separated on Ficoll-Hypaque (Lymphoprep, density 1.077 g/ml; Life Technologies, Paisley, U.K., http://www.lifetechnologies.com) by density centrifugation, and CD34+ or AC133+ cells were isolated using CD34 or AC133 antibody–conjugated superparamagnetic micro-beads and MiniMACS columns (Miltenyi Biotech, Bergisch Gladbach, Germany) as described elsewhere [27]. Briefly, MNCs were incubated with CD34 or AC133 antibody–conjugated microbeads for 30 minutes at 4°C. After incubation, cells were washed in ice-cold PBE (phosphate-buffered saline [PBS] containing 1% bovine serum albumin [wt/vol] and 5 mM EDTA). Cells were passed through a MiniMACS column retained in a magnetic field, and the column was washed with PBE to remove unbound cells. Enriched CD34+ or AC133+ cells were recovered by releasing the magnetic field and flushing cells from the column. Cells were passed through a second column to achieve a purity >90%.

Isolation of G0 and G1 Cells
AC133+ cells were labeled with AC133(2) pure antibody (Miltenyi Biotech) at 10 µl per 106 cells, followed by rabbit anti-mouse biotin at a 1:400 dilution (Dako, Cambridgeshire, U.K.) and finally streptavidin phycoerythrin (PE)-Cy7 conjugate at 10 µl/106 cells (Caltag Laboratories, Burlingame, CA). The cells were washed after each 15-minute incubation period carried out at room temperature and were resuspended in PBE. The control sample had PE-Cy7 added without the AC133(2) pure antibody or rabbit anti-mouse biotin. Cells were subsequently labeled using a method previously described by Gothot et al. [28]. AC133+ cells were washed and then incubated in Hoechst buffer (Hanks balanced salt solution; 0.1% [wt/vol] D-glucose; 20 mM HEPES, 10% [vol/vol] FCS) at a concentration of 5 x 106 cells/ml. Hoechst 33342 dye was added to give a final concentration of 1 µg/ml, and the cells were incubated for 45 minutes at 37°C. After this incubation period, Pyronin Y was added to a final concentration of 0.5 µg/ml, and incubation continued at 37°C for an additional 45 minutes. The cells were then washed once and resuspended in ice-cold Hoechst buffer and sorted on a fluorescence-activated cell sorter (FACS) Vantage equipped with an argon laser providing excitation in the UV spectrum at 357 nm (Stabalite 2017) and an argon laser providing excitation in the visible spectrum (Innova 70C/2, Coherent; Fig. 1Go).



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  		Figure 1. Flow cytometric sorting of G0/G1 cells. A lymphocyte gate was defined according to forward and side scatter, as shown in (A), and cells within this gate were also selected for AC133 expression (C) or CD34 expression (not shown). Simultaneous staining of RNA and DNA with Hoechst and Pyronin Y, respectively, allows cells in G0 to be distinguished from those in G1 by virtue of their RNA content. Cells with no more than "2n" DNA and low RNA content (Pyronin Y fluorescence 220 or less) are gated as G0, and those with greater RNA content (Pyronin Y fluorescence 400 or more) are gated as G1 (B). (D): Cell-cycle analysis based on DNA content alone, where > 99% of the CB cells are in G0/G1 phase of the cell cycle.

 
CD34+ cells were stained with Hoechst and Pyronin Y following the same protocol described above; however, the fluorochrome labeling of the cell-surface marker was carried out at the end of the incubation period, not at the beginning. A total of 10 µl/106 cells of CD34-fluorescein isothiocyanate (FITC) antibody (HPCA-2; Becton, Dickenson) or a mouse immunoglobulin G1 (IgG1) FITC antibody (Becton, Dickenson, Cowley, U.K., http://www.bectondickenson.com) as an isotype-matched control was added 10 minutes before the end of the Pyronin Y incubation. This method of three-color sorting resulted in a purity of CD34+ andAC133+ cells of >97%.

FACS Sorting of CD34+/–AC133+/– Cells
MNCs were resuspended in PBE at 1 x 108 cells/300 µl. Ten microliters<symbols> of the sample were removed and suspended in 100 µl to act as the isotype control. 50 µl of CD34 FITC-conjugated antibody (Becton, Dickenson) and 50 µl of AC133(1) PE-conjugated antibody (Miltenyi Biotec) per 108 cells were added to the sample and incubated for 30 minutes at 4°C. The second aliquot (control) was labeled with 10 µl of mouse IgG1-PE and 10 µl of mouse IgG1-FITC (Becton, Dickenson). After incubation, the cells were washed and resuspended in PBE at 2 x 106 cells/ml and sorted on a FACS Vantage equipped with an argon laser providing excitation at 488 nm. Cells were sorted according to whether they coexpressed CD34 and AC133 or whether they expressed one or none of the cell-surface markers.

Serum-Free Cultures
The G0 or G1 cells were cultured in 24-well plates (Becton, Dickenson) in a volume of 1 ml at 5 x 103 cells/ml in StemSpan serum-free medium (StemCell Technologies, Meylan, France, http://www.stemcell.com). Previous experiments showed similar results when a range of cell concentrations was used (103–104 cells/ml up to 8 weeks of culture, data not shown). Cultures were supplemented with stem cell factor (SCF) at 300 ng/ml, FL-3 at 100 ng/ml, and TPO at 100 ng/ml and maintained at 37°C in 5% CO2 AND 5% O2 in nitrogen. All cytokines were obtained from R & D Systems, Minneapolis. Each week, cultures were demi-depopulated and fed with an equal volume of fresh medium containing SCF, FL-3, and TPO at the concentrations indicated. This method has been shown to give comparable results to doubling the culture volume each week [29]. The harvested cells were counted, and progenitor cell content was assessed in standard clonogenic assays. At specified time points, cells were analyzed for CD34 and AC133 expression by flow cytometry, as described below. Cell morphology was determined after May Grünwald/Giemsa staining.

Progenitor Cell Assays
To assay CFU-GM and erythroid colony-forming cells (BFU-E), 1–10 x 103 harvested cells were plated in a 1-ml mixture containing a final concentration of 30% (vol/vol) FCS, 1% (wt/vol) deionized bovine serum albumin, 10% (vol/vol) 5637 conditioned medium (from the EJ bladder carcinoma cell line), 2 U erythropoietin (EPO), and 1.35% (wt/vol) methylcellulose. Cultures were plated in triplicate. Plates were incubated in a humidified atmosphere of 5% CO2, 5% O2 in nitrogen at 37°C for 14 days. Colonies were scored according to standard criteria [30].

CD34 and AC133 Expression in Cultured Cells
Aliquots of cultured cells were harvested at specified time points, washed, and resuspended in PBS. After a 15-minute incubation at room temperature with a CD34-PE antibody (HPCA-2, Becton, Dickenson) or AC133-PE (Miltenyi Biotech) or a mouse IgG1-PE as an isotype-matched control, cells were washed and analyzed on a FACScan flow cytometer. A minimum of 10,000 events was collected for each sample. Cells were gated on low side scatter, because more granular (high side scatter) cells exhibited autofluorescence, and isotype controls ensured specificity.

Limiting Dilution Assay for Long-Term Culture–Initiating Cells
Freshly sorted cell populations of CD34+ cells in G0 or G1 and AC133+ cells in G0 or G1 were plated in limiting dilution in the long-term culture–initiating cell (LTC-IC) assay [31]. LTC-IC assays were performed as previously described [32]. Briefly, cells were plated in six different dilutions of 30 replicates on preirradiated M210B4-coated 96-well plates. Cells were inoculated at 5–250 cells/ml (1–50 cells/well), which were previously determined as the concentrations giving the maximum information. Medium consisted of Iscove’s modified Dulbecco’s medium at 350 mOsm/kg, 10% FCS (vol/vol), 10% horse serum (vol/vol), and 5 x 10–7 M hydro-cortisone-21-succinate (Sigma). Cultures were maintained for 5 weeks with a weekly change of half of the medium. Medium was then completely removed and replaced with methylcellulose as described above for clonogenic assays. After 14 days, wells were evaluated for the presence or absence of hemopoietic colonies and scored as positive or negative, respectively. LTC-IC frequency was then calculated according to Poisson statistics [33].

StatisticalAnalysis
Results of experimental points obtained from multiple experiments are expressed as the mean ± standard error of the mean. Data were analyzed using a two-tailed paired Student’s t-test or unpaired t-test where appropriate. Probability values < .05 designated significant differences between test points.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CFC and LTC-IC Content of Fresh Cells
The CFC content of freshly sorted CD34+ andAC133+ and G0 and G1 CB cells was assessed before culture (Fig. 2Go). Cells residing in G0 showed a significantly higher colony content than those in G1, which was largely accounted for by the presence of more erythroid colonies in the G0 populations. The mean incidence of CFCs for AC133+ G0 cells was 1 in 2.8 cells, and the mean for CD34+ G0 cells was 1 in 3 cells. When LTC-ICs were investigated, they showed an incidence of 1 per 4.2 AC133+ G0 cells (Table 1Go).



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  		Figure 2. Colony content of purified fresh cord blood cells. The number of colonies produced per 1,000 cells plated is represented. (A): G0 cells have more BFU-E than G1 cells, whether they are from derived CD34+ orAC133+ cells. There are no significant differences in colony-forming units–granulocyte-macrophage content (CFU-GM) (B) (*p < .05 by paired Student’s t-tests; n = 20).

 

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  		Table 1. Incidence of long-term culture–initiating cells (LTC-ICs) in freshly isolated cord blood
 
Expansion of Cord Blood Cells
Freshly sorted cells were seeded into serum-free, stroma-cell free liquid culture in the presence of FL-3, TPO, and SCF, as described in Materials and Methods. These cultures were demi-depopulated and fed on a weekly basis, and the harvested cells were assayed for progenitor content and expression of CD34 and AC133. Initially, extensive expansion of total nucleated cells was seen regardless of whether the cells were originally isolated from G0 or G1 phase of the cell cycle; however, beyond approximately 18 weeks of culture, the G0 cultures began to exhibit superior expansion capabilities compared with their G1 counterparts, which started to decline at approximately week 21. The AC133+ G1 cells showed a maximum fold expansion of 4.4 x 106, and that of CD34+ G1 was 1.3 x 107 (Fig. 3Go). In contrast, up to 6.6 x 108-fold expansion was achieved from AC133+ G0 cells, reaching a plateau at approximately week 27. The proliferative capacity shown by CD34+ G0 cells was approximately 30 times less at approximately 2.4 x 107-fold expansion, plateauing at week 21.



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  		Figure 3. Fold expansion of total nucleated cells. Fold expansion of cells from AC133+ cultures (n = 12 up to week 21; n = 6 thereafter) and from CD34+ cultures (n = 6 up to week 21; n = 4 thereafter) is shown. The weekly totals were determined by taking the number of cells in the harvested proportion (half volume in each well) and making the calculation of how many cells would have been present if weekly demi-depopulation had not occurred (multiply by 2n, in which n is the week of demi-depopulation). The total number of cells was then divided by the starting number to calculate the fold expansion. Open and closed symbols denote G0- and G1-derived cultures, respectively.

 
Expansion of Progenitors
The number of progenitor cells generated in the cultures was assessed weekly by colony-forming assays, and the results are represented on cumulative plots (Figs. 4Go, 5Go). More than 1 million-fold expansion of erythroid colonies was seen in the AC133+ G0 cells, and production of colonies plateaued at approximately 26 weeks (Fig. 4Go). This contrasted with the AC133+ G1 population, which only achieved 140-fold expansion of BFU-E and plateaued much earlier at 8 weeks. A similar pattern was seen with CD34+ G0 cells, but the maximum expansion was only 9.4 x 104-fold, and the cultures plateaued approximately 4 weeks earlier.



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  		Figure 4. Fold expansion of BFU-E. The cumulative production of BFU-E of cells from AC133+ cultures (n = 12) and from CD34+ cultures (n = 6) is shown. Each week, the number of colonies present in an aliquot of harvested cells was assessed by progenitor cell assay. The number of colonies present in the total population was determined by taking into account the total number of cells present if demi-depopulation had not occurred (Fig. 3Go). Fold expansion was calculated by dividing by the number of erythroid colonies in the starting cultures. Because these represent cumulative plots, a plateau indicates that the cultures are no longer producing BFU-E. Open and closed symbols denote G0- and G1-derived cultures, respectively.

 


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  		Figure 5. Fold expansion of CFU-GM. The cumulative production of CFU-GM of cells from AC133+ cultures (n = 12) and from CD34+ cultures (n = 6) is shown. Fold expansion is calculated as for BFU-E (Fig. 4Go). Open and closed symbols denote G0- and G1-derived cultures, respectively.Abbreviation: CFU-GM, colony-forming units–granulocyte-macrophage.

 
When the numbers of granulocyte-macrophage progenitors were examined, the AC133+ G0 cells proved, yet again, to be the population most able to generate progenitor cells; production of CFU-GM was only beginning to plateau at 30 weeks, at which time point the fold expansion reached 1.2 x 108 (Fig. 5Go). In comparison, the corresponding G1 cells produced a maximum fold expansion of 3.7 x 103 and plateaued at 22 weeks. The cultures initiated with CD34+ G0 cells achieved more than a 1 million-fold expansion of CFU-GM but plateaued at 22 weeks.

Expression of CD34 and AC133 on Expanded Cells
We also analyzed the cells produced in culture, using PE-labeled MoAbs, at specified time points for expression of AC133 and CD34 by flow cytometry. Cultures initiated with AC133+ cells were analyzed for AC133, and cultures initiated with CD34+ cells were analyzed for CD34. Our data demonstrated that numbers of AC133+ cells were lower than CD34+ cells after 1 week of culture (27.6 ± 3.0% of cultured cells expressed AC133 in the cultures initiated with AC133+ G0 cells compared with 54.0 ± 10.1% of cultured cells that expressed CD34, n = 8 and 5, respectively); however, this did not translate into a reduction in total numbers of cells or progenitors produced from the AC133+ cultures compared with CD34+ cultures. Figure 6Go shows that at 8 weeks of culture, AC133+ G0 population had produced 390 times the input number of AC133+ cells and the CD34+ G0 population had produced 664 times the input number. G1 cultures from AC133+ and CD34+ populations had produced 47 and 33 times the input numbers, respectively. In cultures started either with AC133+ or CD34+ cells, G0 cultures showed a higher expression of AC133 or CD34, respectively, than those from G1 cultures at all time points. Production of both AC133- and CD34-positive cells declined more rapidly in the G1 cultures than in the G0 cultures.



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  		Figure 6. Fold expansion of CD34+ and AC133+ cells. (A): Production of AC133+ cells expressed as fold expansion compared with input number in the AC133+-initiated cultures (n = 8). (B): Production of CD34+ cells expressed as fold expansion compared with input number in the CD34+-initiated cultures (n = 8 for all time points except weeks 1 and 2, in which n = 5 and n = 6, respectively).

 
Because others have reported that approximately 90% of AC133+ CB cells coexpress CD34 [10], we sought to additionally characterize the subset of AC133+ cells that do not express CD34. Analysis of freshly isolated CB cells (n = 4) showed that 98 ± 0.6% of AC133+ cells coexpress CD34 and 74 ± 3.0% of CD34+ cells coexpress AC133. Fresh CB AC133+CD34– cells have the ability to produce CD34+ cells in culture (after 2 weeks of culture, 7.5 ± 2.5% of cells express CD34 [n = 4] and an average of 13,000 CD34+ cells are generated from 5,000 fresh CB AC133+CD34– cells, which have increased to 22,000 CD34+ cells after 4 weeks). AC133+CD34– CB cells are also capable of generating cells and progenitors for up to 15 weeks in serum-free, stroma cell–free culture, achieving 7.2x 104-fold expansion of total cells and 6 x 104-fold expansion of CFU-GM (n = 4). However,AC133–CD34+ CB cells are unable to generate AC133+ cells in ex vivo culture and do not produce CFCs after 4 weeks (data not shown).

Cell-Cycle Analysis of Cultured Cells
Because cultures initiated with cells originally sorted according to G0 cell-cycle status showed the higher expansion capacity (Figs. 3Go–5Go), we proposed that maintenance of a quiescent population in the culture would be important in maintaining the long-term proliferative capacity. Cultured cells were restained with Hoechst and Pyronin Y at various time points and analyzed by flow cytometry (Fig. 7Go). The first observation is that at 24 hours there were marked differences in the cell-cycle analysis of the G0 and G1 starting populations; unsurprisingly, the initial G0 population lagged behind G1 in entry to the cell cycle (0.2% in S/G2/M versus 10%, respectively) and had a larger proportion of cells remaining in G0 (24% versus 12%; p <.05 for both). However, by 48 hours, approximately 50% of cells were cycling (S/G2/M) in both sets of cultures, and by 3 weeks, approximately 10% of the cells were cycling. The cultures initiated from G0 cells had a larger proportion of cells in G0 compared with cultures initiated from G1 cells at 48 hours (9.6% versus 5.4%) and 3 weeks (34% versus 20%). After 3 weeks of culture, the number of cells in G0 that have been generated from both AC133+ G0 and CD34+ G0 cultures is more than 100-fold the initial input number (Table 2Go).



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  		Figure 7. Cell-cycle analysis of cultured AC133+ cells. Representative DNA (Hoechst)/RNA (Pyronin Y) plots of G0 (left column) and G1 (middle column) cultures are shown at 24 hours (top), 48 hours (middle), and 3 weeks (bottom). At 24 hours, the cells from the G0 cultures remain in G0/G1; however, the cells from the G1 cultures can already be seen to be moving into S/G2/M. The histograms from three experiments (right column) show that significant differences (*p < .05) are demonstrated between the G0 (open bars) and the G1 (closed bars) cultures at this time point, with substantially more cells from G0 cultures remaining in G0 and virtually no progression into S/G2/M. At 48 hours, approximately half of the cells from either starting culture have moved into S/G2/M. At both 48 hours and 3 weeks, there is a trend toward cells from the G0 starting cultures having a greater proportion of their cells within G0.

 

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  		Table 2. Number of G0 cells in culture
 
Morphology of Cultured Cells
Expanded cells were sorted after cell-cycle analysis, and their morphology was examined at 3 weeks of culture by May-Grünwald/Giemsa staining of cytospin preparations.

The cells remaining in G0 and G1 maintain a large proportion of blast cells, whereas those from S/G2/M have a higher proportion of differentiated cells. Examination of the morphology of cultured cells from G0 and G1 starting populations showed that the proportion of blasts present in G0 cultures was higher than in G1. After 12 weeks, cultures initiated with AC133+ cells contained 21.5 ± 1.2% and 9.0 ± 1.3% blasts for G0 and G1 populations, respectively, and cultures initiated with CD34+ cells contained 12.2 ± 2.1% versus 6.2 ± 2.5% for blasts for G0 and G1 populations, respectively (n = 6; p < .05 for both).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that AC133+ G0 cells demonstrated an LTC-IC incidence of 1 in 4.2 cells, had a CFC incidence of 1 in 2.8 cells, and were capable of producing 660 million-fold expansion of nucleated cells and 120 million-fold expansion of CFU-GM over a period of 30 weeks in serum-free, stroma cell–free conditions and were consistently superior to CD34+ G0 cells according to these parameters.

Several studies have indicated that AC133 is an antigen expressed on primitive cells. AC133, like CD34, is expressed on some leukemias [34, 35] and on the teratocarcinoma cell line NT2 [12]. When this cell line is induced to differentiate, it rapidly loses AC133 antigen expression. Few studies have investigated the ex vivo expansion potential of AC133+ cells in stroma cell–free liquid culture [36–40], and none for more than 14 days. Matsumoto et al. [37] showed that AC133+ peripheral blood (PB) cells produced more CFCs after 14 days ex vivo culture than CD34+ PB cells. However, the degree of expansion was approximately 10 times less than we report at 14 days. This may be accounted for by the different sources of hemopoietic cells used (PB as opposed to CB) and by the different growth factor cocktail used (no SCF and lower concentrations of FL-3 and TPO). They also showed that CD34+AC133+ PB cells are enriched in LTC-IC compared with CD34+AC133– cells and are capable of threefold expansion of CFCs after 14 days of serum-free culture, whereas no CFCs were detected in cultures started with CD34+AC133–PB cells [37]. De Wynter et al. [11] examined the differences between CD34+AC133+ and CD34+AC133– cells derived from cord blood. Cultures were supported on BM stroma for 10 weeks and demonstrated that higher numbers of nucleated cells and CFU-GM were generated from cultures started with CD34+AC133+ cells compared with CD34+AC133– cells. NOD/SCID repopulating cells have also been shown to be enriched in the CD34+AC133+ population [10, 11].

AC133 is expressed by 60%–80% of CD34+ CB cells [11, 39], and approximately 90%–98% of AC133+ CB cells express CD34 [10]. Although the capacity to generate progenitors and the incidence of LTC-IC and NOD/SCID repopulating cells are higher in CD34+AC133+ cells, we have demonstrated that the cells responsible for long-term generation of cells and CFCs in vitro are also present in the AC133+CD34– population. Furthermore, we have shown that AC133+CD34– cells have the ability to generate CD34+ cells in culture. These results suggest that at least some AC133+ cells are ancestral to CD34+ cells. Alternatively, they may reflect reversible expression of CD34, which was initially described in the murine system [41] and more recently in human BM transplanted to nude mice [42]. Interestingly, Gallacher et al. [10] demonstrated that CD34–AC133+ CD38–lin– cells have a greater capacity to engraft NOD/ SCID mice than CD34–AC133–CD38–lin– cells. This also suggests that AC133 selection may isolate a more primitive cell population than CD34 selection and may therefore provide not just an alternative to CD34 for the characterization of cells for transplantation, ex vivo expansion, and gene therapy, as Yin et al. [12] suggested, but supports our contention that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34.

The incidence of LTC-IC in AC133+ G0 cells (1 in 4.2) is the highest reported to date and is substantially higher than the published data for LTC-IC detected in AC133+ CB cells, which have not been selected according to cell-cycle status (LTC-IC incidence of 1 in 160). The LTC-IC incidence is also higher than the published data for CD34+ G0 cells from MPB (1:33) [43] and from BM (1:16) [28], which are similar to the 1 in 12 reported here for CD34+ G0 cells from CB (Table 1Go). Although the incidences of CFCs for CD34+ and AC133+ were initially similar, later in the culture period there is lower cumulative cell production from CD34+ G0 cultures, with an approximately 30-fold difference between CD34+ G0 and AC133+ G0 cultures for total nucleated cells, more than 10-fold lower production of BFU-E, and almost 90-fold lower production of CFU-GM.

There is evidence from colony assays, LTC-IC determinations, and NOD/SCID repopulating studies that in BM and MPB, HSCs are enriched in the quiescent (G0) phase of the cell cycle [14, 28, 43, 44]. We have demonstrated that cells initially from CB isolated from G0 have a higher incidence of LTC-IC and superior capacity for generation of CFCs in vitro. The differences in production of progenitors between G0 and G1 cultures were evident earlier (at approximately 2 weeks) than differences in nucleated cells (which did not become apparent until approximately 20 weeks). This restates the phenomenon that production of progenitors is more discriminatory of stem cell function than production of nucleated cells [45]. Although these progenitors were able to develop normally in cultures, i.e., they produced colonies normal in size and cell maturation, the fact that higher numbers of CFCs did not result in higher numbers of total cells is likely to be a reflection of the lack of cytokines needed for full proliferation and maturation of the progenitor cells (for example, erythropoietin and GM-CSF) in the expansion cultures. These results imply that HSCs are also significantly enriched in the quiescent (G0) phase of the cell cycle in CB, whether the cells were originally isolated using CD34 or AC133. However, AC133 selection gave consistently better results than CD34 selection. We propose above that the AC133+ population contains cells ancestral to CD34+ cells. Alternative explanations are that cells are in some way activated by binding antibody to the CD34 antigen, which leads to a reduction in proliferative capacity, or that higher numbers of more mature CD34+ cells produce cytokines that enhance maturation. These different interpretations, however, do not invalidate the proposition that AC133 is preferable to CD34 for the selection of primitive cells.

The duration and degree of expansion of progenitors observed in this serum-free, stroma cell–free system is superior to published data. The use of serum may lead to problems with reproducibility, because most sera contain both inhibitors and stimulators of growth, and constituents can vary, even within the same batch. Moreover, the risks of using animal sera, which may contain allergens or infectious agents, like bovine spongiform encephalitis or other prion-type diseases, for expansion protocols with potential clinical use are unacceptable. Even the use of human serum may present risks. Thus, the use of serum-free conditions is crucial if expanded cells are to be used in the clinical setting. Practical reasons also make the use of a stroma cell–free system preferable.

The extended duration over which G0 cells are able to maintain production of progenitors and the magnitude of such production suggest that HSCs may be undergoing self-renewal. Our results indicate that G0 cells may re-enter G0 state after cell division.

Examination of the morphology of cultured cells revealed that a substantial proportion are blasts (up to 21% at week 12 in G0 cultures), which supports the functional data obtained from the expansion cultures. The higher incidence of LTC-IC, greater CFC content, and superior production of progenitors from AC133+ G0 cultures compared with CD34+ G0 suggest that although cells that express AC133 constitute a smaller proportion of BM, MPB, and CB cells, they may be more important in maintenance of long-term hemopoiesis.

Gan et al. [46] previously showed that ex vivo culture of BM and CB cells on stromal layers maintained or expanded CFC and LTC-IC but SCID repopulating cells (SRCs) declined. However, others have achieved modest expansion of SRCs [29, 47, 48]. When one considers the extensive differences we have observed between G0 and G1 populations, it is perhaps surprising that Wilpshaar et al. [44] only demonstrated a 1.6-fold increase in SRCs in freshly sorted CB CD34+ G0 cells compared with CD34+ G1 cells. Ex vivo culture can modify primitive hemopoietic functions, such as homing and adhesion [49], and it would be interesting to test the function of freshly isolated and expanded CD34+ and AC133+ and G0 and G1 cells in transplantation experiments.

The method of isolating cells according to their cell-cycle status in combination with the AC133 antigen reported here provides a population highly enriched for putative stem cells, which can be used as a target for cell expansion and gene therapy protocols and will also facilitate further genetic characterization of HSCs.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This research was supported by Cancer Research U.K. Erika de Wynter is now at Molecular Medicine Unit, Clinical Sciences Building, St. Jame’s University Hospital, Leeds LS9 &TF, United Kingdom.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received January 14, 2004; accepted for publication March 21, 2004.



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