SEARCH

SEARCH BY CITATION

Keywords:

  • AC133;
  • cord blood;
  • apoptosis;
  • stem cells;
  • cytokines

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

Summary. Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2–4 × 104/ml), mononuclear cells (MNC, 1–2 × 106/ml) and AC133negative cells (AC133neg, 2–4 × 104/ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either ‘K36EG’[c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or ‘TPOFL’ (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133neg populations (K36EG FI: 15·04 ± 5·46; TPOFL FI: 8·59 ± 2·92, P < 0·05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0·01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20·50 ± 2·36; TPOFL: 47·00 ± 7·69; P < 0·05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.

Umbilical cord blood (UCB) raised great hopes as an alternative source of transplantable haemopoietic stem/progenitor cell (HSPC) to adult bone marrow (BM) or growth factor-mobilized leukapheresis harvest (GFMLH). UCB HSPC have been used successfully in over 1500 haemopoietic stem cell transplantations (HSCT) in a range of malignant and non-malignant diseases, including Fanconi's anaemia as well as acute myeloid and lymphoid leukaemias (Gluckman, 2000; Rocha et al, 2000). UCB HSPC harvest exhibits lower donor risk (low prevalence of infectious spread). In use, a lower incidence of graft-versus-host disease (GVHD) and higher levels of CD34+ and AC133+ populations are an advantage (de Wynter et al, 1998; Rocha et al, 2000).

For over a decade, the sialomucin CD34 was the gold standard identification selection marker for early HSPC. Recently, cells expressing the AC133 antigen were considered to be a potent substitute for CD34+ cells. Phenotypic and functional studies revealed UCB AC133+ populations contain higher levels of early HSPC than UCB CD34+ harvested populations (Pasino et al, 2000).

However, slower engraftment and low cell numbers harvestable from a single UCB specimen still limit the use to paediatric HSCT and the international cryobanking programme (Gluckman, 2000). Ex vivo expansion technology may circumvent UCB HSPC limitations and extend the use to adult HSCT, as expansion aims to generate in vitro HSPC amplification with growth factors, while limiting proliferation-induced differentiation (Aglietta et al, 1998). Mechanisms modulating this fine balance between proliferation–differentiation and maintenance–self-renewal are poorly understood.

This study investigated early ex vivo expansion kinetics of UCB AC133+ cells, mononuclear cells (MNC) and AC133negative (AC133neg) cells in stroma-free short-term liquid culture stimulated by two different growth factor combinations: ‘K36EG’[c-Kit ligand (cKitL), interleukin 3 (IL-3), IL-6, erythropoietin and granulocyte colony-stimulating factor (G-CSF)], known to recruit cells into proliferation and differentiation (Aglietta et al, 1998), and ‘TPOFL’ (thrombopoietin and Flt-3 ligand), whose synergism was recently reported to efficiently amplify early HSPC populations (Gilmore et al, 2000).

UCB collection and MNC isolation. UCB specimens were collected from full-term deliveries scheduled for elective caesarean sections following hospital ethical regulations. Samples were diluted 1 in 4 in PBS supplemented with a citrate-based anticoagulant (0·6% ACD-A; Baxter, Maurepas, France) and bovine serum albumin (0·5% fraction V; Sigma Aldrich, UK) at pH 7·4 and referenced as ‘ACD-A buffer’. Diluted UCB was carefully overlaid in a 1:4 ratio onto a research grade Ficoll–Paque solution (d:1·077 g/cm3; Pharmacia Biotech, Uppsala, Sweden) prior to centrifugation (400 g, 30 min, 22°C). The MNC layer was extracted, washed twice in ACD-A buffer, pelleted (400 g, 10 min) before resuspending in ACD-A buffer and cell aliquots taken for cell viability/enumeration using trypan blue (Sigma Aldrich, Poole, UK).

AC133+/neg cell immunomagnetic selection. AC133+/neg cells were obtained from MNC after immunomagnetic separation using the AC133 mini-MACS selection kit (Miltenyi Biotec, Germany): labelling volume 500 μl/108 cells in ACD-A buffer containing Fc receptor-blocking reagent (100 μl, 5 min incubation, 4°C) before adding colloidal super-paramagnetic MACS microbeads conjugated to monoclonal mouse anti-human AC133/1 antibody (100 μl IgG1 isotype, 25 min incubation, 4°C). Cells were then washed (5 ml ACD-A buffer, 400 g, 10 min, 4°C) before resuspended cells in 500 μl ACD-A buffer were applied to a chilled magnetic-activated cell-sorting (MACS) positive-selection column (MS+/RS+) on a magnet. The column was rinsed with cool ACD-A buffer (4 × 500 μl) and the AC133neg cell population retained at 4°C. After magnet removal, AC133+ cells were eluted with 1 ml of cold ACD-A buffer. The AC133+ cell fraction was reapplied to a new column, prior to cell enumeration and viability assays.

Short-term ex vivo expansion cultures. Cells were seeded in duplicate in four separated wells for harvest and enumeration at d 0, 3, 5 and 8 in a stroma-free liquid culture system at the following densities: MNC (1–2 × 106/ml), AC133+ (2–4 × 104/ml) and AC133neg (2–4 × 104/ml). The liquid culture system consisted (in 1 ml total) of Iscove's-modified Dulbecco's medium (IMDM; Life Technologies, Paisley, UK) supplemented with fetal calf serum (FCS) 10% (Sigma) Aldrich and gentamycin (50 μg; Life Technologies). Culture systems were supplemented with three different growth factor conditions, respectively, A: ‘K36EG’[cKitL 20 ng/ml, IL-3 50 ng/ml, IL-6 20 ng/ml, erythropoietin (EPO) 6 U/ml, G-CSF 10 ng/ml]; B: ‘TPOFL’ (thrombopoietin 10 ng/ml, Flt-3 ligand 50 ng/ml); C: no cytokine as a control. At d 0, 3, 5 and 8, CD38, AC133 and CD34 immunophenotyping and apoptotic levels were measured as described below. IL-3, IL-6 and Flt-3 ligand were purchased from R & D systems, Abingdon, UK. G-CSF, TPO, cKitL were a kind gift from Amgen, Thousand Oaks, CA, USA. EPO was generously donated by Ortho Biotech, Raritan, USA.

HSPC immunophenotyping. Cells were incubated in human gammaglobulins [20 min, 4°C, 2% in phosphate-buffered saline (PBS), Sigma Aldrich] to block non-specific Fc receptors. Cells were directly labelled with monoclonal mouse anti-human antibodies (30 min, 4°C): anti-CD38-fluorescein isothiocyanate (FITC)-conjugated (HIT2, BD-Pharmingen, CA, USA), anti-AC133-phycoerythrin (PE)-conjugated (AC133/2, Miltenyi Biotec, Bergish Gladbach, Germany) and anti-CD34-peridinin chlorophyll (PERCP) (HPCA-II, BD-Pharmingen). Cells were then washed twice in staining buffer (400 g, 10 min, 4°C) prior to fixation in paraformaldehyde (1%, BDH, Poole, UK). Fluorescent events were acquired on a Becton Dickinson (CA, USA) FACScan flow cytometer with cellquest software prior to analysis with winmdi software.

Cell survival status. HSPC survival status was assessed using TACS™ annexin V-FITC (AV)/propidium iodide (PI), apoptosis detection kit (R & D Systems) complete with positive, negative controls and compensation tests prior to fluorescence-activated cell sorting (FACS) analysis as described above. This allowed discrimination of three cell conditions: viable cells (AV/PI), apoptotic cells (AV+/PI) and dead/necrotic cells (permeable to propidium iodide (PI+) losing their membrane permeability.

Statistical analysis. When applicable, results are expressed as mean ± standard error of the mean (SEM) from data obtained in a minimum of four separate experiments performed at least in duplicate. Statistical significance was calculated by Student's two tailed t-test for paired data.

AC133+ cell purification

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

UCB MNC immunolabelled against AC133 or CD34 antigens contained a significantly lower frequency of AC133+ cells (0·38% ± 0·03 SEM; range 0·19–0·68) than CD34+ cells (0·57% ± 0·06 SEM; range 0·22–1·31; P = 0·002, n = 28).

However, throughout this study upon AC133+ immunomagnetic cell separation performed on UCB MNC, the mean purity of AC133+ selected cells was 92·70% ± 2·81 SEM (range 84·51–97·15, n = 4) and AC133+ cells mean recovery from MNC was 73·60% ± 5·36 SEM (range 60·36–84·47, n = 4).

AC133+ cells have a higher proliferation potential than MNC and AC133neg cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

MNC, AC133+ or AC133neg populations were cultured for 8 d in stroma-free liquid cultures stimulated by K36EG, TPOFL or no cytokine as a control. A lack of cytokine resulted in no noticeable proliferation for all three populations. In both cytokine-stimulated liquid culture systems, AC133+ cells experienced a higher total nucleated cell fold increase (FI) (Fig 1) (n = 4; K36EG FI: 8·96 ± 3·54; TPOFL FI: 2·61 ± 1·32) than MNC (K36EG FI: 0·70 ± 0·24, P < 0·05; TPOFL FI: 0·19 ± 0·08, P = 0·10) or AC133neg cells (K36EG FI: 2·10 ± 0·70, P < 0·05; TPOFL FI: 0·67 ± 0·22, P = 0·08) (n = 4). As expected, K36EG stimulation resulted in significantly higher AC133+ proliferation when compared with TPOFL synergism (P < 0·05). All three UCB cell populations went through a lag phase between d 3 and d 5, prior to an exponential growth sustained until d 8 for AC133+ cells. The importance of the lag phase was highlighted by recalculation of d 8 expansion FI from the remnant cell numbers at lag phase. AC133+ cells recovered better from the lag phase with significantly higher FI (K36EG FI: 15·04 ± 5·46; TPOFL FI: 8·59 ± 2·92) when compared with MNC (K36EG FI: 7·27 ± 6·44, P < 0·05; TPOFL FI: 0·64 ± 0·24, P < 0·05) and AC133neg cells (K36EG FI: 6·01 ± 2·47, P < 0·05; TPOFL FI: 1·36 ± 0·91, P < 0·05).

image

Figure 1. Eight-day cell proliferation in liquid culture for MNC, AC133+ and AC133neg populations. Results are expressed as mean total nucleated cell fold increase (+SEM) from original cell input at d 0 (n = 4). When stimulated by K36EG (inducing proliferation) or TPOFL (promoting early HSPC maintenance), AC133+ cells proliferated more than the heterogeneous MNC and AC133neg populations (*P < 0·05). No cytokine stimulation induced no cell proliferation.

Download figure to PowerPoint

Cell populations containing AC133+ cells appear to be protected from apoptosis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

Cell survival status was assessed by flow cytometry using Annexin V-FITC (AV) and propidium iodide (PI) (Fig 2). Figure 3 represents variations in the percentages of viable (AV/PI), over 8 d in liquid cultures. Depriving the culture systems of cytokines resulted in decreased viable cell percentages for all three populations. The proliferation lag phase observed between d 3 and d 5 further correlated with a decrease in viable cells percentage, but more markedly so for AC133+ and AC133neg cells than MNC. After 8 d in culture with K36EG or TPOFL, AC133+ cells had a significantly higher proportion of viable cells (K36EG: 91·5 ± 4·7; TPOFL: 87·1 ± 3·2) than AC133neg cells (K36EG: 58·05 ± 16·2, P < 0·05; TPOFL: 42·0 ± 18·5, P < 0·05) (n = 4), whereas MNC showed similar viable cell percentages (K36EG: 91·2 ± 1·7; TPOFL: 88·41 ± 6·2) to AC133+ cells. Moreover, after 8 d in cytokine-stimulated liquid culture, the percentage of apoptotic cells (AV+/PI) within MNC (K36EG: 4·9 ± 1·2; TPOFL: 3·4 ± 1·6) and AC133+ cell (K36EG: 2·6 ± 1·1; TPOFL: 1·7 ± 0·9) populations (both containing cells originally expressing AC133), were significantly lower than within AC133neg cell population (K36EG: 23·0 ± 2·4, P < 0·01; TPOFL: 17·4 ± 1·9, P < 0·01) (n = 4).

image

Figure 2. Example of cell survival status assessed by flow cytometry using annexin V-FITC (AV) and propidium iodide (PI). Cells negative for both products are classified as viable (AV/PI). Early stages of apoptosis involve flipping of phosphatidyl serine (PS) from the inner to the outer cell membrane. Annexin V-FITC binding specifically to newly exposed PS permits detection of early apoptotic events (AV+/PI). Cells permeable to fluorescent dye PI are categorized as necrotic/late apoptotic having lost membrane permeability and cellular integrity (PI+).

Download figure to PowerPoint

image

Figure 3. Percentage variation of viable cells for MNC, AC133+ and AC133neg cell populations over 8 d in liquid culture. Results expressed as mean viable cell percentage (± SEM) of total nucleated cells harvested at d 0, 3, 5 or 8 (n = 4). At d 8 in all culture conditions, MNC and AC133+ cells demonstrated a higher percentage of viable cells than AC133neg cells. Lack of cytokine in culture caused a decrease in viability when compared with systems stimulated by K36EG or TPOFL.

Download figure to PowerPoint

TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

Flow cytometry permitted AC133, CD34 and CD38 antigen co-expression analysis throughout the ex vivo expansion study on all cultured populations (Table I). After 8 d in culture, AC133+ cells produced significantly higher levels of progenitor cells expressing AC133 and/or CD34 antigen, when compared with MNC and AC133neg cells mostly including differentiated cells (P < 0·05 for both MNC and AC133neg cells). K36EG significantly induced more differentiation of AC133+ cells than TPOFL (P < 0·05).

Table I.  Overview of AC133, CD34 and CD38 antigen co-expression amongst the MNC, AC133+ and AC133neg cell populations after 8 d growth factor-stimulated culture.
 Cultured cells
K36EGTPOFL
AC133+MNCAC133negAC133+MNCAC133neg
  1. * P-value: testing difference between MNC or AC133neg cells versus AC133+ cells subphenotypes cultured in each cytokine mix.

  2. P-value: testing the difference between AC133+ cells cultured with K36EG vs TPOFL, n = 4 at least in duplicate.

  3. Resultant subpopulations are classified in two groups: cells expressing AC133 and CD34 (still at a fairly immature stage of haemopoiesis) and cells lacking these two antigen. Figures expressed as mean percentages of total cells harvested at d 8 ±SEM.

%AC133+ and CD34+ cells20·50 ± 2·362·60 ± 0·804·67 ± 2·1947·00 ± 7·695·90 ± 1·963·45 ± 0·58
(*P = 0·01)(*P = 0·02)(P = 0·03)(*P = 0·01)(*P = 0·004)
%AC133negCD34 CD38+79·50 ± 2·3697·40 ± 0·8095·33 ± 2·1953·00 ± 7·6994·10 ± 1·9696·55 ± 0·58
and mature cells (*P = 0·01)(*P = 0·02)(P = 0·03)(*P = 0·01)(*P = 0·004)

To ensure that our culture system supported expansion of the earliest HSPC subsets, a finer subphenotyping analysis was applied to the data. AC133 and CD34 expression endorses immaturity status of HSPC. Previous work from our research group and others (Bhatia, 2001) established a model for a ‘steady state’ stem/progenitor cell compartment hierarchy by subphenotyping both early and developing HSPC. Briefly, using comparative analysis of AC133, CD34 and CD38 on developing cells, it was shown that AC133 expression on HSPC precedes dual AC133–CD34 antigen expression. As differentiation evolves, developing HSPC progressively downregulate AC133 and upregulate CD34+ internal stores to the membrane surface (Pearce et al, 1999). In applying this model to our AC133+ cells expanded for 8 d, it was found that K36EG induced more AC133+ cells to proliferate and differentiate (Fig 4). On the other hand, TPOFL provoked slower AC133+ cell proliferation but maintained a higher proportion of the earliest HSPC, i.e. AC133+CD34CD38 (% of total viable AC133 cell population after 8 d in culture with K36EG: 2·96 ± 1·5 or TPOFL: 4·01 ± 1·82, P > 0·1, n = 4). Within the expanded AC133+ cell population, TPOFL also maintained a significantly higher percentage (34·20 ± 9·00) of CD34+ cells when compared with K36EG culture (11·84 ± 2·38, P < 0·05, n = 4), which after 8 d, favoured CD34 and AC133 antigen downregulation while upregulating CD38 expression. Two other subphenotypes, which may also be of interest for marrow repopulation, were difficult to insert in our ‘steady-state’ haemopoiesis model: (i) AC133+CD34CD38+ (K36EG: 3·05 ± 1·35; TPOFL: 2·50 ± 1·48); (ii) AC133negCD34+ CD38 (K36EG: 4·92 ± 1·35; TPOFL: 7·49 ± 2·48). The latter phenotype shows downregulation of AC133 while CD34 is still expressed. Such cell subpopulations could result from CD34 reversible expression as described by other groups (Sato et al, 1999; Van Bekkum, 2001) or account for the presence of endothelial precursors in UCB (Quirici et al, 2001).

image

Figure 4. Resultant subpopulations generated from the AC133+ cell population cultured ex vivo for 8 d with K36EG or TPOFL. Results expressed as subphenotype percentages of total viable cells at d 8 (+ SEM). TPOFL maintains a higher proportion of early HSPC while K36EG induced AC133+ cell differentiation (n = 4).

Download figure to PowerPoint

Morphological appearance after 8 d in culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

In order to confirm the apoptotic phenotype identified by flow cytometry (AV/PI), cells were harvested after 8 d in culture and cytospun for 10 min at 200 g. Apoptotic cells cause their plasma and nuclear membranes to condense, forming nuclear aggregation and dislocation of the cytoskeleton (Israels & Israels, 1999). Standard haematological staining revealed examples of apoptotic cells showing typical cell disassembly into smaller membrane-bound apoptotic bodies (Fig 5).

image

Figure 5. Morphological examples taken during the apoptotic study. (A) Magnification ×40 displaying apoptotic (1) and necrotic (2) cells among normal progenitors. (B) High power (magnification ×100) oil immersion displaying cell undergoing apoptosis with characteristic nuclear degradation (3) and membrane accumulation and release of nuclear apoptotic bodies (4). Photographed following standard haematological staining.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

UCB provides immediate access to HSPC of early ontogenic origins enriched for AC133+ and CD34+ cells (de Wynter et al, 1998). However, low cell numbers harvested from average UCB collections combined with their slower engraftment pace, when compared with BM and GFMLH HSPC, limit their use in full reconstitution of adult BM. Ex vivo expansion, by amplifying early HSPC numbers available for long-term engraftment while limiting their differentiation, is one strategy to overcome these problems.

Several investigators during preclinical and clinical studies demonstrated the application of CD34 selection to isolate HSPC to be expanded ex vivo (McNiece & Briddell, 2001). However, AC133 was further described as an important marker for the identification of primitive progenitors (Bhatia, 2001) and several studies established the benefits of using AC133-purified HSPC. AC133+ cells showed a higher proliferation potential and a higher LTC-IC frequency than CD34+ cells (de Wynter et al, 1998). Hence, this study provided more insights about the clinical relevance and advantages of UCB AC133+ cells as a starting cell population for HSPC ex vivo expansion strategies.

Our results emphasized the importance of selecting a population enriched for immature HSPC as starting material for ex vivo expansion (Haylock et al, 1992). AC133+ cells were obtained through direct immunomagnetic selection. Although AC133+ cells retain bound reagents, the manufacturer (Miltenyi Biotec, personal communication) notes that this process does not alter purified cell proliferation and survival status. However, little information for such phenomena has yet been reported in the literature. Comparison with other immunomagnetic selection methods, currently under investigation in our laboratory, shows that long-term HSPC proliferation is unlikely to be affected by the direct immunomagnetic selection chosen for this study, and has also been confirmed by Servida et al (1996).

When stimulated by cytokines, AC133+ cells proliferated more than MNC or AC133neg cells that contained cells at late stages of differentiation. Mature cells consume most of the available growth factors, while contributing little to engraftment potential. Hence, early HSPC proliferation within these MNC and AC133neg cell populations is limited if not completely inhibited. Other studies also suggested the release by accessory cells of haemopoietic growth inhibitory cytokines (macrophage inhibitory protein 1-α, tumour necrosis factor α-β or interferon γ), which would restrain early HSPC proliferation (Gilmore et al, 2000).

Our 8 d growth factor-stimulated liquid culture system was applied to help better understand the early kinetics of cytokine-stimulated HSPC growth in order to optimize ex vivo expansion technology on both earlier more immature cells and low cell numbers. This is of extreme importance as maintenance of the required cells in the early stages of ex vivo expansion has been found to be problematic, particularly from umbilical cord blood. This work identified a lag phase, occurring at an early stage of ex vivo expansion, prior to an exponential growth phase for AC133+ cells when stimulated by K36EG or TPOFL cytokine mix. This has also been reported by other investigators using UCB CD34+ cells as a starting population. Recovery from the lag phase took between 6 and 12 d prior to exponential growth (Gilmore et al, 2000). Our results demonstrated that the lag phase in proliferation correlated with a decrease in viable cells and an increase in apoptotic cells. Stroma-free liquid culture systems differ greatly from the physiological environment hosting haemopoietic cells development. The osmotic shock encountered by growing cells seeded in culture appeared to result in increased apoptotic and necrotic events. In addition, the culture system may lack unknown plasma proteins and growth factors contributing to cellular homeostasis in vivo (Koury, 1992). This initial cell loss may be due to delayed HSPC response to the early acting cytokines used in this assay. Additional growth factor feeding after 3 d of culture may be a way to overcome or minimize such a lag phase and accelerate the onset of HSPC exponential growth.

When compared with K36EG stimulation, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher proportion of AC133+ and CD34+ HSPC (P < 0·05), including the earliest haemopoietic cells that we could phenotype (AC133+CD34CD38). This population may be essential for transplantation as Gallacher et al (2000) reported an extremely discrete CD34AC133+ cell population highly enriched for severe combined immunodeficiency-repopulating and progenitor activity. In order to ensure long-term engraftment to patients, ex vivo expansion culture systems must maintain and amplify the amount of early HSPC available for transplantation (McNiece & Briddell 2001). Hence, limiting in vitro production of committed progenitors and differentiated cells is necessary to restore long-lasting haemopoiesis in engrafted recipients but also to modulate further GVHD intensity.

Several groups opted for the TPOFL cytokine combination to successfully expand and maintain early HSPC populations in both short-term and long-term liquid culture, sometimes for over 6 months (Piacibello et al, 1997; Gilmore et al, 2000; Matsumoto et al, 2000). Our ongoing work will further investigate AC133+ cells' potential in ex vivo expansion systems stimulated by TPOFL supplemented with cKitL. Recent studies have combined TPOFL with cKitL in order to enhance HSPC self-renewal and optimize cell survival by inhibiting apoptosis (Kohler et al, 1999; Murray et al, 1999; Lewis & Verfaillie, 2000).

TPOFL synergism not only maintained earlier HSPC within expanded AC133+ cells but also induced less apoptotic events than K36EG stimulation in our tests, although this difference was not statistically significant. Maintaining an early quiescent HSPC pool through self-renewal may require suppression of apoptosis and blockade of their progression through the cell cycle ‘check points’. The latter induce apoptosis when cellular integrity is impaired (Israels & Israels, 1999). In this study, after 8 d of culture, cell populations lacking AC133+ cells were significantly more inclined to undergo apoptosis than AC133+ selected cells or MNC under proliferative conditions. This is in agreement with previous work showing that GFMLH AC133negCD34+ cells ex vivo expanded with TPOFL for 12 d and contained a higher proportion of apoptotic cells than AC133+ CD34+ cells (Matsumoto et al, 2000) and is an important finding for ex vivo expansion development using umbilical cord blood as a starting product. In this study, the AC133neg cell subset contains the majority of unfractionated MNC. One might hypothesize that the difference in cell survival observed between AC133neg cells and MNC was due to MNC being cultured at a concentration 100-fold higher. However, it is important to remember that AC133neg cells contain not only maturing cells but also an important number of CD34+ proliferative cells, which accounts for our choice of seeding concentration. Further, although MNC contain all the required proliferative cells of interest, their unique low concentration (in MNC) requires a higher seeding density. Under cytokine stimulation, neither MNC nor AC133neg cells showed notable proliferation potential when compared with purified AC133+ cells.

Taken together, these results suggest a possible role for AC133 in limiting apoptotic onset. AC133+ cells may either release soluble factors protecting surrounding cells from apoptosis or operate via cell–cell interactions. These methods are well known for importance in controlling HSPC destiny (McNiece & Briddell 2001). The fact that the MNC (which also contain AC133+ cells) cannot sustain proliferation even at a higher seeding density lends further credence to the hypothesis that a critical cell number of HSPC needs to be reached for expansion. Our laser scanning confocal microscopy and transmitted electron microscopy imaging has demonstrated AC133 antigen distribution to isolated membrane protrusions as supported by studies from our group and others (unpublished observations). We found the AC133 antigen was located to extensive membrane pseudopodia, which inferred its possible involvement in cell–cell communication (McGuckin et al, 1999; Corbeil et al, 2000). This is strongly supported by the presence of a leucine-zipper motif on the second extracellular loop of the AC133 antigen, as leucine-zippers are known to cause protein homo/heterodimerization (Fry et al, 1999). AC133 involvement in intercellular communication may provide a role in prevention of apoptosis through bio-mechanisms yet to be identified.

In conclusion, TPOFL synergism demonstrated potential for AC133+ HSPC ex vivo expansion, inducing slow proliferation, maintenance of early HSPC as well as promoting HSPC survival status. The TPOFL combination and the AC133+ population appear to be promising partners to initially expand purified UCB HSPC ex vivo.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References

We thank Dr Jenny Tooze from St George's Hospital, Department of Haematology, London, UK for her precious assistance with flow cytometry. We are grateful to Mr Pretish Raja for technical assistance with staining of prepared cells in Fig 5. We are also very grateful to St George's Hospital Delivery Suite staff for their help in collecting umbilical cord blood samples.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. AC133+ cell purification
  6. AC133+ cells have a higher proliferation potential than MNC and AC133neg cells
  7. Cell populations containing AC133+ cells appear to be protected from apoptosis
  8. TPOFL ex vivo expansion of UCB AC133+ cells maintains a higher proportion of early HSPC than K36EG
  9. Morphological appearance after 8 d in culture
  10. Discussion
  11. Acknowledgments
  12. References
  • Aglietta, M., Bertolini, F., Carlo-Stella, C., De Vincentis, A., Lanata, L., Lemoli, R.M., Olivieri, A., Siena, S. & Zanon, P. (1998) Ex vivo expansion of hematopoietic cells and their clinical use. Haematologica, 83, 824848.
  • Bhatia, M. (2001) AC133 expression in human stem cells. Leukemia, 15, 16851688.
  • Corbeil, D., Roper, K., Hellwig, A., Tavian, M., Miraglia, S., Watt, S.M., Peault, B., Buck, D.W. & Huttner, W.B. (2000) The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. Journal of Biological Chemistry, 275, 55125520.
  • Fry, A.M., Arnaud, L. & Nigg, E.A. (1999) Activity of human centrosomal kinase, Nek2, depends on unusual leucine zipper dimerization motif. Journal of Biological Chemistry, 274, 1630416310.
  • Gallacher, L., Murdoch, B., Wu, D.M., Karanu, F.N., Keeney, M. & Bhatia, M. (2000) Isolation and characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hemopoietic stem cells using cell surface markers AC133 and CD7. Blood, 95, 28132820.
  • Gilmore, G.L., Depasquale, D.K., Lister, J. & Shadduck, R.K. (2000) Ex-vivo expansion of human umbilical cord blood CD34+ hemopoietic stem cells. Experimental Hematology, 28, 12971305.
  • Gluckman, E. (2000) Current status of Umbilical Cord Blood haemopoietic stem cell transplantation. Experimental Hematology, 28, 11971245.
  • Haylock, D.N., To, L.B., Dowse, T.L., Juttner, C.A. & Simmons, P.J. (1992) Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage. Blood, 80, 1405/1a.
  • Israels, G. & Israels, D. (1999) Apoptosis. Oncologist, 4, 332339.
  • Kohler, T., Plettig, R., Wetzstein, W., Schaffer, B., Ordemann, R., Nagels, H.O., Ehninger, G. & Bornhäuser, M. (1999) Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels. Stem Cells, 17, 1924.
  • Koury, M.J. (1992) Programmed cell death (apoptosis) in hematopoiesis. Experimental Hematology, 20, 391394.
  • Lewis, I.D. & Verfaillie, C.M. (2000) Multi-lineage expansion potential of primitive hematopoietic progenitors: Superiority of umbilical cord blood compared to mobilized peripheral blood. Experimental Hematology, 28, 10871095.
  • McGuckin, C.P., Pettengell, R., Martin, F., Jones, K., Gordon-Smith, E.C. & Pearce, D. (1999) Is AC133 involved in direct cell to cell communication? Experimental Hematology, 28, 160.
  • McNiece, I. & Briddell, R. (2001) Ex-vivo expansion of haemopoietic progenitor cells and mature cells. Experimental Hematology, 28, 311.
  • Matsumoto, K., Yasui, K., Yamashita, N., Horie, Y., Yamada, T., Tani, Y., Shibata, H. & Nakano, T. (2000) In vitro proliferation potential of AC133 positive cells in peripheral blood. Stem Cells, 18, 196203.
  • Murray, L.J., Young, J.C., Osborne, L.J., Luens, K.M., Scollay, R. & Hill, B.L. (1999) Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ and recruit primitive CD34+Thy-1+ cells into rapid division. Experimental Hematology, 27, 10191028.
  • Pasino, M., Lanza, T., Marotta, F., Scarso, L., De Biasio, P., Amato, S., Corcione, A., Pistoia, V. & Mori, P.G. (2000) Flow cytometric and functional characterization of AC133+ cells from human umbilical cord blood. British Journal of Haematology, 108, 793800.
  • Pearce, D., Pettengell, R., Gordon-Smith, E.C. & McGuckin, C.P. (1999) Intracellular Analysis of AC133+ cell subsets. Blood, 94, 132b.
  • Piacibello, W., Sanavio, F., Garetto, L., Severino, A., Bergandi, D., Ferrario, J., Fagioli, F., Berger, M. & Aglietta, M. (1997) Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood, 89, 26442653.
  • Quirici, N., Soligo, D., Caneva, L., Servida, F., Bossolasco, P. & Lambertenghi Deliliers, G. (2001) Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells. British Journal of Haematology, 115, 186194.
  • Rocha, V., Wagner, J.E., Sobocinski, K., Klein, J., Zhang, M.J., Horowitz, M.M. & Gluckman, E. (2000) Graft versus host disease in children who have received a cord blood or bone marrow transplant from an HLA identical sibling. New England Journal of Medicine, 342, 18461853.
  • Sato, T., Laver, J.H. & Ogawa, M. (1999) Reversible expression of CD34 by murine hematopoietic stem cells. Blood, 94, 25482552.
  • Servida, F., Soligo, D., Caneva, L., Bertolini, F., De Harven, E., Campiglio, S., Corsini, C. & Lambertenghi Deliliers, G. (1996) Functional and morphological characterization of immunomagnetically selected CD34+ Hematopoietic Progenitor Cells. Stem Cells, 14, 430438.
  • Van Bekkum, D.W. (2001) New Stem Cell Workshop. Stem Cells, 19, 260262.
  • De Wynter, E.A., Buck, D., Hart, C., Heywood, R., Coutinho, L.H., Clayton, A., Rafferty, J.A., Burt, D., Guenechea, G., Bueren, J.A., Gagen, D., Fairbairn, L.J., Lord, B.I. & Testa, N.G. (1998) CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors. Stem Cells, 16, 387396.