The plant mannose-binding lectin NTL preserves cord blood
haematopoietic stem/progenitor cells in long-term culture and
enhances their ex vivo expansion
Umbilical cord blood is an important source of haematopoi-etic stem cells for the treatment of malignancy, immunological and genetic blood disorders. To date, more than 6000 cord blood transplantations have been performed and 400 000 units stored worldwide (Brunstein et al, 2007). Although cord blood harbours good quality haematopoietic stem cells in terms of self-renewal and clonogenic capacity, its clinical application to the patients of large body weight and outcomes of transplan-tation have been limited by the number of stem cells in a cord blood unit (Gluckman et al, 2005). Efforts to overcome this shortfall have been investigated through ex vivo expansion in the presence of promoting factors with or without stromal cell support (Sphall et al, 2002; Jaroscak et al, 2003), and utiliza-tion of multiple units of cord blood for a single transplantautiliza-tion
(Majhall et al, 2006). To date, the mechanisms and clinical success of these strategies have not been well established. Although the lifelong supply of haematopoietic cells has been sustained by their stem cells in the bone marrow (BM) niche, effects of known cytokines on ex vivo expansion have been limited, with data showing promotion of differentiation and proliferation at the expense of self-renewable stem cells (Bhatia et al, 1997; McNiece et al, 2002).
Colucci et al (1999) reported that a mannose-binding flt-3 receptor-interacting lectin (FRIL) purified from a dicotyledon hyacinth bean (Dolichos lablab) has the ability to preserve primitive haematopoietic progenitor cells in suspension cul-ture for 1 month without serum or growth factor supplement. FRIL also supports prolonged in vitro maintenance of cord Karen Li,1Vincent E. C. Ooi,2Carmen
Ka Yee Chuen,1Audrey Carmen Lam,1
Linda Shiou Mei Ooi,2Xiao Bing Zhang, 1Kam Sze Tsang,3Lawrence Chi Ming
Chiu,2Kathy Yuen Yee Chan,1Chi Kong
Li,1Tai Fai Fok,1Patrick Man Pan Yuen1and Pak Cheung Ng1 1Li Ka Shing Institute of Health Sciences, Department of Paediatrics,2Department of Biology, and3Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China
Received 29 July 2007; accepted for publication 10 August 2007
Correspondence: K. Li, Department of Paediatrics, The Chinese University of Hong Kong 6th Fl, Clinical Sciences Block Prince of Wales Hospital, Shatin NT, Hong Kong, China. E-mail: [email protected]
Summary
Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis, on prolonged
maintenance and expansion of cord blood CD34+cells. Our results showed
that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free
culture for 35 d. The effect of NTL on the ex vivo expansion of CD34+cells in
the presence of stem cell factor, thrombopoietin (TPO) and FL was also
investigated. NTL-enhanced expansion of early progenitors (CD34+,
CD34+CD38), mixed colony-forming units and CFU-GEMM) and
committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe
combined immunodeficient mice, increased engraftment of human CD45+
cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications
in various cell therapy strategies, such as the clinical expansion of CD34+cells
for transplantation.
Keywords: cord blood CD34+cells, ex vivo expansion, flt-3 ligand,
mannose-binding lectin NTL, severe combined immunodeficient-repopulating cells.
blood CD34+CD38)/severe combined immunodeficient (SCID) repopulating stem cells. The suggested mechanism includes the preservation of CD34+ cells in the G
0/G1 phase
(Kollet et al, 2000) and modulation of cell cycle regulatory gene HTm4 and HTm4S expressions (Xie et al, 2004). In the present study, we investigated a novel mannose-binding lectin NTL on prolonged maintenance of enriched cord blood CD34+cells in a serum-free, cytokine-free culture and ex vivo expansion of these cells in the presence of early-acting cytokines. NTL was purified from the leaves of a monocot Chinese daffodil, Narcissus tazetta var. chinensis, which belongs to the family Amaryllidaceae (Ooi et al, 1998, 2001). NTL has a molecular mass of 26 kDa and exists as an unglycosylated homodimer (Ooi et al, 2001). It does not exert potent cytotoxicity against mouse (L929 and ROS) or human (hu) (SPC-A1, MKN-28 and M7609) cancer cell lines (Ooi et al, 2000). In mouse, intraperitoneal injection of NTL modulates the gene expression of immuno-regulatory cytokines such as interferon-c, transforming growth factor-b and stem cell factor (SCF) in macrophages and/or spleen cells (Ooi et al, 2002). In the present study, our results showed that NTL preserved haematopoietic stem and progenitor cells after 35 d of culture in a serum- and cytokine-free condition, and enhanced ex vivo expansion of SCID-repopulating cells.
Materials and methods
Purification of Narcissus tazetta lectins NTL
Lectins from the leaves of the Chinese daffodil, Narcissus tazetta var. chinensis, was prepared as described previously (Ooi et al, 1998, 2001), using diethylamino ethanol-cellulose affinity column chromatography (Sigma, St Louis, MO, USA) on Affi-gel Blue gel (Pharmacia, Uppsala, Sweden) and mannose–agarose (Sigma), chromatography and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12 (Sigma). Haemagglutination activity was determined using a serial twofold dilution of the lectin solution in microtitre U-bottomed plates (50 ll) that was mixed with 50 ll of a 2% suspension of rabbit erythrocytes in pH 7Æ2 phosphate-buffered saline (PBS) at room temperature. Specific activity is the number of haemagglutination units per mg protein. The purity of NTL was estimated to be >99% by FPLC-gel filtration, PD10 desalting column (Bio-Rad, Hercules, CA, USA) and sodium dodecyl sulphate polyacrylamide gel elec-trophoresis (SDS-PAGE). The molecular mass of the purified lectins was determined to be about 26 kDa by gel filtration and 13 kDa by SDS-PAGE. The NTL is therefore a mannose-binding, non-glycoprotein dimer with a molecular subunit of 13 kDa. The presence of carbohydrates (glycoproteins) in the lectin was tested by Fuchsin-sulfite (Sigma) staining after electrophoresis in polyacrylamide slab gel containing 0Æ1% SDS. The cDNA-derived amino acid sequence of NTL was determined by molecular cloning (Ooi et al, 2001). The primary structure contains a mature polypeptide consisting
of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr–Gly. There are two fixed position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulfide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One-third of the deduced amino acid composition consisted of glycine, leucine and asparagine.
Collection of cord blood CD34+cells
Blood samples were collected from umbilical cord veins during normal full-term, vaginal deliveries and processed within 24 h. Mononuclear cells (MNC) were prepared by density gradient centrifugation (Ficoll Hypaque 1Æ077 g/ml; Amersham Phar-macia, Uppsala, Sweden). CD34+cells were enriched using the
VarioMACS Isolation Kit (Miltenyi Biotec Inc., Gladbach, Germany) as described previously (Li et al, 2005, 2006). Informed consent was obtained from the mothers for all blood sample collections and the study was approved by the Ethics Committee for Clinical Research of The Chinese University of Hong Kong.
Long-term preservation of stem/progenitor cells in serum-and cytokine-free culture
The MNC at 2 · 105/ml (n = 8) or enriched CD34+ cells (n = 5) at 2 · 104/ml were cultured in 5 ml X-Vivo 10 medium (Biowhittaker; Walkersville, MD, USA) in 25 cm2
flasks (Corning; New York, NT, USA), with or without NTL (200 ng/ml), or flt-3 ligand (FL, 40 ng/ml). The concentra-tion of NTL used was optimized previously in a dose-effect pilot study. All cytokines were purchased from Peprotech (Rocky Hill, NJ, USA), and culture reagents from Gibco (Grand Island, NY, USA) unless specified otherwise. The cultures were maintained at 37C and 5% CO2 in a fully
humidified atmosphere without medium change. At day 14, 21, 28 and 35, total nucleated cell (TNC) counts, cell viability and haematopoietic colony forming unit (CFU) assays were performed.
Ex vivo expansion of cord blood CD34+cells
Enriched CD34+cells (n = 16) at 2 · 104/ml were cultured in
Iscove’s modified Dulbecco’s medium and 10% fetal calf serum (FCS) in 24-well culture plates (Becton Dickinson, Franklin Lakes, NJ, USA). The cultures contained thrombo-poietin (TPO, 50 ng/ml), stem cell factor (SCF, 50 ng/ml) and FL (80 ng/ml), with or without NTL (200 ng/ml). At day 4 and 8, cell cultures were split into three portions, with fresh medium and cytokines added. TNC counts, flow cytometric analysis of progenitor cells and CFU assays were performed on day 0, 8 and 12. Cells at day 0 and expanded cells at day 12 were infused into sublethally irradiated non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice for the analysis of NOD/SCID-repopulating cells.
NTL Preserves and Expands CD34 Cells
Flow cytometric analysis of haematopoietic stem/ progenitor cells
Enriched CD34+cells or expanded cells were stained with CD34-fluorescein isothiocyanate (FITC), CD38-phycoerythrin (PE), CD61-FITC (Dako, Copenhagen, Denmark), CD41-PE (Dako) and respective isotype controls for 20 min. All antibodies and cytometric reagents were purchased from Becton Dickinson-Pharmingen (BD, San Jose, CA, USA), unless specified otherwise. The cells were then washed and resuspended in PBS with 0Æ5% bovine serum albumin (BSA; Sigma). 7-Amino-actinomycin D was added to the cells prior to flow cytometric acquisition for the purpose of gating out dead cells, using a FACS Calibur flow cytometer and the CellQuest software (BD).
Colony-forming unit assay
Granulocyte–macrophage colony-forming units (CFU-GM), erythroid burst-forming units/colony-forming units (BFU/
CFU-E) and mixed colony-forming units (CFU-GEMM) were cultured in methylcellulose (1%) supplemented with FCS (30%), 1% BSA, 0Æ1 mmol/l b-mercaptoethanol, 3 IU/ml erythropoietin (Cilag, Zug, Switzerland), 10 ng/ml granulo-cyte–macrophage colony-stimulating factor (GM-CSF; Sandoz, Basele, Switzerland), 10 ng/ml interleukin-3 and 50 ng/ml SCF. Enriched CD34+ cells or expanded cells at 3 · 103/ml were seeded in triplicate. Colonies were scored after culturing for 14 d. Megakaryocyte colony forming units (CFU-MK) were assayed using the plasma clot system as described previously (Li et al, 2005, 2006). CFU-MK was identified as a cluster of three or more strongly stained CD61-FITC (Dako) positive cells examined by fluorescence microscopy.
Engraftment of expanded cells in NOD/SCID mice The NOD/LtSZ-scid/scid mice were obtained from The Walter and Eliza Hall Institute of Medical Research (Melbourne, Vic., Australia), and bred in the Laboratory Animal Services Centre
0 100 200 300 400 500 600 700 800 900 (A) (B) (C) (D)
Total CFU per 1
× 10 5 CD34+ cells * * * * ** ** ** 0 20 40 60 80 100 120 140 CFU-GEMM per 1 × 10 5 CD34+ cells * * ** ** * 0 100 200 300 400 500 600 700 Total CFU per 1 × 10 6 MNC ** ** * ** ** ** **** ** 0 10 20 30 40 50 60 70 80 D14 D21 D28 D35 Duration of culture (d) D14 D21 D28 D35 Duration of culture (d) D14 D21 D28 D35 Duration of culture (d) D14 D21 D28 D35 Duration of culture (d) CFU-GEMM per 1 × 10 6 MNC Control NTL200 FL40 FL + NTL200 Control NTL200 FL40 FL + NTL200 Control NTL200 FL40 FL + NTL200 Control NTL200 FL40 FL + NTL200 ** * ** ** * **
Fig 1. NTL preserved stem/progenitor cells in serum- and cytokine-free cultures. Enriched CD34+cells (n = 5; A and B) or cord blood mononuclear cells (n = 8; C and D) were cultured in X-VIVO-10 medium for 14, 21, 28 or 35 d without medium change. Colony-forming units (CFU) assays demonstrated that the presence of NTL significantly increased total CFU (A and C) and CFU-GEMM (B and D) in one or more time points. Results are presented as mean ± standard error of the mean. *P < 0Æ05; **P < 0Æ01 compared with respective control cultures without NTL.
at The Chinese University of Hong Kong. Mice at 8–10 weeks of age (n = 90) were exposed to 280–320 cGy total body irradiation from a 137Cs source (Gammacell-1000 Elite
Irra-diator; MDS Nordion, Kanata, ON, Canada). In each inde-pendent experiment (n = 15), 3 · 104enriched CD34+cells at
day 0 and cells expanded for 12 d (progenies of 3 · 104cells), in the presence or absence of NTL were infused into sex- and age-matched mice. To prevent the loss of data because of animal mortality, two mice were assigned to each treatment group and the engraftment parameters averaged as a single datum for analysis, as described previously (Su et al, 2002). Mice were killed 6 weeks post-transplantation. All procedures were approved by the Animal Research Ethics Committee, The Chinese University of Hong Kong.
The engraftment of huCD45+ cells and subsets in the BM, spleen and peripheral blood (PB) was quantified. For flow cytometric analysis, red blood cells were lysed with 0Æ83% ammonium chloride and washed with PBS/0Æ1% BSA. The cells were incubated with mouse IgG and 5% human serum (Gibco) before adding monoclonal antibody specific for huCD45 (PC5; Immunotech, Marseille, France), CD34-FITC and propidium iodide (PI; 10 lg/ml; Sigma) for 20 min. Seventy thousand events were acquired. BM samples that contained >1% human cells were further immunophenotyped using anti-human antibodies CD19-PE, CD14-PE, CD33-PE, CD61-PE (Dako) and their isotypic controls. Non-viable cells (PI positive) were gated out during data analysis. Human CFU in these BM samples were also analysed, using methylcellulose culture and scored after 14 d. As described previously, this culture duration was selective for huCFU assay and did not support murine CFU formation, which normally took 7 d (Su et al, 2002).
Statistical analysis
Treatment groups were compared by analysis of variance and paired t-test or Wilcoxon Sign Rank test, depending on data distribution, using the SigmaStat software (Jandel Scientific Software; San Rafael, CA, USA). We compared survival rates of NOD/SCID mice using the Fisher’s exact test. A P-value of £0Æ05 was considered as statistically significant. All values were expressed as mean ± standard error of the mean.
Results
NTL preserved stem/progenitor cells in serum- and cytokine-free culture
In the absence of serum and cytokine, all subsets of haemat-opoietic stem/progenitor cells (total CFU, CFU-GM, CFU-E and CFU-GEMM) were significantly decreased with increased duration of culture (Fig 1). In control cultures, total and differential CFU were extensively reduced, compared with their respective counterparts at day 0 (<20% at day 14 and <5% at day 35; data not shown). The early progenitor colonies
CFU-GEMM were undetectable in all day 35 cultures, with either MNC (n = 8) or enriched CD34+ (n = 5) cells as the
starting cell population (Fig 1B and D). The presence of NTL at 200 ng/ml significantly increased total CFU and CFU-GEMM at one or more time points, compared with the respective control cultures (Fig 1, *P < 0Æ05 and **P < 0Æ01). Similar effects were observed on the concentrations of BFU/ CFU-E and CFU-GM in culture (data not shown). The addition of FL at 40 ng/ml or FL + NTL preserved total and differential CFU at a comparable manner.
NTL promoted ex vivo expansion of enriched CD34+cells
The cell viability was high after 12 d of ex vivo culture with (95Æ0 ± 0Æ90%; n = 16) or without (93Æ0 ± 0Æ94%) the addi-tion of NTL. In the presence of TPO, SCF and FL (TSF), efficient expansions were observed in TNC at day 8 (116 ± 20Æ2-fold) and day 12 (424 ± 68Æ8-fold); as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays (Fig 2). The presence of NTL significantly increased TNC (148 ± 24Æ5-fold at day 8 and 572 ± 91Æ9-fold at day 12; P < 0Æ01) and the expansion of early progenitor cells (CD34+, CD34+CD38) and CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (Fig 2; **P < 0Æ01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+cells in the presence of NTL (8Æ58 ± 2Æ14 · 105 vs. 7Æ30 ± 1Æ82 · 105 cells/ml, P < 0Æ001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in
0 50 100 150 200 250
CD34+ CD34+CD38– CFU-GM BFU/CFU-E CFU-MK CFU-GEMM
Fold increase D8 TSF D8 TSF + NTL D12 TSF D12 TSF + NTL ** ** ** ** ** ** ** ** ** ** ** **
Fig 2. Effects of NTL on ex vivo expansion of cord blood CD34+ cells. Enriched cord blood CD34+cells at 2 · 104/ml were cultured in the presence of thrombopoietin (T, 50 ng/ml), stem cell factor (S, 50 ng/ml) and flt-3 ligand (F, 80 ng/ml), with or without NTL (200 ng/ml) for 8 or 12 d. Medium change and cell dilution (1:3) were performed on day 4 and 8. The Y-axis represents the fold increase of individual progenitor subsets relative to those in the seeding popula-tion at day 0. NTL significantly enhanced the expansion of various stem and progenitor cell sub-populations as determined by flow cytometry and colony-forming units assay. Results are presented as mean ± standard error of the mean and n = 16. **P < 0Æ01 when compared with respective TSF cultures without NTL at day 8 or 12.
NTL Preserves and Expands CD34 Cells
the proportion of CD34+cells, CD34+CD38)cells (Fig 3A and B) and the density of CFU-GM, BFU/CFU-E and CFU-GEMM (Fig 3C). The proportions of megakaryocytic progenitors CFU-MK and CD61+CD41+ cells were not affected by the
addition of NTL.
Six weeks after enriched CD34+cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/ SCID mice, there was no difference in the mortality rates of
animals in the day 0 (10%), day 12 TSF (13Æ3%) and day 12 TSF + NTL (10%) group. HuCD45+ cells were detectable in
the BM, spleen and PB of the mice (Fig 4A). In the BM, there were engraftments of human haematopoietic cells of the early (CD34+), myeloid (CD33+and CD14+), B-lymphoid (CD19+)
and megakaryocytic (CD61+) lineages (Fig 4B). In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of huCD45+cells in the BM (19Æ3%
0 1 2 3 4 5 6 7 (A) (B) (C) CD34+ CD34+CD38–
Proportion of expanded cells (%)
D8 TSF D8 TSF + NTL D12 TSF D12 TSF + NTL ** * ** 1·09% 0·93% a 1·8% 1·82% b 100 101 102 FL1-height 103 104 1010 0 0 10 1 10 2 10 3 10 4 101 102 FL1-height FL2-height 10 0 10 1 10 2 10 3 10 4 FL2-height 103 104 0 20 40 60 80 100 120
CFU-GM BFU/CFU-E CFU-MK CFU-GEMM Total CFU
CFU per 3000 cells
D8 TSF D8 TSF + NTL D12 TSF D12 TSF + NTL ** * ** ** * * **
Fig 3. NTL increased the proportion of stem/progenitor cells in ex vivo expansion culture. Enriched CD34+ cells were cultured in the presence of thrombopoietin (T, 50 ng/ml), stem cell factor (S, 50 ng/ml) and flt-3 ligand (F, 80 ng/ml), with or without NTL (200 ng/ml) for 8 or 12 d. NTL significantly increased the proportions of (A) CD34+and CD34+CD38)cell subsets as demonstrated by (B) flow cytometry (CD34-FITC/CD38-PE staining, a = D12 TSF, b = D12 TSF + NTL) and (C) densities of progenitors of the myeloid [colony-forming units (CFU)-GM], erythroid (BFU/ CFU-E) and early lineages (CFU-GEMM). Results are presented as mean ± standard error of the mean and n = 16. *P < 0Æ05, **P < 0Æ01 compared with respective TSF cultures without NTL at day 8 or 12.
vs. 11Æ5%; P = 0Æ03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0Æ07, n = 14). There was no significant difference
between engraftment of fresh (day 0) CD34+ cells and expanded cells with or without NTL. The BM of the three groups of NOD/SCID mice did not show any difference in the proportion of human myeloid (CD33+and CD14+), erythroid
(BFU/CFU-E), lymphoid (CD19+), megakaryocytic (CD61+)
or early progenitor cells (CFU-GEMM) (Fig 4B and C).
Discussion
The results of this study demonstrated that NTL possessed the activity of preserving haematopoietic stem and progenitor cells in long-term culture in the absence of serum and growth factors. Whilst the early progenitor CFU-GEMM became undetectable after culturing 1 · 105 CD34+ cells or 1 · 106 MNC for 35 d, the presence of NTL significantly maintained small quantities (18Æ0 ± 9Æ60/1 · 105 CD34+ cells or 14Æ6 ± 4Æ97/1 · 106 MNC seeded) of CFU-GEMM in these cultures (Fig 1). Our data are in line with those reported by Colucci et al (1999) on FRIL, where CFU were detectable in FRIL-supplemented cultures for 29 d. Our results showed that FL at 40 ng/ml also exerted progenitor preservation activities, but the presence of both NTL and FL did not give additional or synergistic effects at day 14, 21, 28 or 35 d of culture. FL has been reported to induce proliferation of quiescent CD34+CD38) cells and maintain progenitor cells in vitro for >60 d of culture (Shah et al, 1996). However, in their study, FL was added either in the presence of BM stromal cells or other growth factors. Our data on FL were different from those of Colucci et al (1999), who did not observe CFU formation at 21 and 29 d of culture. One possible reason for the discrepancy of results on FL could be the absence of FCS and the slightly fewer seeding cells (80% of those in our study) in their methylcellulose culture system, resulting in the compromised sensitivity of detecting low numbers of CFU.
In the ex vivo expansion study, the cytokine combination of TPO, SCF and FL in QBSF-60 serum-free medium significantly expanded CD34+ cells to early and lineage-committed pro-genitors after 8 d of culture. The numbers of various cell subsets were further increased at day 12 (Fig 2). However, the proportions of early progenitors, in terms of the percentage of expanded cells and CFU/3000 cells, were generally reduced at day 12 (Fig 3). These observations were in agreement with studies that prolonged ex vivo culture might promote differ-entiation and possibly at the expense of early progenitors (Bhatia et al, 1997; Mo¨best et al, 1999). The presence of NTL significantly increased the numbers of all subsets of stem and progenitor cells. It is significant that the expansion not only resulted from cell proliferation, but also increased proportions of early progenitor CD34+ cells, CD34+CD38) cells and
densities of multi-lineage CFU (Fig 3) at both day 8 and 12 of culture. The promoting effects of NTL on the expansion of SCID-repopulating cells after 12 d of culture were confirmed by the increased engraftment of huCD45+cells in the BM of these animals 6 weeks post-transplantation, compared with cultures containing only TSF. The preservation of stem and
0 5 10 15 20 25 30 (A) (B) (C) BM Spleen PB
HuCD45+ cell engraftment (%)
*
0 5 10 15 20 25 30 35 40 45 CD34 CD14 CD33 CD19 CD61Cell engraftment (% CD45+ cells)
0 2 4 6 8 10 12 14 16 18
CFU-GM BFU/CFU-E CFU-GEMM Total CFU
CFU in BM (per 2 × 10 5 cells) Day 0 TSF TSF + NTL Day 0 TSF TSF + NTL Day 0 TSF TSF + NTL
Fig 4. Engraftment of human haematopoietic cells in non-obese dia-betic (NOD)/severe combined immunodeficient (SCID) mice. Day 0 enriched CD34+cells and cells expanded for 12 d in the presence of TSF, with or without NTL, were infused in sub-lethally irradiated NOD/SCID mice. (A) After 6 weeks, a significant increase in huCD45+ cell engraftment was observed in BM of animals that received expanded cells in the NTL group compared with those without NTL (P = 0Æ03, n = 15). (B and C) Multilineage engraftment of human progenitor [CD34+and colony-forming units (CFU)-GEMM], myeloid (CD14+, CD33+and CFU-GM), lymphoid (CD19+) and erythroid (BFU/CFU-E) cells was observed in bone marrow of animals that received day 0 and expanded cells, but the proportions of these subset were not affected by NTL.
NTL Preserves and Expands CD34 Cells
progenitor cells by NTL was reflected by the similar numbers of NOD/SCID repopulating cells in animals that received non-expanded cells and 12-d non-expanded cells (Fig 4A). Mo¨best et al (1999) reported that ex vivo expansion of PB CD34+cells for
7 d significantly reduced long-term culture initiating cells and NOD/SCID repopulating cells. Bhatia et al (1997) achieved two- to fourfold increase in SCID-repopulating cells after culturing cord blood CD34+cells for 4 d. However, after 9 d of culture, all SCID-repopulating cells were lost, despite further increases of total cells, CFU and CD34+cells. In our study, the presence of NTL in a 12-d expansion preserved SCID-repopulating cells. NTL appeared to possess the functions of preserving early progenitor cells for long-term engraftment and simultaneously enhancing early-promoting cytokines on the expansion of multilineage progenitors of the myeloid, erythroid and megakaryocytic lineages. We provided evidence for the first time that FL also has such dual activities.
The mechanism of NTL or FRIL on haematopoietic progenitor cells remains unclear. In the study of Kollet et al (2000), cell cycle analysis was performed after enriched CD34+
cells were cultured for 3 d in the presence of various combinations of cytokines with or without FRIL. However, the postculture cell population would inevitably be heteroge-neous and contained various subsets of early and lineage-committed CD34+cells. So, it remains uncertain which subsets of CD34+cells or even CD34) progenitors had the cell cycle profile affected by FRIL. A similar difficulty of interpretation also appeared in the study of Xie et al (2004) who cultured CD34+ cells for 14 d. In our long-term culture experiments, NTL has preserved the survival of a small population of early (CFU-GEMM) as well as committed progenitors (CFU-GM and BFU/CFU-E). During ex vivo expansion, NTL enhanced the proliferation of committed progenitors as well as SCID-repopulation cells. We speculate that the preservation of early stem cells and promotion of expansion are not necessarily
contradictory functions, as effects of NTL on stem/progenitor cells at different cell cycle status or stage of differentiation could vary. In addition, the regulation could be further modified in the presence of known early acting cytokines TPO, FL and SCF.
Lectins are a class of extensively investigated proteins with carbohydrate-binding specificity, occurring in plants, animals and microorganisms. They have been used in studies of blood grouping, erythrocyte polyagglutination, lymphocyte subpop-ulation, cell fractionation and histochemistry of normal and pathological conditions (Gabius et al, 1993; Bouwman et al, 2006). The physiological role of lectins as plant defence proteins has been expounded by Chrispeels and Raikhel (1991). Monocot lectins exhibit similarities in amino acid sequence to each other but obvious differences from dicot lectins (Richardson et al, 1984). The sequence comparison of NTL and FRIL showed 10Æ2% amino acid identity (Fig 5) (Corpet, 1988) and both peptides contain putative functional/ structural sites, such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation (de Castro et al, 2006). It is uncertain whether the monocot lectin NTL and dicot lectin FRIL exert similar regulatory mechanism on haematopoietic stem and progenitor cells. FRIL has not been shown to enhance ex vivo expansion of CD34+ cells. Kollet et al (2000) reported that FRIL did not expand cord blood CD34+ cells when added alone for 13 d, whereas the addition of SCF, FL, IL-6 and sIL-6R at day 6 or day 10 significantly expanded progenitor levels. Using125I-FRIL and125I-FL binding study on 3T3 fibroblasts transfected with the FL-3 tyrosine kinase receptor, Moore et al (2000) demonstrated that FRIL and FL bound to distinct non-overlapping sites on Flt-3. FL-3 is known to selectively express on CD34+ cells (Small et al, 1994). In our experiment, NTL
and FL did not exhibit notable additive or synergistic effects on long-term maintenance of haematopoietic progenitor cells
Fig 5. Amino acid sequence comparison between NTL and flt-3 receptor-interacting lectin (FRIL). Amino acid sequence alignment of NTL and FRIL (GenBank accession No. AF067417) was compared using the MultAlin software. NTL and FRIL have 10% homology as calculated by dividing the number identical residues (underlined) by the number of amino acids in the longer of the two sequences.
after 14–35 d of culture, despite the relatively low concentra-tion of FL present. Further study is required to investigate whether the activity of NTL is mediated via a cytokine receptor or by binding to a mannose moiety on the stem cell membrane.
In summary, our data show that NTL is a plant protein that has the capacity to preserve haematopoetic stem/progenitor cells in prolonged liquid culture and enhance early acting cytokines on expansion of multilineage progenitors and SCID-repopulating cells. These properties have prompted NTL a potential candidate for ex vivo expansion of haematopoitic stem cells for rescue of patients from prolonged neutropenia and thrombocytopenia post-transplant. NTL could also be developed for use in ex vivo maintenance of stem/progenitor cells for gene transfection, in vivo protection of these cells during cancer treatment and other novel strategies of cellular therapy (Brunstein et al, 2007).
Acknowledgements
This project was supported by the Li Ka Shing Institute of Health Sciences, Strategic Grant SRP2/02 and Direct Grant CUHK, The Chinese University of Hong Kong.
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