original research report€¦ · Cell morphology evaluation and culture conditions Fresh BM-MNC...

original research report

Mesenchymal stem cells promoteleukaemic cells aberrant phenotype fromB-cell acute lymphoblastic leukaemiaViviana M Rodrıguez-Pardo a, Jose A Aristizabal a, Diana Jaimes a, Sandra M Quijano a,b,Iliana de los Reyes b,c, Marıa Victoria Herrera b,c, Julio Solano b,c, Jean Paul Vernot d,e,*

a Grupo de Inmunobiologıa y Biologıa Celular, Facultad de Ciencias, Pontificia Universidad Javeriana, Cra 7a, No. 40-62, Bogota

D.C., Colombia, b Hospital Universitario San Ignacio, Pontificia Universidad Javeriana, Cra 7a, No. 40-62, Bogota D.C., Colombia, c Centro

Javeriano de Oncologıa, Pontificia Universidad Javeriana, Cra 7a, No. 40-62, Bogota D.C., Colombia, d Grupo de Fisiologıa Celular y

Molecular, Facultad de Medicina, Universidad Nacional de Colombia, Carrera 30, No. 45-03, Bogota D.C., Colombia, e Instituto de

Investigaciones Biomedicas, Facultad de Medicina, Universidad Nacional de Colombia, Carrera 30, No. 45-03, Bogota D.C., Colombia

* Corresponding author at: Grupo de Fisiologıa Celular y Molecular, Departamento de Ciencias Fisiologicas, Facultad de Medicina,

Universidad Nacional de Colombia, Carrera 30, No. 45-03, Edificio 471, Bogota D.C., Colombia. Tel.: +57 1 3165466;

fax: +57 1 5300936 Æ [email protected] Æ Accepted for publication 28 September 2013

Hematol Oncol Stem Cell Ther 2013; 6(3–4): 89–100

ª 2013 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.DOI: http://dx.doi.org/10.1016/j.hemonc.2013.09.002

BACKGROUND AND OBJECTIVES: The role of bone marrow-mesenchymal stem cells (BM-MSC) in leukae-

mic cell control is controversial. The purpose of this work was to evaluate BM-MSC role regarding the viability,

proliferation and immunophenotype of normal B-cell precursors from control (Ct) patients and leukaemic cells

from B-acute lymphoblastic leukaemia (B-ALL) patients.

PATIENTS AND METHODS: BM-MSC were isolated and characterised from voluntary donors. Mononuclear

cells isolated from Ct and B-ALL bone marrow samples were cultured in the presence or absence of

BM-MSC for 7 days. Cell viability was determined with LIVE/DEAD and proliferation index evaluated by CFSE

labelling. Cell population immunophenotypes were characterised by estimating CD19, CD10, CD20 and CD45

antigens by flow cytometry.

RESULTS: After co-culture, B-ALL cells exhibited higher viability (20–40%) as compared to just cells (3–10%).

Ct and B-ALL absolute cell counts were higher in the presence of BM-MSC (Ct: 25/mm3 cf 8/mm3, B-ALL: 15/

mm3 cf 3/mm3). Normal B-cell subpopulations in co-culture had increased expression of CD19 and CD10 (Pre-

pre B) and CD45 and CD20 antigens (Pre-B). B-ALL cells co-cultured with BM-MSC showed an increase in

CD19 and CD20, although the greatest increase was observed in the CD10 antigen.

CONCLUSIONS: Lymphoid cell maintenance, at early stages of differentiation, was significantly promoted by

BM-MSC in normal and leukaemic cells. Co-cultures also modulated the expression of antigens associated with

the B-ALL asynchronous phenotype as CD10 co-expressed with CD19 and CD20. To our knowledge, this is the

first time that CD10, CD19 and CD20 leukaemic antigens have been reported as being regulated by BM-MSC.

Haematopoiesis is a physiological processwhich depends on hematopoietic stem cell(HSC) ability to self-renew, proliferate or

differentiate.1,2 Since Schofield’s niche was proposed30 years ago, researchers have learned that more than10 cell populations may interact with HSC throughadhesion molecules and/or different soluble factors.3

Bone marrow mesenchymal stem cells (BM-MSC)are particularly important hematopoietic regulators

Hematol Oncol Stem Cell Ther 6(3–4) Fourth Quarter 2013 hemoncstem.edmgr.

due to their capacity to differentiate into specialisedstromal cells and produce soluble factors promotingprimitive hematopoietic cell maintenance.4–6 Thiscapacity has been extensively used for in vitro HSCexpansion, preserving stemness properties, that is,self-renewal and differentiation potential.7,8 This hasbeen useful in preserving myeloid populations but ithas been not fully investigated in lymphoid progeni-tors.9 Also, few studies dealt with human lymphocyte

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http://dx.doi.org/10.1016/j.hemonc.2013.09.002

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original research report BM MSC IN LEUKAEMIC CONTROL

immunophenotypic changes in vitro co-cultured withBM-MSC.10 In spite of this, the differentiation pat-tern of committed progenitors such as the B-cell line-age has been accurately dilucidated.10–12

B-cell lineage hierarchical organisation begins withPre-pre B-cells, where CD19 is first expressed to-gether with CD10, CD34, CD79a and nuclear termi-nal deoxynucleotidyl transferase (TdT) markers.These cells then differentiate into Pre-B cells express-ing cytoplasmic Igl (cy-Igl); they up-regulate CD45without CD34 expression. After this phase, B-cellswill lose CD10 expression whilst preserving CD19and CD45 and gaining CD20 expression.13 The com-bination of CD10 and CD20 with CD19 (pan-B-cellmarker) is recommended for analysing normal B-celldifferentiation to identify sequential maturation stages(Pre-Pre B, Pre-B, immature and mature B-cells).13,14

Similarly, several studies have shown that BM-MSC can also interact with leukaemic stem cells(LSC) and exert an inhibitory effect on K562 cell lineproliferation through Wnt-inhibitor Dickkopf-1(DKK-1) or by inhibiting cell cycle progression.15,16

Additionally, BM-MSC can inhibit the proliferationof leukaemic cells from patients having chronic mye-loid leukaemia (CML).17 However, it also has beenestablished that BM-MSC could have leukaemogenicactivity since BM-MSC do not inhibit Jurkat T-cellproliferation,18 but are known to have an anti-apopto-tic effect on acute promyelocytic leukaemia cells19 andto promote leukaemic cell survival.20 Furthermore,BM-MSC have been shown to play a protective roleregarding leukaemic cells isolated from acute myeloidleukaemia (AML) and CML from apoptosis inducedby FI700 (FLT3 inhibitor)21 and imatinib,22 respec-tively. Also, BM-MSC induce resistance to apoptosisin acute lymphoid leukaemia (ALL) cells exposed toasparagine depletion.23 Nevertheless, opposite effectshave been observed in non-haematologicaltumours.24,25 Bergfeld and De Clerck have debatedwhether mesenchymal cells are cancer cell friends orfoes, since their activity depends on their origin(BM, adipose tissue, cord blood), differentiation state(STRO-1+CD56+TNAP�/dim21or total stromalcells) and the type of leukaemia (acute or chronic).24

ALL is currently the most frequent malignancyoccurring in children worldwide, constituting 44%of children’s cancers from which 80% have a B-cellprecursor immunophenotype.26,27 The World HealthOrganization (WHO) recommends that leukaemiclymphoblasts must express TdT, HLA-DR, CD19,CD79a and CD10 in B-ALL diagnosis by flowcytometry, with variable CD20 and CD22 expres-sion.28 Several studies have dealt with the effect of

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BM-MSC on B-ALL cell viability3,29 and drugresistance;23,30 however, their role in B-ALL immuno-phenotype regulation has only been established inBLIN-2 cell lines, unfortunately without completecharacterisation of stromal cells.31,32

The present study has evaluated the effect ofBM-MSC on cell viability, proliferation and theimmunophenotype of primary leukaemic cells isolatedfrom B-ALL patients compared to cells isolated frompatients not suffering from haematological neoplasia.This could be useful since most studies have beenperformed with in vitro established cell lines andmay not reflect the real situation in vivo.

MATERIALS AND METHODS

Isolation and characterisation of BM-MSC

BM aspirates were obtained from the iliac crest of vol-unteer donors after signing the informed consentform given by the ethical committee of Hospital Uni-versitario San Ignacio. BM-MSC were isolated andcultured as previously published6,33 and they wereused in passages 3–5 for co-culture assays. BM-MSC phenotype assessed by flow cytometry with fol-lowing monoclonal antibodies (MAbs): FITC mouseanti-human CD73 (clone AD2, BD Pharmingen�),PE mouse anti-human CD105 (clone SN6, Invitro-gen), PerCP mouse anti-human CD45 (clone 2D1,BD Biosciences) and APC mouse anti-humanCD34 (clone 581, BD Pharmingen�). Data was ac-quired in a FACS Calibur cytometer (BD Biosciences,San Jose, CA, USA). For data analysis PAINT-A-GATE software (BDB) was used. BM-MSCfunctional assay were performed according toprevious reports6 with StemPro Osteogenesis,Adipogenesis and Chondrogenesis DifferentiationKits (Invitrogen).

Immunophenotype evaluation of controls and

patients with B-LLA

Patients with B-ALL (n = 6) were initially diagnosedat the Hospital Universitario San Ignacio’s FlowCytometry Service in Bogotá (Colombia, SouthAmerica). B-ALL diagnosis was established accordingto the WHO criteria.28,34 The B-ALL immunophe-notype in all cases was CD19+, CD34+, TdT+CD10+ with CD20 asynchronous expression in fourof the six samples. Percentage of infiltrated leukaemicblasts was elevated with an average of 84.6% (interval:73.2–92.1%). The mean percentage of normal BMcells in all samples was 15%.

After morphological and immunophenotypic diag-nosis, BM samples were sent to our laboratory, where

l Oncol Stem Cell Ther 6(3–4) Fourth Quarter 2013 hemoncstem.edmgr.com

Figure 1. Morphology of BM-MSC. BM-MSC displayed a fibroblast-like morphology after 8 (A) and 15 (B) days in culture (Olympus CK2, magnification 10·. Scale bar:100 lm). Photographs were taken with a Sony Cybershot DSC-W7 digital camera, and colours were corrected with Adobe Photoshop.

BM MSC IN LEUKAEMIC CONTROL original research report

mononuclear cells (MNC) were isolated by Ficolldensity gradient centrifugation (Histopaqued = 1.077 g/cm3, Sigma–Aldrich, USA) and frozenat �70 �C (Ct and B-ALL samples were frozen fora maximum period of 30 days). After cell thawing,immunophenotype was repeated (here on, basalimmunophenotype) with the following MAbs panel:FITC mouse anti-human CD19 (clone HIB19, BDPharmingen�), PE mouse anti-human CD10 (cloneHI10a, BD Biosciences), PerCP mouse anti-human

Figure 2. BM-MSC immunophenotype. Histograms showing antigen expresCD34 (hematopoietic antigens); BM Sample 01 (A–D) and 02 (E–H). Dashe


CD45 (clone 2D1, BD Biosciences) and APC mouseanti-human CD20 (cloneL27, BD Biosciences).

Control patients (Ct) (n = 6) BM samples wereobtained from volunteer donors with confirmednon-malignant blood disorders and evaluated in thesame way. For flow cytometry analysis, BM-MNCfrom Ct and B-ALL patients were selected using theCD19/side scatter (SSC) dot-plot and CD10/sidescatter (SSC) dot-plot, respectively. Data wasacquired in a FACS Aria-II flow cytometer

sion in freshly BM-MSC: From left to right CD105, CD73 (mesenchymal antigens) and CD45 andd line: antigen expression; solid line: auto-fluorescence control.

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Figure 3. BM-MSC multdefined media for 2–3 wsafranin ``O'' staining (5

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(BD Biosciences, San Jose, CA, USA) and analysed inmultiparameter PAINT-A-GATE software (BDBioscience).

Cell morphology evaluation and culture conditions

Fresh BM-MNC samples from Ct and B-ALL werecytospun and stained with Wright’s dye. Photographswere taken with a Sony Cybershot DSC-W7digitalcamera and the colours corrected with Adobe Photo-shop software.

After cell thawing, BM-MNC (Ct or B-ALL) werecultured in two experimental conditions: (i) 2 · 104

cells were plated in direct contact with confluentBM-MSC feeder cells in RPMI medium; and (ii)

ipotency assay. Cultured BM-MSC were induced to differentiate into adipeeks. Differentiation was evidenced by lipid vacuole detection by Sudan

·) (C and D) and calcium deposits were detected by Von Kossa staining (

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2 · 104 cells were plated without BM-MSC. Cultureswere maintained in a humidified atmosphere at 37 �Cfor 7 days.

Viability and absolute count of Ct and B-ALL cells

Ct and B-ALL BM-MNC viability were evaluated be-fore and after culture using LIVE/DEAD FixableAqua Dead Cell Stain Kit (405 nm excitation –Molecular Probes). Cells were counted by flow cytom-etry using CountBright� Absolute Counting Beads(Molecular Probes). Absolute cell numbers per sam-ple volume were calculated using the following for-mula: (number of cell events)/(number of beadsevents) · (beads/50 ll)/(volume of sample).

ocytes (A and B), chondrocytes (C and D) and osteoblasts (E and F) inblack staining (10·) (A and B); positivity for glycosaminoglycans with

5·) (E and F) (Olympus CK2, Scale bar: 100 lm).


Figure 4. Cell morphology from one Ct and two B-ALL BM samples. Microscopic evaluation of three BM samples, as examples: (A) Ct, (B) B-ALL (Pre-pre B), (C) B-ALL (Pre-B)(scale bar: 30 lm) (Olympus CK2, magnification 100·).

Figure 5. Cell viability from Ct and B-ALL patient samples. Percentage of viable cells assessed by flow cytometry in (A) Ct and (B) B-ALL patients in basal conditions (beforeculture) compared to cells cultured for 7 days in the absence (�) or presence (+) of BM-MSC.


Cell division estimated by carboxyfluorescein

diacetate succinimidyl ester (CFSE) labelling

Proliferation assays for CFSE stained BM-MNCfrom Ct and B-ALL cells were performed as previ-ously described.6 A fraction of BM-MNC was serumdeprived for 48 h to establish the highest CFSE meanfluorescence intensity (cell with no proliferation).Both CFSE+ cells were cultured as described above.Decrease in mean fluorescence intensity (MFI) ofCFSE was determined using the cytometer Facs-IIAria (BD Biosciences). Data analysis was performedwith the application ‘‘proliferation analysis’’ available


FlowJo 7.6.5 program (TreeStar, Inc) to calculatethe proliferation index (PI).

Evaluation of Ct and B-ALL cells

immunophenotype after co-cultivation

Evaluation of cells immunophenotype was carryoutafter 7 days of culture employing the same antibodiesand software used for the basal phenotype estimation.Reliable discrimination between BM-MSC and Ct orB-ALL cells was possible according to (i) their differ-ent forward-scatter (FSC) and side-scatter (SSC) sig-nals, and (ii) cells discrimination for antigen

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Figure 7. Ct and B-ALLcompared to cells lackin

Figure 6. Absolute cellwith BM-MSC(+). The m

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expressions. Mean fluorescence intensity (MFI) wasanalysed for each antigen after 7 days of culture.

Hierarchical clustering analysis

The hierarchical clustering analysis was carried outwith the MFI information for each marker and the ra-tio between the MFI values for each marker in B-ALLneoplastic cells and the MFI value for the same mar-ker in Ct. Clustering was performed by median cen-tring and normalisation of the fluorescence ratios. Alogarithmic (base 2) transformation was applied tothe ratio values in individual data sets. The resultingnormalised log 2 ratios were used for the Clusterand Tree View programs according to procedurespreviously described.35

Statistical analysis

The distribution of the data was analysed using theShapiro-Wilk test. Statistical tests were used fornon-parametric data. To compare culture conditions

BM samples proliferative index. Significant differences in the PI of (A) Ctg BM-MSC *(p < 0.05).

counts from Ct and B-ALL BM samples. Comparative analysis in absolute nean value is indicated by the horizontal line.

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(Ct or B-ALL) the Wilcoxon signed-rank test wasused using Statistix 6.0 and GraphPad Prism 5.0for graphics. Results were considered significant whenp value was below 0.05.

RESULTS

BM-MSC characterisation

Following International Society of Cellular Therapyrecommendations,36 BM-MSC were characterisedby morphology (Figure 1A and B) and immunophe-notype (Figure 2). All experiments were performedwith BM-MSC isolated from two independent donors(M01 and M02). BM-MSC were negative for CD34and CD45 and more than 98% were positive forCD73 and CD105 antigens (Figure 2). Differentia-tion assays showed cells with lipid vacuoles (adipo-genic lineage), glycosaminoglycans (chondrocytes)and alkaline phosphatase activity (osteoblasts)(Figure 3).

and (B) B-ALL cells were observed after 7 days culture in co-culture as

umbers of (A) Ct and (B) B-ALL cells after 7 days culture without (�) or


Figure 8. Ct and B-ALL cell basal immunophenotype. FSC and SSC bivariate dot plot beads (red dots in R1 region) and cells (R2 region) were gated from representativesamples (A and F). Antigens expression is indicated (B–E) for Ct (C42 sample) and (G–J) for B-ALL (L15 sample). Panel E shows normal B-cell lineage in Ct: (1) Pre-pre-B, (2)Pre-B and (3) B-cells.

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Figure 9. Ct cells immunophenotype analysis. Antigen expression in basal and in the different culture conditions (7 days of culture without (�) and with (+) BM-MSC): (A–C)CD19, (D–F) CD45 (G–I) CD10 and (J–L) CD20 at different stages of B-cell differentiation, Pre-pre B (upper line), Pre-B (middle line) and B-cells (bottom line).

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Morphological characterisation of Ct and B-LLA

cells

Fresh BM-MNC cell samples from Ct included myel-ocytes, promonocytes, granulocytes and erythroid pre-cursors (Figure 4A). In contrast, BM-MNC samplesfrom B-ALL were highly infiltrated (see Materialsand methods) with blasts cells having prominentnucleoli and basophilic-agranular cytoplasm (Fig-ure 4B and C; two patients’ samples).

Cell viability, absolute counts and proliferation

index before and after co-cultures

Cell cultures were done with and without BM-MSC.After 7 days of culture viability, percentage in Ct andB-ALL cells without BM-MSC had significantly de-creased as compared to basal cell viability (before cul-ture) (p < 0.05); in contrast, normal and leukaemic

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cells co-cultured with BM-MSC showed higher viabil-ity compared to cultures without BM-MSC(p < 0.05) (Figure 5A and B).

Ct and B-ALL cells absolute counts were esti-mated after 7 days of culture with and without BM-MSC. More cells were observed in Ct samples whenco-cultured (Figure 6A). Similar results were ob-served in B-ALL cells (Figure 6B). In both Ct andB-ALL samples, the proliferation index (PI) was high-er when co-cultured with BM-MSC (Figure 7A andB) (p < 0.05).

Immunophenotypic analysis of Ct and B-ALL

patient cells

Basal immunophenotype was analysed in all samples.Identification of B-cell precursors from Ct (Figure 8A)was based on CD45 expression in CD19+ cells


(Figure 8B–E). B-ALL leukaemic cells show homoge-neous FSC and low SSC signals (Figure 8F), CD19(Figure 8G), bright CD10 (Figure 8I), asynchronousCD20 (Figure 8J) with low CD45 expression(Figure 8G and H), consistent with a leukaemicimmunophenotype.37
After 7 days of culture, three differentiation stagescould be distinguished in Ct B-lineage cells immuno-phenotype: Pre-pre-B, Pre-B and B-cells13,38

(Figure 8E). Therefore, the immunophenotype analy-sis was made over these subpopulations. In Ct cellsco-cultured with BM-MSC, CD19 and CD10 expres-sions had increased in Pre-pre B subpopulation(Figure 9A–G). CD45 and CD20 expression had in-creased in Pre-B subpopulation (Figure 9E–K), whileonly CD19 expression decreased in B-cells(Figure 9C).

Concerning B-ALL cell immunophenotypeanalysis, CD19 expression had increased followingBM-MSC co-culture, compared to cells withoutBM-MSC (Figure 10A). CD45 expression did notchange after the co-culture (Figure 10B). The greatestincrease was in CD10 (Figure 10C) and CD20(Figure 10D) antigens.

For a global assessment of immunophenotypepatterns between Ct and B-ALL cells a hierarchicalclustering analysis was done after culture. Figure 11shows the clusters at different experimental

Figure 10. B-ALL cells immunophenotype analysis. Antigen expression in b(A) CD19, (B) CD45, (C) CD10 and (D) CD20. p Values are indicated.


conditions, confirming CD19, CD10 and CD20overexpression in B-ALL cells after co-culture withBM-MSC. Hierarchical clustering analysis revealedan association between Pre-pre B-cells and B-ALLin basal conditions (Figure 11A) and, after co-culture,with BM-MSC (Figure 11C). Ct and B-ALL analysisrevealed no association between cell groups afterculturing without BM-MSC (Figure 11B).

DISCUSSION

Several authors have demonstrated leukaemic cell via-bility maintenance by BM-MSC.23,29,39 Zhang et al.demonstrated that LSC isolated from CML patientscan produce cytokines such as G-CSF which alterstromal cell CXCL12 secretion and HSC normalgrowth but favouring leukaemic cell proliferation, thisbeing a bidirectional interaction between LSC andstromal cells.40

Our results regarding BM-MSC influence of leu-kaemic cell viability maintenance agreed with these re-ports. Herein, we have demonstrated that BM-MSCcells from volunteer donors promoted cell survival ofnormal as well as leukaemic cells (B-ALL), even afterthawing and culturing for 7 days (Figure 5). Thisshowed that BM-MSC had a pro-viability effect onthis type of leukaemic cell. We also found that BM-MSC did not inhibit Ct and B-ALL cell proliferation;

asal and in the different culture conditions (7 days of culture without (�) and with (+) BM-MSC):

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Figure 11. Hierarchical cluster analysis of markers differentially expressed in B-ALL cases compared to Ct populations. (A) Basal, (B) without BM-MSC and (C) co-culture(dotted line indicates the association between Pre-pre B and B-ALL cells). The columns show normalised log2 ratios for MFI values calculated for each analysed marker.Protein expression levels are represented by colour codes. Red indicates over-expression respecting the mean; and green represents under-expression compared to the meanvalue. Colour intensity denotes deviation magnitude (scale extends from ratio �3.00 to +3.00 log2 units).

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conversely, the absolute counts (Figure 6) and PI(Figure 7) increased after co-cultivation. Notably,our results with BM-MSC isolated from two differentindividuals (Figure 2) were very similar (data notshown), indicating that the source of bone marrowcells did not affect the outcome.

Recent reports have proved that mesenchymal cellsisolated from the umbilical cord can inhibit B-lym-phocyte differentiation.41 Our results showed thatBM-MSC did affect the immunophenotype of normalB lineage cells (Ct), increasing CD19 and CD10expression in Pre-pre B (Figure 9 A–G). It was strik-ing that the CD20 antigen expression was increasedafter co-culture with BM-MSC in Pre-B cells because

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during this stage these cells initiate CD20 expression,whilst pre-pre-B cells do not normally express it dur-ing this differentiation stage.

Tabera et al have demonstrated that BM-MSC in-duces a decrease in CD19 antigen expression in nor-mal lymphoid cells;11 however, herein we have shownthat it changes according to lymphoid cell differentia-tion stage (Figure 9A–C), because CD19 expressiononly decreased in B-cells (Figure 9C). It is known thatCD19 antigen is involved in B-cell lineage develop-ment, differentiation, activation42 and survival.43 Itcan thus be assumed that BM-MSC promote earlylymphoid progenitor viability (Pre-pre B) by increas-ing CD19 expression as part of lymphopoiesis


homeostasis, whilst BM-MSC has an immunomodu-lating effect in more differentiated cells (B-cells) bydecreasing CD19 antigen expression.
The immunophenotype changes of B-ALL cells in-duced by BM-MSC were more dramatic. After7 days, B-ALL cells cultured with BM-MSC in-creased CD19, CD10 and CD20 compared to base-line and without co-culture with BM-MSC. CD10antigen is a member of the membrane-bound zinc-dependent endopeptidases, a protein family regulatingdifferent cellular processes by peptide-substrate cleav-age which can inhibit cellular differentiation.44 Also,CD10 has been used as a cell surface marker for stemcell population identification in breast cancer45 andfor sphere-forming cells, suggesting that this proteincan identify stem/progenitor populations. This find-ing is important since BM-MSC increased CD10expression in cell populations during early stages ofdifferentiation (Pre-pre-B and B-ALL cells), whichmight indicate that BM-MSC has a role in regulatingmore primitive hematopoietic progenitors and this, inturn, could be involved in leukaemogenesis.

BM-MSC also increased CD20 expression inB-ALL. This antigen is a calcium-permeable cationchannel known to accelerate the G0/G1 progressioninduced by insulin-like growth factor-I (IGF-1).46

Interestingly, BM-MSC produce IGF-147 inducingleukaemic cells to increase CD20 expression, asobserved here. Interestingly, CD20 expression inB-ALL is asynchronous, so it would have beenexpected that BM-MSC could also favour anasynchronous phenotype.

Previous studies have shown that the bone marrowmicroenvironment provides higher viability and resis-tance to chemotherapy for leukaemic cells,48–50 there-by increasing the likelihood of relapse in patients withB-ALL.51 It has been observed that increased CD20expression is associated with a shorter time toremission and disease survival.52,53 The efficiency of


monoclonal antibody therapy, such as rituximab(anti-CD20), has shown that this antigen is importantin B-ALL pathophysiology. The same could be con-sidered concerning the CD19 antigen, since the useof blinatumomab (anti-CD19) has also led to promis-ing results.54 Moreover, increased CD10 expressionhas been considered a good prognosis factor inchildren having B-ALL,44,55 although others haveconsidered that its prognostic value should not beconsidered independently56 and that it should beanalysed within the context of the other antigens.This study has clearly demonstrated that BM-MSCincreased CD10, CD20 and CD19 expression inblasts isolated from patients having B-ALL. Sinceblast infiltration was high in our patients, we believethat haematogones and the contribution of otherprimitive normal cells to the immunophenotype musthave been very low, or non-existent. It is likely thatthis BM-MSC-induced antigen over-expression is ablast protection-inducing mechanism, probablyresponsible for increased relapse.

CONFLICT OF INTEREST

None declared.

AcknowledgementsThe authors would like to thank the patients who volun-tarily participated in the study; Mr. Jason Garry forproof-reading and revising the manuscript; Prof. ClaudiaCifuentes, Pontificia Universidad Javeriana for criticallyreviewing the manuscript; Dr. Rafael Pérez and Dr.Efrain Leal, Hospital Universitario San Ignacio, for theirsupport in collecting the bone marrow samples; Dr. Alber-to Orfao for permission to use Cluster and Tree-view soft-ware; and the clinical laboratory staff of the HospitalUniversitario San Ignacio for their assistance in samplecollection.

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