Mesenchymal stem cells from Shwachman-Diamond syndrome patients display normal functions and do not contribute to hematological defects

Abstract

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34 ⁺ -sorted cells, SDS-MSCs were able to sustain CD34⁺ cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients.

Citation: Blood Cancer Journal (2012) 2, e94; doi:10.1038/bcj.2012.40& 2012 Macmillan Publishers Limited All rights reserved 2044-5385/12

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ORIGINAL ARTICLE

Mesenchymal stem cells from ShwachmanDiamond syndrome patients display normal functions and do not contribute to hematological defects

V Andr1, D Longoni2, S Bresolin3, C Cappuzzello1, E Dander1, M Galbiati1, C Bugarin1, A Di Meglio4, E Nicolis5, E Maserati6, M Serani7, AJ Warren8, G te Kronnie3, G Cazzaniga1, L Sainati4, M Cipolli5, A Biondi2,9 and G DAmico1,9

ShwachmanDiamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufciency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, signicantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34-sorted cells, SDS-MSCs were able to sustain CD34 cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients.

Blood Cancer Journal (2012) 2, e94; doi:http://dx.doi.org/10.1038/bcj.2012.40

Web End =10.1038/bcj.2012.40 ; published online 12 October 2012

Keywords: ShwachmanDiamond syndrome; mesenchymal stem cells; bone marrow failure; SBDS

INTRODUCTIONShwachmanDiamond syndrome (SDS) is a rare autosomal recessive inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufciency. In almost all affected patients, the disorder is characterized by a broad range of additional clinical features, including hematological alteration, with neutropenia as the most common deciency, frequently associated with anemia, thrombocytopenia, defects in the lymphoid lineage, increased levels of fetal hemoglobin and aplasia. Development of the latter disorder suggests that the hematopoietic defect may arise at the level of an early hematopoietic stem cell (HSC) or in the BM stroma. In addition, similarly to other marrow failure syndromes, SDS patients show an increased risk of myelodisplastic syndrome (MDS) and malignant transformation, in particular to acute myeloid leukemia. Although SDS represents an important model for understanding genetic determinants in hematopoiesis and in developmental biology, the mechanisms underlying the BM dysfunction observed in SDS is not fully understood. In 1999, Dror and Freedman1 reported a general marrow defects in SDS patients with abnormal BM stroma in terms of ability to produce fat clusters and to support and maintain hematopoiesis.

The key component of BM stroma are mesenchymal stem cells (MSCs), which represent the pivotal organizers for the generation,

maintenance and plasticity of the HSC niche by supporting the proliferation and differentiation of HSCs and their progenies. MSCs generate a number of stromal cells, which have been shown to have an impact on HSC behavior, including adipocytes,2 preosteoblast,3 osteoblasts and chondrocytes.4 The hematopoietic microenvironment controls the formation of blood cells through the release of cytokines and extracellular matrix molecules.

Recently, Raaijmakers et al.5 demonstrated that targeted deletion of miRNA processing endonuclease Dicer1 in osteoprogenitor cells in the BM could induce complex secondary changes in the organization of the hematopoietic lineages, including the development of independent genetic mutations and frank leukemia. This model, for the rst time, candidates MSCs as new actors in the multi-hit process of oncogenesis, as an expression of the involvement of hematopoietic microenvironment in the pathophysiology of the disease. Several studies demonstrated the presence of chromosome alterations in MSCs derived from MDS or leukemic patients,6,7 suggesting that unstable MSCs may facilitate the expansion of malignant cells. Whether MSC alterations inuence hematological disorders and how such alterations contribute to the progression of the disease remains controversial.

In this study, for the rst time, we deeply characterized MSCs from SDS patients (SDS-MSCs) by investigating their possible

1Centro Ricerca M Tettamanti, Clinica Pediatrica Universit degli Studi di Milano-Bicocca, Ospedale San Gerardo, Monza, Italy; 2Clinica Pediatrica, Universit degli Studi di Milano-Bicocca, Ospedale San Gerardo, Monza, Italy; 3Laboratorio di Oncoematologia, Universit di Padova, Padova, Italy; 4Clinica Pediatrica, Universit di Padova, Padova, Italy; 5Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 6Dipartimento di Medicina Clinica e Sperimentale, Universit delllnsubria, Varese, Italy; 7Dulbecco Telethon Institute at Centro Ricerca Tettamanti, Clinica Pediatrica Universit degli Studi di Milano-Bicocca, Monza, Italy and 8Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. Correspondence: Dr G DAmico, Centro Ricerca M Tettamanti, Clinica Pediatrica Universit di Milano-Bicocca, Ospedale San Gerardo, Via Pergolesi 33 20900, Monza 20052, Italy.

E-mail: mailto:[email protected]

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9These authors contributed equally to this work.

Received 14 September 2012; accepted 18 September 2012

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defects in supporting hematopoiesis and their potential role in promoting malignant transformation.

MATERIAL AND METHODSPatientsBM samples, derived from 27 out of 80 SDS patients enrolled in the SDS

Italian registry, were collected from exceeding diagnostic specimens. Table 1 shows the main clinical and genetic characteristics of all SDS patients enrolled in this study. All patients were diagnosed as SDS based on clinical criteria, including clear evidence of both hematological and exocrine pancreatic dysfunction and were positive for the SBDS gene mutation. The median age at enrollment was 9 years (range 127). All patients did not show any tumor evidence at the time of MSC generation and at 1 year of follow-up. The severity of neutropenia was determined according to absolute neutrophil count (ANC) (ANC o1500 mild

neutropenia, ANC o500 severe neutropenia, ANC o200 very severe

neutropenia). BM samples from 25 healthy donors (HDs) were obtained from collection bags, discarded after BM infusion. Informed, written consent approved by local ethical committee was obtained in all cases.

Isolation and culture of MSCsMononuclear cells were isolated from BM using Ficoll-Paque PLUS (GE

Healthcare, Waukesha, WI, USA) and seeded at 16 104 cells per cm2 in

Dulbeccos Modied Eagle Medium low glucose (Lonza, Basel, Switzerland) supplemented with 10% of fetal calf serum (Biosera, Ringmer, UK) at 37 1C with 5% CO2. After 24 h, nonadherent cells were removed. Adherent cells were trypsinized after reaching 7080% conuence, and seeded at0.3 104 cells per cm2. MSCs were used for the experiments between

passage (P)3 and P5.

To study MSC growth pattern, adherent cells were trypsinized and counted by 0.4% trypan blue exclusion method (dead cells were stained by trypan blue and thus excluded) at different time points. The number of population doubling was calculated from P2, when the cell population is

homogeneous in terms of morphology and phenotype, up to P5, with the following formula: population doubling t log 2/(logNt logN0).8

Phenotypic characterization of SDS-MSCsTo characterize expanded MSCs, the following monoclonal antibodies were used, according to the manufacturers instructions: allophycocyanin-labeled anti-CD11b (Biolegend, San Diego, CA, USA); phycoerythrin (PE)-labeled anti-CD14 (eBioscience, San Diego, CA, USA); peridinin chlorophyll protein-labeled anti-CD19 (Becton Dickinson (BD), Franklin Lakes, NJ, USA); uorescein isothiocyanate-labeled anti-CD34 (IQ products, Groningen, The Netherlands); PE-labeled anti-CD45 (BD); PE-labeled anti-CD73 (BD); PE-labeled anti-CD90 (eBioscience); PE-labeled anti-CD105 (eBioscience); uorescein isothiocyanate-labeled anti-HLA-ABC (BD) and PE-labeled anti-HLA-DR (BD). Samples were acquired by FACScalibur (BD) and analyzed by CellQuest Software (Becton Dickinson, Inc., Oakville, ON, Canada).

Multilineage differentiationAdipogenic and osteogenic differentiating ability of MSCs was determined at P5 as previously described.9 Briey, osteogenic differentiation was evaluated by Alizarin Red (Sigma-Aldrich, St Louis, MO, USA) staining and adipogenic differentiation by Oil Red O (Sigma-Aldrich) staining. Quantitative reverse transcriptase PCR was used to determine the expression of the following genes involved in the differentiation process: leptin (LEP), lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPAR-gamma), alkaline phosphatase (ALKP), osteocalcin (OCN) and osteopontin (OPN). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene. All quantitative reverse transcriptase PCR analysis were performed on LightCycler 480 (Roche, Basel, Switzerland) using Universal Probe Library probes. Samples were run in triplicate. Gene expression relative to GAPDH was quantied by the 2 DDCt method10 and indicated as fold change. Chondrogenic differentiation was determined as previously described.9 Accumulation of cartilage matrix, proteoglycans and glycosaminoglycans was evaluated by hematoxylin-eosin and safranin/fast green staining.

Table 1. Clinical and genetic characteristic of the 27 patients enrolled in the study

Patients Sex Age at study (years)

ANC/ml Infections SBDS mutations Karyotype

UPN1 F 7 1100 NO c.258 2T4C c.183_184TA4CT 46,XX

UPN2 F 3 1770 NO c.258 2T4C c.183_184TA4CT 46,XX[20]

UPN3 F 4 1820 NO c.258 2T4C c.183_184TA4CT 46,XX,i(7)(q10)[28]/46,XX[1]

UPN4 F 3 1440 NO c.258 2T4C c.183_184TA4CT 46,XX[7]

UPN5 F 1 260 NO c.258 2T4C c.183_184TA4CT 46,XY[23]

UPN6 F 8 100 NO c.258 2T4C c.183_184TA4CT 46,XX,i(7)(q10)[5]/46,XX[16]

UPN7 M 6 1600 URTI c.258 2T4C c.183_184TA4CT;c.258 2T4C 46,XY[15]

UPN8 F 1 260 NO c.258 2T4C c.183_184TA4CT;c.258 2T4C 46,XX[17]

UPN9 M 18 380 NO c.258 2T4C c.183_184TA4CT 46,XY[20]

UPN10 F 4 1100 NO c.258 2T4C c.183_184TA4CT 46,XY[9]

UPN11 M 4 2400 NO c.258 2T4C c.258 2T4C ND

UPN12 M 19 950 NO c.258 2T4C c.624 1G4C 46,XY[13]

UPN13 M 27 1120 NO c.258 2T4C c.183_184TA4CT 46,XY[20]

UPN14 F 9 1000 NO c.258 2T4C c.258 2T4C 46,XX[23]

UPN15 M 20 250 NO c.258 2T4C c.183_184TA4CT 46,XY[24]

UPN16 M 10 2350 NO c.258 2T4C c.258 2T4C ND

UPN17 F 6 1620 NO c.258 2T4C c.183_184TA4CT 46,XX[24]

UPN18 F 7 1760 ND c.258 2T4C c.183_184TA4CT 46,XX[22]

UPN19 M 3 2960 NO c.258 2T4C c.183_184TA4CT 46,XY[26]

UPN20 F 15 278 NO c.258 2T4C c.6524CT ND

UPN21 M 18 1500 NO c.258 2T4C c.183_184TA4CT 46,XY,i(7)(q10)[1]/46,XY[6]

UPN22 M 11 310 ND c.258 2T4C c.183_184TA4CT 46,XY,i(7)(q10)[4]/46,XY[16]

UPN23 M 13 800 ND c.258 2T4C c.183_184TA4CT;c.258 2T4C 46,XY,del(20)(q11,21q13,32)[6]/46,

XY [7](q10)[1]/46,XY[6]

UPN24 F 12 ND NO c.258 2T4C c.352A4G 46,XX[10]

UPN25 M 3 820 ND c.258 2T4C c.183_184TA4CT 46,XY[9]

UPN26 F 7 ND NO c.258 2T4C c.183_184TA4CT 46,XX[31]

UPN27 M 7 ND ND c.258 2T4C c.183_184TA4CT;c.258 2T4C 46,XY[17] Abbreviations: ANC, absolute neutrophil count; F, female; M, male; ND, not detected; PB, pheripheral blood; UPN, unique patient number; URTI, upper respiratory tract infection. All mutations are described according to the mutation nomenclature (www.hgvs.org/mutnomen).

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Proliferation assayPeripheral blood mononuclear cells were stimulated with 5 mg/ml of phytohemagglutinin (PHA) (Irvine Scientic, Santa Ana, CA, USA) and cocultured with different doses of irradiated (35 Gray) MSCs in 96-well plates. After 48 h of coculture cells were pulsed for 16 h with [3 H]-thymidine at 1 mCi per well (PerkinElmer, Waltham, MA, USA) and then harvested. [3 H]-thymidine incorporation was measured using Multipurpose Scintillation Counter (Beckman Coulter, Brea, CA, USA).

Neutrophil and MSC cocultureIn order to obtain a pure and minimally activated cell population, neutrophils were isolated from peripheral blood of HDs under endotoxin-free conditions following the protocol described by Boyum.11 After purication, the mean neutrophil purity was 95%, as determined by morphological and phenotypic analyses. Neutrophils were then layered on MSC or cultured alone. At 24 and 48 h, cultured neutrophils were recovered, counted and analyzed for viability by Apoptosis/Necrosis detection kit (Enzo Life Sciences, Farmingdale, NY, USA) and owcytometry analysis. Supernatants from the coculture were used to evaluate the amount of interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (eBioscience), according to the manufacturers instruction.

HSC and MSC cocultureCD34 cells were isolated from BM of HD by anti-CD34 antibody-conjugated magnetic beads, according to the manufacturers instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34 cells were suspended in complete Roswell Park Memorial Institute medium and plated at a density of 5 104 cells per cm2 on a conuent irradiated (35

Gray) MSC layer in 48-well plate. Phenotype and short-time colony assay of CD34 cells were assessed weekly. A total of 103 cells were resuspended in Iscoves Modied Dulbeccos Media (Euroclone, Pero, Italy) and plated in 10% methylcellulose-based medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 1% of pen-strep, L-glutamine, 2 U of erythropoietin (Roche) and 10% 5637 conditioned medium. The conditioned medium from 5637 tumor cell line was used as a source of colony-stimulating factor at a nal concentration of 10%. After 21 days of incubation at 37 1C in a fully humidied atmosphere of 5% CO2, colony-forming unit were scored under an inverted microscope. Aggregates of 450 cells were evaluated as colonies.

Western blot analysisMSCs (1.5 106) were collected at P5 and were lysed in radioimmuno-

precipitation assay buffer as already described.12 A total of 50 mg of protein was loaded per lane on a 10% sodium dodecyl sulfate-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane (Life Technologies, Paisley, UK). The following antibodies were used: goat polyclonal anti-SBDS (1/800) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-b-actin (loading control, 1/20000) (Sigma-Aldrich)

in PBST/5% of milk.

Karyotype and uorescence in situ hybridization (FISH) analysis of SDS-MSCs

MSCs were cultured in medium supplemented with 10 ml/ml of colcemid (10 g/ml, Sigma) for up to 6 h. Conventional cytogenetics was performed by the standard procedures and QFQ (Q-bands by uorescence using quinacrine) banding. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature (2009).13 FISH

analysis was performed on MSCs using LSI D20S108 probe (Vysis, Downers Grove, IL, USA) and D7S486/CEP7 FISH Probe Kit (Vysis) and on BM patients using D7S486/CEP7 FISH Probe Kit (Vysis) and BAC RP11-17F3 (20q12). The FISH cut-off for each probe was determined analyzing MSCs derived from HDs. The probes cut-off level was 6% for LSI D20S108 and 2% for D7S486/ CEP7. At least 200 nuclei were analyzed for each case using Isis software(v.5.4.9; MetaSystems, Altlussheim, Germany) and Imager Z1 (Zeiss, Oberkocken, Germany) epiuorescence microscope equipped with 4,6-diamidino-2-phenylindole, uorescein isothiocyanate and TEXAS RED lters.

Statistical analysisAll values are expressed as means.d. or s.e. To evaluate statistical signicance between the groups, Students t-test was used. A P-value o0.05 was considered to be statistically signicant.

RESULTSEvaluation of MSC-typical features in cells expanded from BM of

SDS patientsMSCs were obtained from BM of 27 SDS patients and were characterized for their proliferating ability, antigen expression and differentiation into mesengenic lineages. SDS-MSCs displayed a typical broblast-like morphology, similarly to HD-MSCs (data not shown). Flow-cytometric analysis showed that SDS-MSCs, as well as HD-MSCs, were negative for hematopoietic (for example, CD34, CD45 and MHC class II), lymphoid (for example, CD19) and myeloid (for example, CD11b and MHC class II) markers and positive for stromal cell-associated markers (for example, CD73, CD90 and CD105) (Figure 1a). Adipocytic, osteoblastic and chondrocytic differentiation assays were performed for SDS-MSCs. SDS-MSCs were able to differentiate into osteoblasts, as demonstrated by the histological detection of calcium deposition by Alizarin Red staining (Figure 1b, on the left). Osteogenic differentiation was also conrmed by real-time PCR, under osteogenic permissive condition at day 14 of culture. Expression of ALKP, OCN and OPN osteogenic genes resulted similar in SDSMSCs and HD-MSCs (Supplementary Figure 1A). In addition, SDSMSCs were able to differentiate into adipocytes, as revealed by the formation of lipid droplets visualized by Oil Red O staining (Figure 1b, in the middle). Similarly to HD-MSCs, SDS-MSCs increased the expression of PPAR-g, LEP and LPL during the adipogenic differentiation process (Supplementary Figure 1B). Concerning the chondrogenic differentiation, cultured pellets became free-oating within the rst 24 h of induction. During the differentiation process, biosynthesis and deposition of extracellular matrix components, such as sulfated proteoglycans, contributed to increase cell pellet size, as demonstrated by hematoxylin-eosin (Figure 1b, on the right), safranin and fast green staining (inner panel).

To evaluate their proliferating ability, SDS-MSCs were subcultured when they reached 7080% of conuence and population doubling time was calculated at each passage from P2 to P5. Proliferation rate was comparable at each time point considered (Students t-test 40.05) between SDS- and HD-MSCs (Figure 1c).

Interestingly, cell passaging reduced the proliferation rate in all MSCs tested. In particular, 4 days (1.2 for SDS-MSCs and 1.4 for HD-MSCs) were necessary to double cell number of both SDS-MSCs and HD-MSCs at P2, whereas population doubling was 4.31.4 days for SDS-MSCs and 3.70.9 days for HD-MSCs at P5.

We then evaluated the immunoregulatory activity of SDS-MSCs by assessing their ability to inhibit lymphocyte proliferation induced by mitogenic stimuli (that is, PHA). Peripheral blood mononuclear cells from HD were stimulated with PHA in the presence or absence of various concentrations of irradiated MSCs. SDS-MSCs were able to strongly suppress lymphocyte proliferation in a dose-dependent manner, with a 58% of inhibition observed at 1:32 SDS-MSC/peripheral blood mononuclear cell ratio (range, 2499%), similarly to HD-MSCs (Figure 1d).

Functional characterization of SDS-MSCsWith the aim to evaluate the functional properties of SDS-MSCs, we analyzed their capability to preserve neutrophils from apoptosis and to maintain the viability of CD34 cells. Neutrophils are very sensitive cells and once released from the BM they circulate in the peripheral blood with a short half-life. We investigated the effects of MSCs on spontaneous neutrophil apoptosis, by culturing HD neutrophils in the absence or presence of SDS-MSCs. Because Raffaghello et al.14 demonstrated that inhibition of neutrophil apoptosis occurred at a MSC:neutrophil ratio ranging from 1:1 to 1:500, we used a MSC:neutrophil ratio lower than 1:50 for all the coculture experiments. As shown in Figure 2a, after 24 h only 3% of neutrophils cultured with medium

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CD34 CD45 CD73 CD90 CD105

CD19

HLA-ABC

HLA-DR

CD11b

CD14

Alizarin red Oil red O H&E

25

6

HD SDS

20

Days for PD

3H Tdr uptake

(x103 CPM)

4

15

** *

10

2

5

0 P2-P3

P3-P4

P4-P5

Passages

0 - 1:1 1:2 1:4 1:8 1:16 1:32

MSC/PMBC ratio

Figure 1. Isolation and expansion of SDS-MSCs. MSCs were isolated from bone marrow of SDS patients and cultured in Dulbeccos Modied Eagle Medium with 10% of fetal calf serum. (a) Fluorescence-activated cell sorting (FACS) analysis of SDS-MSCs. Isotype control staining (white area under continuous line) versus the specic antibody staining prole (gray area) is shown. Histograms from 1 representative experiment out of 27 performed are shown. (b) Mesengenic differentiation of SDS-MSCs. To detect the osteogenic differentiation, cells were stained for calcium deposition with Alizarin Red (on the left) and adipogenic differentiation was evaluated through the morphological appearance of fat droplets with Oil Red O (in the middle). Cell pellet was treated with chondrogenic medium for 21 days, sections of cell pellet were stained with hematoxylin-eoxin (on the right) and Safranin O (inner panel), which binds proteoglycans and glycosaminoglycans. Canon PowerShot G5, magnication 10. One experiment out of twenty-seven performed is shown. (c) Expansion curve of SDS-MSCs. The graph represents the

means.e. of 25 HD-SDS versus 27 SDS-MSC. Population doubling (PD) t log 2/(logNt-logN0), where t represents culture time, N0 and Nt

represent cell number before and after seeding, respectively. (d) Lymphocyte proliferation in the presence or absence of SDS-MSCs. Peripheral blood mononuclear cells (PBMCs) were stimulated with PHA in the absence ( ) or presence of different concentration of MSCs in 96-well

plates. After 48 h, cells were pulsed for 16 h with [3H]-thymidine at 1 mCi per well (PerkinElmer) and then harvested. The graph represents the means.e. of eight experiments (**Po0.01 and *Po0.05, versus PBMC stimulated with PHA in absence of MSC).

alone resulted viable. This percentage further decreased after 48 h of culture to 1% of viable cells. In contrast, HD-MSCs and SDSMSCs were comparably able to support the viability of neutrophils at both 24 and 48 h (Figure 2a). In particular, at 24 h, 7.6% (range 316%) and 13% (range 423%) of neutrophils were alive (double negative cells) when cocultured with HD-MSCs or SDS-MSCs, respectively. It has been demonstrated that IL-6 is constitutively produced by MSCs and has a crucial role in inhibiting neutrophil-programmed cell death. For this reason, we evaluated IL-6 concentration in supernatants recovered from SDS-MSCs and neutrophil coculture. SDS-MSCs were able to produce IL-6 in a time-dependent manner (Figure 2b). In particular, IL-6 production increased after 72 h of culture with both SDS- and HD-MSCs, with no statistically signicant differences between the two groups.

In order to assess the ability of SDS-MSCs to support and maintain hematopoiesis, we took advantage of long-term cultures to mimic the interactions between the hematopoietic progenitors and the microenvironment of the BM stem cell niche in vitro. With this aim, CD34-sorted cells were cocultured with MSCs from both patients and controls. Total number of nonadherent cells was calculated during the rst 3 weeks of coculture and was found to be similar. After an initial decline at day 7, the total number increased after 14 days of coculture (Figure 3a). As expected, after 21 days, the percentage of CD34 cells diminished up to 20% (Figure 3b). Importantly, SDS-MSCs as well as HD-MSCs were able to preserve the stemness of recovered cells, as evaluated by their ability to form CFU-GM (colony forming unit-granulocyte/macrophage) colonies in methylcellulose. No statistical differences in the number of CFU-GM

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25

120

HD SDS

**

20

% viable neutrophils

100

15

*

80

*

10

60

5

40

0

- HD SDS - HD SDS

20

24h 48h

0

25

7 14 21

HD

SDS

Days of co-culture

20

Day 7

IL-6 ng/ml

15

83%

89%

17%

13%

10

HD

5

0

CD34

6 24 48 72

Hours of co-culture

Figure 2. (a) Evaluation of neutrophil viability cultured with SDSMSCs. Neutrophils from HDs were cultured alone or in the presence of HD-MSCs and SDS-MSCs at 24 and 48 h. Viable neutrophils were evaluated by annexin V and 7-AAD (7-aminoactinomycin D) staining as percentage of double negative cells on recovered cells at each time point analyzed. The mean percentage of viable cells evaluated in 10 independent experiments is shown. (b) IL-6 secretion after neutrophils and SDS-MSCs coculture. Quantication of IL-6 in the supernatants was detected by enzyme-linked immunosorbent assay at different time points. The graph represents the means.e. of 10 experiments (Students t-test 40.05).

Day 21

SDS

50

HD SDS

40

n of CFU-GM

n of Nonadherent cells (X103 )

30

20

colonies (Figure 3c) obtained from CD34 cells cocultured with both SDS-MSCs and HD-MSCs were observed.

Analysis of SBDS protein in SDS-MSCsIt has been demonstrated that SBDS protein shows low expression levels in all hematopoietic cell lineages from SDS patients.15 In order to assess the level of SBDS protein in SDS-MSCs, we performed western blot analysis on lysates from seven SDS-MSCs and eight HD-MSCs. In line with previous studies on BM,15 SBDS protein was undetectable in all SDS-MSCs (lanes 13, Figure 4). On the contrary, a variable amount of protein was detected in HD-MSC (lanes 48).

Karyotype and FISH analysis of SDS-MSCsTo study the potential role of MSCs in the predisposition to leukemia or MDS of SDS patients, we focused on the cytogenetic characterization of SDS-MSCs. With this aim, we performed conventional cytogenetic and FISH analysis to explore genomic alterations in MSCs obtained from 4 out of 5 patients carrying chromosomal abnormalities, and to compare these with the aberrations observed in the BM of the same patients.

Karyotype and FISH analysis were performed in SDS-MSCs obtained from UPN3, UPN6, UPN21 and UPN23, in the BM sample and in MSCs from ve healthy volunteers. The median number of MSC passages was 5. The culture time ranged from 29 to 35 days.

FISH analysis demonstrated that in none of the patients, SDSMSCs displayed the chromosomal abnormality (n 0/200 nuclei

analyzed) observed in the respective BM sample (UPN3: i(7)(q10), n 124/200; UPN6: i(7)(q10), n 49/549; UPN21: i(7)(q10), n 30/

300 and UPN23: del(20)(q11), n 162/601). Figure 5a shows the

FISH analysis of a representative experiment.

10

0

7 14 21

Days of culture

Figure 3. (a) Nonadherent cell numbers from long-term culture with SDS-MSCs. In all, 5 104 CD34 cells from HDs were plated in wells

containing irradiated MSCs (dotted line). Cells were harvested from individual wells after 7, 14 and 21 days. Data represent the mean of eight experiments performeds.e., Students t-test 40.05. (b) FACS analysis of nonadherent cells obtained after long-term culture. Percentage of CD34 cells from HD-MSCs or SDS-MSCs cocultures was shown. The experiment is representative of eight performed. (c) Number of CFU-GM derived from long-term culture nonadherent cells, plated weekly in short-term clonogenic assays. After 7, 14 and 21 days of coculture, 1000 of nonadherent cells were seeded in wells containing methylcellulose. Colonies were scored as CFU-GM using an inverted microscope after 21 days. Data represent the mean of eight experiments performeds.e. (Students t-test 40.05).

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1 2 3 4 5 6 7 8

SBDS

[afii9826]-actin

30 kDa

43 kDa

Figure 4. Detection of SBDS protein in SDS-MSCs by western blot. Proteins extracted from MSCs were analyzed by immunoblotting with the SBDS antiserum. b-actin protein level served as a loading control. SBDS protein is absent in SDS-MSCs. Shwachman patients are lanes 1, 2 and 3 (UPN16, UPN1 and UPN11, respectively); controls are lanes 4, 5, 6, 7 and 8. Molecular size markers are indicated on the left in kilodaltons.

BM MSC

UPN 3

UPN 23

CEP7

D7S486

CEP7

D7S486

RP11-17F3 D20S108

Figure 5. (a) FISH analysis performed on SDS-MSCs. The LSI D7S486/ CEP7 (Vysis) and BAC RP11-17F3 probes were used to search for isochromosome 7q and 20q arm deletion in BM samples: UPN3 showed hybridization pattern consistent with the presence of isochromosome 7q (two green signals and three red signals); UPN23 revealed deletion of 20q (one aqua signal). SDS-MSCs derived from patients were tested for the same abnormalities using LSI D7S486/ CEP7 (Vysis) and LSI D20S108 (Vysis) probes, and they did not reveal any aberrations.

Cytogenetic analysis of SDS-MSCs (20 metaphases in all cases) did not show any of the chromosomal alterations observed in the BM (UPN3: i(7)(q10), n 28/29; UPN6: i(7)(q10), n 5/21; UPN21:

i(7)(q10), n 1/7; and UPN23: del(20)(q11), n 6/13) (Table 1). No

other abnormalities, aneuploidy or polyploidy were observed in SDS-MSCs. Chromosome analysis of HD-MSCs showed normal diploid karyotype, with no aneuploidy, polyploidy or structural abnormalities (data not shown).

DISCUSSIONSDS is an autosomal recessive disorder characterized by hemato-logical defects, exocrine pancreatic insufciency and skeletal deformities. Patients may display a wide range of hematological abnormalities, but the number of neutrophils in peripheral blood samples is reduced (neutropenia) in all individuals affected. Using long-term cultures of marrow stromal cells from SDS and unaffected subjects, Dror and Freedman1 demonstrated that patients show dysfunction of the BM stroma. In particular, a reduced ability to support and maintain hematopoiesis and a stem cell defect were observed. Until now, the role of MSCs in the

pathophysiology of the inefcient hematopoiesis observed in SDS is still unclear.

In the present study, we isolated and characterized MSCs from the BM of 27 patients. We demonstrated that SDS-MSCs are similar to HD-MSCs in terms of morphology and growth kinetics. Furthermore, ow-cytometry data demonstrated the expression of several cell-surface antigens, such as CD73, CD90 and CD105, commonly found on HD-MSCs. Moreover, SDS-MSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes, under appropriate induction conditions. Finally, we demonstrated that SDS-MSCs strongly inhibit the proliferation of PHA-activated lymphocytes. Overall, these results demonstrated that cells with typical MSC features can be successfully obtained from the BM of SDS patients.

After characterizing SDS-MSCs, we tried to better understand their potential role in the hematopoietic insufciency observed in SDS patients. Stromal impairment in SDS can be related to either the reduced expression of hematopoietic cytokines, the altered production of extracellular matrix or the abnormal expression of adhesion molecules necessary for the interaction between hematopoietic progenitors and stromal cells. We cocultured SDS-MSCs with CD34 cells and, in our culture condition, we observed that SDS-MSCs were able to support the viability and the stemness potential of CD34 cells as well as HD-MSCs. The altered function of SDS stroma reported by Dror1 was demonstrated by in vitro experiments using BM stroma in toto as an integral unit of various cell types. In our experiments, we used isolated MSCs as precursors of a number of stromal cells, which have been shown to have an impact on HSC behavior. Despite their essential role in organizing and maintaining the HSC niche, our data suggest that MSCs themselves do not seem to be responsible for the BM failure typical of SDS.

We also analyzed the effect of SDS-MSCs on neutrophils and we demonstrated that, after coculture, neutrophils were protected from apoptosis. As demonstrated by Raffaghello et al.13 for normal MSCs, SDS-MSCs produced IL-6, a cytokine involved in prevention of excessive or inappropriate neutrophil activation. Overall, these results indicate that SDS-MSCs act in vitro just like their normal counterparts. Further in vivo studies mirroring the whole architecture of the HSC niche are needed to comprehend the possible MSC defects in SDS patients.

Patients analyzed in our study were diagnosed with SDS based on clinical criteria and the diagnosis was supplemented by positive SBDS gene mutation tests. In agreement with previous reports, the 258 2T4C and 183-1844CT mutations were the

most common in our cohort. Woloszynek et al.16 detected the SBDS protein in samples from family members with normal SBDS alleles but not in patients. Full-length SBDS protein was not detected in leukocytes of SDS patients with the most common SBDS mutations, consistent with a loss-of-function mechanism. We demonstrated that, even though SBDS gene transcript expression did not differ signicantly between patients and controls (data not shown), SBDS protein was not detectable in SDS-MSCs. Recent studies in yeast and patient BM cells show that SBDS gene is involved in RNA metabolism and ribosome biogenesis.17,18 SBDS is an essential gene in embryogenesis and it is also implicated in cell division and cellular stress response.19,20 Further studies are needed to better comprehend the mechanisms compensating the absence of SBDS protein.

Similar to other marrow failure syndromes, SDS patients are cancer prone and have a risk of developing myelodysplastic syndrome and/or leukemia. Raaijmakers et al.,5 using transgenic mice, showed that genetic alteration of cells belonging to the BM microenvironment can induce MDS with ineffective hematopoiesis and with occasional transformation to acute myeloid leukemia. Although it is generally agreed that the marrow microenvironment has a role in the biology of hematological diseases, the mechanisms by which the stromal

Blood Cancer Journal & 2012 Macmillan Publishers Limited

Mesenchymal stem cells in ShwachmanDiamond syndrome V Andr et al

7

compartment promote the malignant transformation is unclear. Different groups6,7,2124 have shown the extensive variability of the aberrations, such as balanced and unbalanced translocations, hypodiploidy, deletions and whole-chromosome gains in MSCs obtained from leukemic patients. In view of these data, genetic alterations in MSCs may indicate a common origin and a potential interaction of MSCs in leukemia development and/or progression. To study the potential implication of MSCs in predisposing or promoting leukemia or MDS evolution in SDS patients, we analyzed their genetic features. In spite of having chromosomal abnormalities in the BM counterpart, we did not nd any chromosomal alteration in SDS-MSCs. Probably, the most common chromosomal alterations [i(7)(q10) or del(20)(q11)] observed in SDS patients occurred in a more mature, already committed, progenitor instead of involving the mesenchymal compartment. The fact that SDS-MSC did not carry the same anomaly observed in the BM of patients does not exclude the presence of other genetic alteration; however, whole-genome single-nucleotide polymorphism array did not show any copy number aberration in MSC from the SDS patient tested. These results suggest that SDS-MSCs patients may be nonmalignant cells.

All SDS patients did not show any tumor evidence at the time of MSC generation and in 1 year follow-up. It would be of interest to continuously follow the patients and, in case of tumor evolution, evaluate the MSC genetic status at the time and early before the progression.

We described, for the rst time at our knowledge, the isolation, expansion and functional characterization of SDS-MSCs. Our results showed that SDS-MSCs were similar to normal BM-derived MSCs in terms of morphology, growth property, surface epitopes and differentiation ability. Moreover, SDS-MSCs had normal karyotype and may be nonmalignant. Despite their pivotal role in the BM niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients. Our data give a new insight into biology of SDS and might have a positive impact of the management of HSC transplantation in SDS patients, by sustaining HSC transplantation despite the supposed risk of failure due to stroma abnormalities.

CONFLICT OF INTEREST

The authors declare no conict of interest.

4 Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC et al.

Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003; 425: 841846.

5 Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA et al.

Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia.Nature 2010; 464: 852857.

6 Lopez-Villar O, Garcia JL, Sanchez-Guijo FM, Robledo C, Villaron EM, Hernandez-Campo P et al. Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specic genetic prole for the 5q- syndrome. Leukemia 2009; 23: 664672.

7 Menendez P, Catalina P, Rodriguez R, Melen GJ, Bueno C, Arriero M et al. Bone marrow mesenchymal stem cells from infants with MLL-AF4 acute leukemia

harbor and express the MLL-AF4 fusion gene. J Exp Med 2009; 206: 31313141.8 Rallapalli S, Bishi DK, Verma RS, Cherian KM, Guhathakurta S. A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells.Biotechnol Lett 2009; 31: 18431850.9 Gatto F, Redaelli D, Salvade A, Marzorati S, Sacchetti B, Ferina C et al. Hurler disease bone marrow stromal cells exhibit altered ability to support osteoclast formation. Stem Cells Dev 2012; 21: 14661477.10 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402408.11 Boyum A. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1g gravity eld. Scand J Clin Lab Invest Suppl 1968; 97: 5176.12 Fazio G, Palmi C, Rolink A, Biondi A, Cazzaniga G. PAX5/TEL acts as a transcriptional repressor causing down-modulation of CD19, enhances migration to CXCL12, and confers survival advantage in pre-BI cells. Cancer Res 2008; 68: 181189.13 Shaffer LG, Slovak ML, Campbell LJ (eds). International System for Human Cytogenetic Nomenclature. S Karger AG: Basel, 2009.14 Raffaghello L, Bianchi G, Bertolotto M, Montecucco F, Busca A, Dallegri F et al.Human mesenchymal stem cells inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche. Stem Cells 2008; 26: 151162.15 Wong TE, Calicchio ML, Fleming MD, Shimamura A, Harris MH. SBDS protein expression patterns in the bone marrow. Pediatr Blood Cancer 2010; 55: 546549.16 Woloszynek JR, Rothbaum RJ, Rawls AS, Minx PJ, Wilson RK, Mason PJ et al.Mutations of the SBDS gene are present in most patients with ShwachmanDiamond syndrome. Blood 2004; 104: 35883590.17 Finch AJ, Hilcenko C, Basse N, Drynan LF, Goyenechea B, Menne TF et al.Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes ShwachmanDiamond syndrome. Genes Dev 2011; 25: 917929.18 Wong CC, Traynor D, Basse N, Kay RR, Warren AJ. Defective ribosome assembly in ShwachmanDiamond syndrome. Blood 2011; 118: 43054312.19 Orelio C, Verkuijlen P, Geissler J, van den Berg TK, Kuijpers TW. SBDS expression and localization at the mitotic spindle in human myeloid progenitors. PLoS One 2009; 4: e7084.20 Stepanovic V, Wessels D, Goldman FD, Geiger J, Soll DR. The chemotaxis defect of ShwachmanDiamond syndrome leukocytes. Cell Motil Cytoskeleton 2004; 57: 158174.21 Blau O, Hofmann WK, Baldus CD, Thiel G, Serbent V, Schumann E et al.Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia.Exp Hematol 2007; 35: 221229.22 Flores-Figueroa E, Arana-Trejo RM, Gutierrez-Espindola G, Perez-Cabrera A, Mayani H. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization. Leuk Res 2005; 29: 215224.23 Klaus M, Stavroulaki E, Kastrinaki MC, Fragioudaki P, Giannikou K, Psyllaki M et al.Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes.Stem Cells Dev 2010; 19: 10431054.24 Roela RA, Carraro DM, Brentani HP, Kaiano JH, Simao DF, Guarnieiro R et al. Gene stage-specic expression in the microenvironment of pediatric myelodysplastic syndromes. Leuk Res 2007; 31: 579589.

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ACKNOWLEDGEMENTS

We thank Associazione Italiana Sindrome di Swhachman (AISS), Fondazione Matilde Tettamanti, Comitato Maria Letizia Verga and Comitato Stefano Verri for their generous and continuous support. This work has been partly supported by grants from AISS, Associazione Italiana Ricerca sul Cancro (AIRC) and Ministero della Salute.

REFERENCES

1 Dror Y, Freedman MH. ShwachmanDiamond syndrome: an inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty marrow microenvironment. Blood 1999; 94: 30483054.

2 Naveiras O, Nardi V, Wenzel PL, Hauschka PV, Fahey F, Daley GQ. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature 2009; 460: 259263.

3 Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 2009; 457: 97101.

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