Bone marrow mesenchymal stem cells from aplastic anemia patients preserve functional and ...

32 Pages·2014·3.1 MB·Spanish

Published Ahead of Print on April 11, 2014, as doi:10.3324/haematol.2014.103580.
Copyright 2014 Ferrata Storti Foundation.
Bone marrow mesenchymal stem cells from aplastic anemia
patients preserve functional and immune properties and do not
contribute to the pathogenesis of the disease
by Clara Bueno, Mar Roldan, Eduardo Anguita, Damia Romero-Moya,
Beatriz Martín-Antonio, Michael Rosu-Myles, Consuelo del Cañizo, Francisco Campos,
Regina García, Maite Gómez-Casares, Jose Luis Fuster, Manuel Jurado, Mario Delgado,
and Pablo Menendez
Haematologica 2014 [Epub ahead of print]
Citation: Bueno C, Roldan M, Anguita E, Romero-Moya D, Martín-Antonio B, Rosu-Myles M,
del Cañizo C, Campos F, García R, Gómez-Casares M, Fuster JL, Jurado M, Delgado M,
and Menendez P. Bone marrow mesenchymal stem cells from aplastic anemia patients preserve functional
and immune properties and do not contribute to the pathogenesis of the disease.
Haematologica. 2014; 99:xxx
doi:10.3324/haematol.2014.103580
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Bone marrow mesenchymal stem cells from aplastic anemia patients preserve functional
and immune properties and do not contribute to the pathogenesis of the disease

Clara Bueno , Mar Roldan , Eduardo Anguita , Damia Romero-Moya , Beatriz Martín-Antonio ,
1* 2* 3 1 1
Michael Rosu-Myles , Consuelo del Cañizo , Francisco Campos , Regina García , Maite Gómez-
4 5 6 7
Casares , Jose Luis Fuster , Manuel Jurado , Mario Delgado , Pablo Menendez
8 9 10 11 1,12

Josep Carreras Leukemia Research Institute. Cell Therapy Program of the University of Barcelona.
1
Faculty of Medicine. Barcelona. Spain. GENYO. Centre for Genomics and Oncological Research:
2
Pfizer / University of Granada / Andalusian Regional Government. Granada. Spain. Servicio de
3
Hematología, Hospital Clínico San Carlos. Madrid. Spain. Biologics and Genetic Therapies
4
Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada.
Department of Hematology. University Hospital of Salamanca and Institute of Biomedical Research
5
of Salamanca (IBSAL). Salamanca. Spain. Department of Neurology, Neurovascular Area, Clinical
6
Neurosciences Research Laboratory, Hospital Clínico-Health Research Institute of Santiago de
Compostela, Spain. Servicio de Hematología, Hospital Clínico de Málaga. Málaga. Spain. Servicio
7 8
de Hematología, Hospital Universitario Insular Materno-Infantil. Las Palmas de Gran Canaria. Spain.
Sección de Oncohematología Pediátrica, Hospital Virgen de Arrixaca. Murcia. Spain. Servicio de
9 10
Hematología, Hospital Universitario Virgen de las Nieves, Granada, Spain. Instituto de
11
Parasitología y Biomedicina López-Neyra, CSIC, Granada. Spain. Instituciò Catalana de Reserca i
12
Estudis Avançats (ICREA).

Running Title: BM-MSCs in Aplastic Anemia

Abstract Word Count: 196
MS Word Count: 2600
References: 48
Nº Figures/tables: 6

Correspondence should be addressed to:
Pablo Menendez Ph.D, MBA
Josep Carreras Leukemia Research Institute.
Faculty of Medicine. University of Barcelona. Carrer de Casanova 143. 08036. Barcelona. Spain.
Phone: [phone]
Fax: [phone]
Email: [email protected]

*Clara Bueno and Mar Roldan equally contributed to this work.
1
ABSTRACT
Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral
pancitopenia and marrow hipoplasia. The majority of aplastic anemia cases remain idiopathic, and
hematopoietic stem cell deficiency and impaired immune responses are hallmark underlying
mechanisms causative for the bone marrow failure in aplastic anemia. Mesenchymal stem/stromal
cells constitute an essential component of the bone marrow hematopoietic microenvironment
because of their immunomodulatory properties and their ability to support hematopoiesis, and they
have been involved in the pathogenesis of several hematological malignances. We have addressed
whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the aplastic
anemia pathogenesis. Here, we report that mesenchymal stem cell cultures can be established from
the bone marrow of aplastic anemia patients and display the same phenotype and differentiation
potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic
anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34+
cells, and preserve their immunosuppressive and anti-inflammatory properties. These data
demonstrates that bone marrow mesenchymal stem cells from aplastic anemia do not exert impaired
functional and immunological properties, suggesting that they do not seem to contribute to the
pathogenesis of the disease.

Keywords: Aplastic Anemia, bone marrow-MSCs, immune properties, hematopoiesis

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INTRODUCTION
Aplastic anemia (AA) is a rare and life-threatening heterogeneous bone marrow (BM) failure disorder
characterized by peripheral pancitopenia and marrow hypoplasia (1, 2). The majority AA cases are
idiopathic, unknown primary etiology (1-3). In some patients, a drug or infection has been implicated
in the etiology of AA although it is unclear why only some individuals are susceptible (4-7). In ~15%
of patients the disease is inherited/congenital like Fanconi Anemia (1, 3). The main suggested
underlying mechanism in AA is a primary hematopoietic stem cell (HSC) deficiency or a secondary
HSC defect due to abnormal balance between HSC death and differentiation (3, 8). Importantly,
pathological autoimmune responses also seem to be involved in AA BM failure, according to the
good responses to immunosuppressive treatments (1, 9).

Mesenchymal stem/stromal cells (MSCs) are rare BM multipotent cells that constitute a source of
progenitors for mesodermic tissues (10). MSCs have emerged as excellent candidates for clinical
applications thanks to their immunomodulatory properties and their ability to support hematopoiesis
(11-13). Importantly, MSCs are an essential component of the BM hematopoietic microenvironment.
The BM hematopoietic microenvironment regulates the homeostasis of hematopoiesis through the
production and secretion of cytokines and extracellular matrix molecules (14). Furthermore, the BM
hematopoietic microenvironment plays a role in the pathogenesis of a variety of hematological
malignances including acute lymphoblastic (15) and myeloblastic leukemias (16), multiple myeloma
(17), lymphomas (18), chronic myeloid leukemia (19) or myelodysplastic syndromes (16, 20).

Because HSC failure and impaired immune responses underlie the pathogenesis of AA, it is
plausible that BM-MSCs may also contribute, directly or indirectly, to the AA pathogenesis. However,
there is almost no information on whether the functional and immunological properties of BM-MSCs
are impaired in AA patients or, their potential contribution to the pathogenesis of the disease. Here,
3
we report that BM-MSCs from AA patients display the same phenotype and differentiation potential
as their counterparts from normal BM, support in vitro homeostasis and in vivo repopulating function
of CD34+ HSPCs, and fully preserve immunosuppressive and anti-inflammatory properties. These
data demonstrates that BM-MSCs from AA patients do not show impaired functional and
immunological properties, suggesting that they do not contribute to the pathogenesis of the disease.

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METHODS
Patients
BM samples from nine newly diagnosed AA patients were studied. Diagnosis of AA was based on
the UK treatment guidelines (1). Seven normal bone marrow (NBM) samples were obtained from
healthy volunteers and used as controls. Table 1 summarizes the main hematological parameters of
each group. Extended clinical/biological information is provided in supplementary methods.

Isolation and expansion BM-MSCs
Mononuclear cells (MNCs) from BM were isolated by Ficoll-Paque and seeded at 3x10 cells/cm .
4 2
After 24h, non-adherent cells were discarded and fresh medium added. When cultures achieved
>85% density, adherent cells were trypsinized and replated at 5x10 cells/cm (15, 21).
3 2

Characterization of MSC cultures
The flow cytometry immunophenotype and osteogenic and adipogenic differentiation of BM-MSCs
was analyzed as previously described(21-24).

Cord blood (CB) collection and CD34+ cell isolation
CB units from healthy neonates were obtained from local hospitals following approval from our local
Ethics Board Committee. MNCs were isolated using Ficoll-Hypaque and CD34+ cells purified using
the CD34 MicroBead kit and the AutoMACSPro. The purity was consistently >95% (25-27).

Co-culture of BM-MSCs and CB-CD34+ cells and in vitro analyses of CD34+ cells homeostasis
CD34+ cells were co-cultured on irradiated normal-BM-MSCs or AA-BM-MSCs on serum-free media
supplemented with SCF, FLT3L and IL3. In vitro analyses were performed with CD34+ cells without
MSC co-culture, as a base-line control for CD34-MSCs co-cultures. Growth kinetics, CD34
5
phenotype, apoptosis, cell cycle analysis and clonogenic progenitor assays were performed as
detailed (26, 28, 29).

Mice xenotransplantation and analysis of engraftment
NOD/LtSz-scidIL2Rγ−/− mice (NSG) were housed under sterile conditions. The Animal Care
Committee of our University approved animal protocols. Mice at 8-12 weeks of age were sublethally
irradiated before intra-BM transplantation (26, 30). 1x10 CD34+ cells that had been cultured on
5
normal or AA-BM-MSCs were transplanted. CD34+ cells not cultured with MSC were transplanted as
a control for CD34-MSCs co-cultures. Mice were killed 7 weeks after transplantation and human
chimerism analyzed by flow cytometry in the injected and the contralateral tibia, spleen, liver and PB
(26, 29).

Assessment of the immunosuppressive response in human T cells
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. To establish
mixed lymphocyte cultures (MLC), responder PBMCs (1x10 ) from donor A were incubated with
5
1x10 allogeneic HLA-mismatched mitomycin C-treated stimulator PBMCs from donor B in the
5
presence/absence of 2x10 normal-BM-MSCs or AA-BM-MSCs. Cells were pulsed with 2.5 Ci/well
4
μ
[ H]-thymidine for the last 12h, harvested onto membranes and proliferation was determined by
3
measuring [ H]-thymidine uptake. After 48h, IL-2, TNF- and IFN- were determined by ELISA (13).
3
α γ

Determination of anti-inflammatory activity
Synovial membrane cells (SMCs) were obtained from patients with rheumatoid arthritis. SMCs
(2x10 ) were stimulated with TNF- (20 ng/ml) for 24h in the presence/absence of 1x10 normal BM-
5 5
α
MSCs (n=7) or AA BM-MSCs (n=7) (13). Extracellular matrix-degrading activities were determined
as determined(13). The content of MMP1 was determined in supernatants by ELISA. SMCs were
6
stimulated with lipopolysaccharide (LPS; 1 µg/ml) in the presence/absence of 1x10 normal BM-
5
MSCs (n=7) or AA BM-MSCs (n=7). After 48h, culture supernatants were assayed for TNF- by
α
ELISA.

7
RESULTS
BM-MSCs from AA patients display normal phenotype and differentiation potential
MSC cultures were successfully established and expanded from the BM of nine patients with AA and
seven age-matched healthy donors for further investigations. Table 1 depicts the main biological-
hematological features of both groups. Established AA-BM-MSC cultures were consistently devoid of
contaminating hematopoietic cells being negative for CD45, CD34, HLA-DR, CD19 and CD14 but
express common MSC markers including CD90, CD73, CD105, CD44 (Figure 1). They displayed
typical fibroblastoid morphology (Figure 2). To further characterize MSCs from AA patients,
adipogenic and osteoblastic differentiation assays were performed at early MSC passage (p3-p5)
(Figure 2A) (15, 31). Osteoblastic and adipogenic differentiation was achieved with a similar
efficiency than those of normal BM-MSCs (Figure 2A). Osteoblastic and adipogenic differentiation
potential was further analyzed by qRT-PCR. Upon osteogenic differentiation, the expression of the
master osteogenic markers osteocalcin, AP and osterix was almost identical in normal BM-MSCs
and AA-BM-MSCs. Similarly, upon adipogenic differentiation, the expression of the late adipogenic
transcription factors PPAR and CEBP was similar between BM-MSCs and AA-BM-MSCs (Figure
α
2B). Thus, BM-MSCs derived from AA patients are phenotypically and functionally similar to those
from healthy donors.

BM-MSCs from AA patients support the in vitro homeostasis and the in vivo repopulating
function of CB-CD34+ cells
Whether AA-BM-MSCs can maintain the homeostasis of purified CB-CD34+ cells was analyzed in
vitro by co-culturing CB-CD34+ cells with early passage (p4-p8) BM-MSCs. CB-CD34+ cells
expanded equally in BM-MSCs cultures from AA patients or healthy donors (Figure 3A). In the
absence of MSC support, CD34+ cells grow slightly slower (Figure 3A). Apoptosis (~2-6%; Figure
3B) and cycling status (~50%; Figure 3C) of CB-CD34+ cells revealed no differences when co-
8
cultured either on normal- or AA-BM-MSCs, supporting a similar expansion of CB-CD34+ cells in any
of the BM-MSC co-cultures (Figure 3A). In contrast, the slower growth of CD34+ cells maintained in
the absence of MSCs was accompanied of a higher apoptotic rate (~18% by day 12; Figure 3B). We
then analyzed the in vitro differentiation kinetics of CB-CD34+ cells by tracing the loss of the CD34
antigen, and found that CD34+ cells progressively disappeared within ~23 days on either normal or
AA BM-MSC co-cultures (Figure 3D). Interestingly, in the absence of MSC support the differentiation
of CD34+ cells was more pronounced. We next tested whether co-culture of CB-CD34+ cells with
AA-BM-MSCs impacts their hematopoietic progenitor (HPC) and/or hematopoietic stem (HSC) cell
function. We utilized in vitro clonogenic CFU assays as a read-out for HPC function. Equal numbers
of CB-CD34+ cells that had been cultured for 2 or 4 days with normal or AA BM-MSCs were plated
in CFU assays and hematopoietic colonies were counted after 14 days (Figure 3E,F). Scoring of
CFUs revealed that co-culture with AA-BM-MSCs did not influence either the clonogenic ability or the
colony phenotype of CB-CD34+ cells. The CFU capacity of CD34+ cells that have not been
previously cultured on MSCs was slightly diminished (p>0.05).

Xenotransplantation assays into NSG mice were undertaken as an in vivo read-out for SCID-
repopulating HSC function. 1x10 CB-CD34+ cells that had been cultured for 4 days with normal or
5
AA BM-MSCs were transplanted intratibia and mice were sacrificed 7 weeks later for chimerism
analysis in multiple hematopoietic organs. Human multilineage reconstitution was determined by flow
cytometry using anti-CD45, anti-HLA-ABC, anti-CD19, anti-CD33 and anti-CD34 (Figure 4A).
CD34+ cells co-cultured on either normal or AA BM-MSCs displayed similar levels of engraftment
(54% vs 61%; p>0.05. Figure 4B). The migration ability of CD34+ cells was assessed by analyzing
the level of chimerism in the injected tibiae, CL, spleen, liver and PB (Figure 4B). Co-culture with
either normal or AA BM-MSCs did not influence the migration capacity of CB-CD34+ HSPCs as
demonstrated by the similar capacity to colonize other hematopoietic tissues in all the animals
9