SDS is an autosomal recessive disorder characterized by hematological defects, exocrine pancreatic insufficiency 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 inefficient 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, flow-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 insufficiency 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+2T>
C and 183-184>
CT 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 significantly 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
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.
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 compartment promote the malignant transformation is unclear. Different groups6, 7, 21, 22, 23, 24
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 find 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 first 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.