Besides IL RCC and RAEB t MDS

Besides IL6 (RCC- and RAEB(t)-MDS), DKK3 (RCC-MDS), CRLF1 and DAPK1 (RAEB(t)-MDS) were differentially expressed by MSCs of healthy controls versus MDS. DKK3 and CRLF1 have been associated with increased cell survival by suppressing apoptosis in MSCs and neuroblastoma cells, respectively (Song et al., 2006; Looyenga et al., 2013). Differential expression of these genes did not correlate with MSC expansion rates (data not shown). In addition, increased CRLF1 in combination with IL-6 has been described in idiopathic pulmonary fibrosis causing inflammation, but suppression of fibrosis (Kass et al., 2012). DAPK1 down-regulation, associated with malignant transformation, has been described in the hematopoietic histone methyltransferase in adult RAEB(t)-MDS potentially attributing to aberrant methylation (Raval et al., 2007; Qian et al., 2010; Wu et al., 2011; Claus et al., 2012; Karlic et al., 2013). We demonstrate a similar expression profile in our MSCs, with expression in RCC-MDS being similar to HC-MSCs, and down-regulation in RAEB(t)-MSCs. After successful HSCT, DAPK1 expression was normalized. The allogeneic HSCT procedure leading to elimination of derailed cells and restoration of hematopoiesis through donor HPCs might contribute to normalization of the stromal environment in the hematopoietic niche, including MSCs of recipient origin. Analysis of total mRNA expression profiles not only revealed differences between RCC and RAEB(t)/MDR-AML in children, but also enabled us to specifically focus on genes previously reported to be differentially expressed in adult MDS. Genes of interest included micro-RNAs reported by Santamaria et al. (2012) as well as genes encoding cytokines, their receptors, chemokines and adhesion molecules. In contrast to what has been described for adult MDS, AURKA, AURKB, SCF, G-CSF, GM-CSF, CXCL12, Dicer1 and Drosha were not differentially expressed in our cohort of pediatric MDS patients compared to healthy controls. Lack of differences in expression levels of Dicer1, Drosha and CXCL12 was further confirmed by RT-PCR (data not shown). This supports the current understanding that pediatric and adult MDS are two different diseases as previous studies have highlighted the differences between adult and pediatric MDS, e.g., in response to treatment and rarity and prognostic value of (epi-)genetic mutations in the hematopoietic compartment (Hasle et al., 2004; Glaubach et al., 2014; Hirabayashi et al., 2012). Besides IL-6, genes included in the clustered analysis did not encode for molecules known to be involved in MSC signaling as reviewed by Le Blanc and Mougiakakos (2012). Studying the pathogenesis of MDS has been complicated by the poor engraftment of human MDS HPCs in immunodeficient mice (Thanopoulou et al., 2004). However, co-transplantation of stromal cells and intramedullary transplantation of hematopoietic cells have led to increased engraftment (Kerbauy et al., 2004). Knockout models resulting in an MDS-like phenotype or the of use of scaffolds with patient-derived MSC to resemble the human bone-marrow microenvironment might be instrumental in further exploring the potentially functional implications of these differences in future studies (Raaijmakers et al., 2010; Groen et al., 2012; Walkley et al., 2007).
Acknowledgments The authors would like to acknowledge the medical, nursing and associated non-medical personnel of our referring centers and the Pediatric Stem Cell Transplantation Unit of the Leiden University Medical Center for the excellent clinical care offered to the patients included in this study. The study would not have been possible without the collaborations within the Dutch Children Oncology Group. We are grateful for the support from the Sequencing Analysis Support Core, in particular dr. H. Mei, and the Leiden Genomic Technology Center of the Leiden University Medical Center. We thank dr. J. Wijnen for the chimerism analysis. This study was supported by a grant from KIKA, Dutch Children Cancer-Free Foundation (Grant 38).