Coexistence of aberrant hematopoietic and stromal elements in myelodysplastic syndromes

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Abstract

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders related to hematopoietic stem and progenitor cell dysfunction. Several studies have shown the role of the bone marrow microenvironment in regulating hematopoietic stem, and progenitor function and their individual abnormalities have been associated with disease pathogenesis. In this study, we simultaneously evaluated hematopoietic stem cells (HSC), hematopoietic stem progenitor cells (HSPCs) and different stromal elements in a cohort of patients with MDS-refractory cytopenia with multilineage dysplasia (RCMD). Karyotyping of these patients revealed variable chromosomal abnormalities in 73.33% of patients. Long-term HSC and lineage-negative CD34 + CD38 − cells were reduced while among the HPCs, there was an expansion of common myeloid progenitor and loss of granulocyte-monocyte progenitors. Interestingly, loss of HSCs was accompanied by aberrant frequencies of endothelial (ECs) (CD31 + CD45  CD71 −) and mesenchymal stem cells (MSCs) (CD31  CD45  71 −) and its subsets associated with HSC niche. We further demonstrate down-regulation of HSC maintenance genes such as Cxcl12, VEGF in mesenchymal cells and a parallel upregulation in endothelial cells. Altogether we report for the first time quantitative and qualitative de novo changes in hematopoietic stem and its associated niche in a cohort of MDS-RCMD patients. These findings further reinforce the role of different components of the bone marrow microenvironment in MDS pathogenesis and emphasize the need for comprehensive simultaneous evaluation of all niche elements in such studies.

Introduction

Myelodysplastic syndromes are a heterogeneous group of disorders characterized by ineffective hematopoiesis resulting in cytopenias of one or more lineages and a variable risk of progression to acute myeloid leukemia [1]. The pathogenesis of MDS is not well defined, but the presence of dysplastic myeloid elements in the bone marrow and specific cytogenetic abnormalities or mutations are found in 60–70% of cases [1]. Chromosomal aberrations in MDS can vary from simple to complex, the most frequent being del (5q), monosomy 7, del (7q), trisomy 8, and del (20q). Other aberrations such as del (17p), del (12p), del (11q23) and the loss of chromosome Y also occur albeit at lower frequencies [2], [3]. These changes were attributed to an abnormal hematopoietic stem cell (HSC) since > 95% of cytogenetic abnormalities were observed in the most primitive HSCs and when transplanted in a xenotransplantation model were able to initiate clonal MDS [4], [5], [6], [7], [8], [9]. Given that the bone marrow niche hosts and maintains hematopoietic stem cells and that patients with MDS have a compromised hematopoiesis, it is likely that abnormalities of the niche could contribute to disease pathogenesis [10]. This concept is supported by the presence of cytogenetic abnormalities, impaired capacity to support hematopoiesis and aberrant generation of key niche factors such as Angptl4, Vegfa and Lif in cultured mesenchymal stromal cells (MSCs) [11], [12], [13], [14]. Further proof of the role of MSC in pathogenesis of MDS came from a recent study wherein a cohort of low-risk MDS patients, co-injection of CD34 + cells with the corresponding in vitro cultured MSCs demonstrated dramatic improvement in engraftment with a myeloid bias, suggesting that bone marrow stromal cells provide key auxiliary signals for MDS pathogenesis [11]. Upregulation of proangiogenic cytokine-like Vegfa, an increase in endothelial colony forming cells (ECFCs), and aberrant angiogenesis in MDS bone marrow suggest that other stromal elements could also contribute to disease pathogenesis [15], [16], [17]. Taken together, these profound alterations at the molecular and functional level suggest that abnormalities may exist in multiple components of the bone marrow or the HSC niche. So far these studies have mostly evaluated only one component either in vitro expanded cells or bone marrow sections. It is likely this isolated approach could have a skewed or masked response from other stromal elements in vivo.

To study the role of different stromal elements in these bone marrow samples, we employed mesenchymal subset selection strategy based on the CD146 and CD271 expression. Since in situ localization studies in human bone marrow showed CD146 is expressed on MSCs located in the perivascular region, but it is absent on MSCs resident in the bone-lining location, while cells coexpressing CD271 and CD146 are present in the sub-endothelial layer of sinusoids. CD271 + CD146 − MSCs reside in the trabecular bone-lining endosteal niche [18].

This study was undertaken to simultaneously evaluate abnormalities in the HSC and myeloid progenitors along with the other components of the stroma in a cohort of MDS patients with refractory cytopenia with multilineage dysplasia (RCMD).

Section snippets

Patients and bone marrow samples

The study included 17 patients (14 with RCMD, 3 with RA/RARS) and 20 age-matched controls with ‘normal’ marrow obtained for other diagnostic purposes (see Table 1, Table 2 for details). We selected the controls with morphologically normal marrow in diseases, where no known abnormalities exist in the hematopoietic stem cell or the stroma (see Table 1 for details). The study was approved by the Institutional Review board of the Christian Medical College, Vellore, India. Mononuclear cells were

Loss of LT-HSCs in RCMD patients

By employing specific-defined markers in the method section above for LT-HSCs and HSPCs, we compared frequencies of highly fractionated LT-HSCs, HSPCs and committed progenitors among patients with RCMD and refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) with age-matched controls (Table 1, Fig. 1a). Phenotypic enumeration of LT-HSCs and HSPC1 in RA/RARS did not show significant change (p = NS) in their frequencies when compared to controls (Supporting information, Fig. S1A).

Discussion

Our data on comprehensive evaluation of HSC and stromal components in the bone marrow of patients with MDS RCMD demonstrates that aberrations are present in several of these elements in the same patient. As shown in our data and consistent with earlier reports [7], [8], RCMD patients had a 11-fold loss of LT-HSCs and 7-fold loss of the corresponding HSPC1 compared to controls. The concurrent analysis also revealed no significant alteration in frequency of HSPC2 and HSPC3 populations between

Acknowledgments

We would like to thankfully acknowledge Maulana Azad National Fellowship, University Grants Commission (UGC), Government of India to Salar Abbas. The authors also acknowledge technical support from core facilities of Centre for Stem Cell Research (CSCR), A unit of Institute for Stem Cell Biology and Regenerative Medicine (inStem), Government of India. This work was supported in part from Fluid Research Grant from Christian Medical College, Vellore, Tamil Nadu, India (CMC Res/2/2011 (IRB Min.

Conflict of interest

None.

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