Myelodysplastic Stem Cells Research Articles

Stem Cells in Myelodysplastic Syndromes and Acute Myeloid Leukemia: First Cousins or Unrelated Entities?

Myelodysplastic syndromes (MDSs) are associated with a significant risk of transformation to acute myeloid leukemia (AML), supported by alterations affecting malignant stem cells. This review focuses on the metabolic, phenotypic and genetic characteristics underlying this dynamic evolution, from myelodysplastic stem cells (MDS-SCs) to leukemic stem cells (LSCs). MDS-SCs are more likely to be derived from healthy hematopoietic stem cells (HSCs), whereas LSCs may originate from healthy progenitors, mostly LMPP (lymphoid-primed multipotential progenitors). Moreover, overexpression of CD123 and CLL1 markers by LSCs and MDS-SCs in high risk-MDS [HR-MDS] has led to exciting therapeutic applications. Single-cell sequencing has suggested that clonal evolution in the stem cell compartment was non-linear during MDS initiation and progression to AML, with pre-MDS-SC acquiring distinct additional mutations in parallel, that drive either MDS blast production or AML transformation. In AML and HR-MDS, common metabolic alterations have been identified in malignant stem cells, including activation of the protein machinery and dependence on oxidative phosphorylation. Targeting these metabolic abnormalities could prevent HR-MDS from progressing to AML. Strikingly, in low risk-MDS-SC, the expression of ribosomal proteins is decreased, which may be accompanied by a reduction in protein synthesis.

Ectopic humanized mesenchymal niche in mice enables robust engraftment of myelodysplastic stem cells.

Myelodysplastic syndrome (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SCs) in mice by implantation of human mesenchymal cell-seeded scaffolds. Critically for modelling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Non-invasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data shows that human microenvironment but not mouse is essential to MDS-SCs homing and engraftment. Notably, the alternative niche provided by healthy donor MSCs enhanced engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice, and offers insights into human-specific determinants of MDS-SC microenvironment.

Revert the SIRT: Normalizing SIRT1 Activity in Myelodysplastic Stem Cells.

Myelodysplastic syndromes are hematologic malignancies with few treatment options and a propensity to transform to acute myeloid leukemia. In this issue of Cell Stem Cell, Sun etal. (2018) report that low SIRT1 levels in myelodysplastic stem cells contribute to aberrant self-renewal through enabling hyperacetylation and reduced activity of TET2.

Del(5q) Myelodysplastic Stem Cells Exhibit Their Clonal Advantage Via Increased Adhesion to the Microenvironment

Abstract 790Lenalidomide has emerged as a very effective therapy for del(5q) myelodysplasia. Its mechanism of action, however, has hitherto remained elusive. Interestingly, the more primitive hematopoietic compartment seems to possess a clonal advantage where del(5q) HSC are able to outcompete remaining normal HSC. We have previously demonstrated that lenalidomide is able to abrogate this clonal advantage and found that lenalidomide restored expression of the matricellular protein SPARC, a gene located within the commonly deleted region on chromosome 5q.We hypothesized that the decreased expression of SPARC in del(5q) HSC leads to increased adhesion of HSC to their respective niche cells, translating to increased rates of proliferation, partly explaining the competitive advantage against non-del(5q) HSC.We conducted a prospective study analyzing the del(5q) HSC/progenitor compartment from 23 patients before, during and after (refractory phase) lenalidomide, in order to test whether a hematopoietic stem cell (HSC)-intrinsic decrease of SPARC explains the why and how a clone of cells inherently defective at spawning functioning cellular descendants is not selected against, but rather exhibits a clonal advantage. In addition, we studied whether treatment with lenalidomide induced changes in the microenvironment these cells reside in.We analyzed cell cycle distribution, frequency of apoptosis, and expression of adhesion markers on normal and del(5q) HSPC by multi-parameter flow cytometry. These experiments revealed a slight increase in proliferation of del(5q) versus normal HSC, as well as complex changes in the expression of adhesion markers in HSC of patients treated with lenalidomide.We studied the functional adhesion of normal and del(5q) HSPC to defined matrix components of the microenvironment and observed that HSPC from del(5q) patients exhibited stronger adhesion than normal bone marrow cells to fibronectin and VCAM-1. Recombinant SPARC protein abrogated adhesion to VCAM-1 specifically in a subset of patients, while having no significant effect on normal HSPC.To study whether SPARC plays a role in the clonal dominance, we used lentiviral transduction to overexpress SPARC in HSPC and found that increased expression of SPARC led to severely reduced engraftment in NSG-mice.We also analyzed how lenalidomide impacts the microenvironmental niche. To this end, we compared the gene expression profile of mesenchymal stem cells (MSC) obtained from del(5q) patients by DNA microarray and found only 36 genes to be differentially expressed by more than 2-fold, with 15 and 21 genes up- or downregulated in del(5q) MSC, respectively. Using longitudinal bone marrow biopsies from 5 patients on and off treatment, we analyzed whether lenalidomide induced changes in the frequency of candidate niche cells such as mesenchymal stem cells (MSC), macrophages, endothelium and megakaryocytes, by using immunohistochemical markers for the aforementioned cells. Lenalidomide induced no significant changes in the number of nestin+ MSC but seemed to decrease the number of Factor-VIII+ megakaryocytes.Taken together, these studies suggest that decreased expression of SPARC leads to increased adhesion of del(5q) HSC/progenitor cells to defined components of the microenvironment and may explain why del(5q) HSC are able to outcompete the remaining healthy HSC. Our studies implicate that lenalidomide is able to abrogate this clonal advantage partly via its increase in SPARC expression with a consecutive decrease in adhesion. Disclosures:No relevant conflicts of interest to declare.

Iron Overload and Haematopoiesis in MDS: Does Blood Transfusion Promote Progression to AML?

Introduction: The biology of myelodysplastic syndrome (MDS) is poorly understood, and treatment options are limited. Thus, most MDS patients require chronic red blood cell transfusion, and many develop secondary iron overload. Although the pathophysiological consequences of iron overload to the heart, liver, and endocrine organs have been well characterized, its effects on haematopoiesis have not been studied. However, it has been observed that chelation therapy in iron-overloaded MDS patients may result in reduction of transfusion requirements, and recent studies have suggested a correlation between the use of iron chelation therapy and improvement in leukaemia-free survival in MDS.At the cellular level, iron toxicity is mediated in large part via the generation of reactive oxygen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haematopoietic stem cells, and that ROS cause DNA damage and promote the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature.Hypothesis: We hypothesize that iron overload secondary to transfusion leads to increased levels of intracellular ROS in early haematopoeitic cells in MDS. The increase in intracellular ROS in MDS would be predicted to lead further impairment of haematopoiesis via stem cell exhaustion and while promoting accumulation of DNA damage by myelodysplastic stem cells and early progenitors, thus accelerating progression of MDS to acute leukaemia.Results: To test this hypothesis, we examined the relationship between transfusion-related iron overload and ROS content of CD34+ bone marrow cells in MDS. ROS content was measured in CD34+ cells by flow cytometry in bone marrow aspirates from 34 consecutive MDS patients (CMML=4, MDS/MPD=2, RA=4, RARS=3, RCMD=2, RAEB 1=6, RAEB 2=12, RAEB-t/AML=1). The patients represented a wide range of prior transfusion burden (0->300 units PRBC) and serum ferritin levels (11->10000 μg/L). ROS was strongly correlated with serum ferritin concentration for patients with iron overload (serum ferritin >1000 μg/L; n=14, R=0.733, p<0.005). The correlation between ROS and ferritin level was even stronger in the subset of patients with RAEB 1 or RAEB 2 and iron overload (n=11, R=0.838, p<0.005). In contrast, no correlation between ROS and ferritin level was demonstrated for patients with serum ferritin <1000 μg/L (n=20). Importantly, iron chelation therapy was associated with a reduction in CD34+ cell ROS content in one patient.To assess the effect of iron overload on normal stem cell and progenitor function, we established a mouse model of subacute bone marrow iron overload. B6D2F1 mice were loaded with iron dextran by intraperitoneal injection (150mg total iron load over 21 days), and sacrificed three days after the end of iron loading. Iron staining of tissue sections confirmed iron deposition in the bone marrow, liver, and myocardium. The development of splenomegaly was noted in iron-loaded animals. Flow cytometric analysis revealed increased apoptosis of bone marrow cells in iron loaded mice based on annexin V+/7 AAD-staining (6.26±0.96% versus 3.54±0.99% for control mice, paired student's t-Test p<0.005). However, ROS content in CD117+ progenitors of iron loaded mice was similar to control mice. Thus, subacute iron loading in mice increases apoptosis but does not alter the ROS content of HSCs; we postulate that chronic iron overload is required to achieve this effect.Conclusions: These results establish a relationship between CD34+ cell ROS content and serum ferritin concentration in MDS patients with iron overload, and indicate that iron chelation therapy in this patient population reverses this ROS accumulation. The physiological consequences of this relationship are currently being investigated in this patient set by haematopoietic colony assays and assessment of DNA damage in CD34+ cells. Nonethelesss, these data may have key implications for the deployment of iron chelation therapy in MDS patients, and may explain the association between the use of iron chelation and improved leukaemia-free survival in MDS.

Comparative analysis of hypermethylation of cell cycle control and DNA-mismatch repair genes in low-density and CD34+ bone marrow cells from patients with myelodysplastic syndrome

Hypermethylation of CpG islands within the promoter region is one of the mechanisms by which genes are inactivated and may be one of the reason for silencing of cell cycle control or DNA-mismatch repair genes in myelodysplastic syndrome (MDS). Since the function of cell cycle control genes including the cyclin-dependent kinase inhibitors known as p15 INK4b and p16 INK4a, as well as p14 ARF which blocks MDM-2 (an inhibitor of p53), the retinoblastoma (RB1) protein and the mismatch repair gene MGMT is critical for hematopoietic proliferation and differentiation, we performed methylation specific polymerase chain reaction (MSP) in low-density, non-adherent bone marrow cells from 49 patients with MDS. In addition, expression of p15 INK4b and RB1 was analysed by quantitative real-time PCR. From selected patients, we analyzed the methylation pattern of cell cycle control genes in CD34+ bone marrow cells. Thirty-nine of 49 cases (80%) had at least one of five genes methylated in our MDS samples by analysing low-density non-adherent bone marrow cells. The frequency of p15 INK4b methylation was 34 of 49 samples (69%). The incidence of methylation of both p14 ARF and p16 INK4a was four of 49 (8%). RB1 gene was methylated in seven samples (14%) and each patient had RA. Interestingly, none of these genes were methylated in the purified CD34+ hematopoietic stem cells from the MDS patients. Furthermore, all our RARS patients had a methylated p15 INK4b promoter correlating with non-detectable expression of this gene in bone marrow cells from those patients. These results indicate that hypermethylation of cell cycle control genes in MDS may occur late during the differentiation of myelodysplastic stem cells.