Feedback Signals in Myelodysplastic Syndromes: Increased Self-Renewal of the Malignant Clone Suppresses Normal Hematopoiesis
Myelodysplastic syndromes are clonal disorders which are characterized by ineffective hematopoiesis , peripheral cytopenia and a high risk of disease progression towards acute myeloid leukemia ( AML ) 1–3 ., They arise from an aberrant HSC that gains growth advantage over normal hematopoiesis resulting in clonal expansion 4 , 5 ., The pathogenesis of this disease is still unclear , and no curative treatment has been developed with the exception of stem cell transplantation 6–8 ., So far , research has particularly focused on cell-intrinsic modification of MDS cells: mutations and molecular aberrations have been identified which seem to increase proliferation of the malignant clone 9 , 10 ., On the other hand , defects might also emerge as a result of an abnormal microenvironment 11–13 ., Mesenchymal stromal cells ( MSCs ) show intrinsic growth deficiency in MDS 14 and fail to support hematopoiesis 13 ., It has been suggested that MDS is also associated with increased apoptosis rates of normal bone marrow cells 3 , 15 ., So far , the mechanisms that suppress normal hematopoiesis remain unclear , as there is no evidence that the bone marrow niche is completely filled by the malignant clone 4 , 16 ., Self-renewal and differentiation of HSCs need to be tightly controlled according to the physiological needs 17 ., For this purpose , feedback signals may either be derived from the immediate bone marrow microenvironment or by systemically released factors ., The highest self-renewal rate is expected for the long-term repopulating HSCs ( LT-HSCs ) which predominantly remain dormant under steady-state conditions 18 ., Yet , self-renewal and differentiation are also prerequisites of short-term repopulating stem cells ( ST-HSCs ) , multipotent progenitor cells ( MPPs ) , committed progenitor cells ( CPCs ) and precursors 19 , 20 ., In analogy , cells derived from the aberrant MDS clone may also display a hierarchy of self-renewal and differentiation: this is in line with the concept of cancer stem cells – or tumor initiating cells – which then reveal further differentiation and heterogeneity 21 , 22 ., It is generally anticipated that proliferation rates are higher in malignant cells ., On the other hand , several mutations seem to affect the self-renewal in MDS 23 , 24 – yet , this is difficult to study under in vivo conditions ., Mathematical modeling is a powerful tool to study interaction of different cell types and the impact of feedback signals 18 , 25 , 26 ., Based on the biological context several models have been proposed to study the impact of feedback signals on system stability and regenerative properties ., Theoretical and experimental studies on the olfactory epithelium 27 , 28 as well as theoretical considerations of self-renewing cell lineages 29 demonstrate the necessity of feedback signals for system stability and efficient regeneration ., We have recently proposed mathematical models describing activation of the HSC-pool upon hematopoietic stem cell transplantation ( HSCT ) ., These models indicated that feedback signals for self-renewal and proliferation are important ., In particular , the increased self-renewal rates of immature cells facilitate efficient hematopoietic reconstitution 18 , 30 ., Similar results have been obtained for the olfactory epithelium 27 ., Subsequently , we have shown that patient serum obtained during aplasia after HSCT has impact on hematopoietic progenitor cells ( HPCs ) in vitro: it significantly increased proliferation , maintenance of the primitive immunophenotype and expansion of colony forming units ( CFUs ) 31 ., These findings supported the notion that systemically released factors contribute to regulation of stem cell function ., In the current work , we conceived a mathematical model to simulate development of MDS with particular focus on self-renewal and proliferation of the aberrant clone ., MDS is a very heterogeneous disease ., Furthermore , multiple mutations contribute to a complex clonal hierarchy during disease progression and many parameters are so far not well defined in specific cellular subpopulations ., In this regard , we aimed for a conceptional approximation how the malignant clone interferes with normal hematopoiesis – irrespective of specific MDS subtypes or hematopoietic cell lineages as well as multiple mutation scenarios ., Existence of the proposed feedback signals was then substantiated using serum of MDS patients ., The use of all human materials was performed after written consent and according to the guidelines approved by the local Ethic Committees: CD34+ cells were isolated from umbilical cord blood ( CB; Permit Number: EK187/08; RWTH Aachen University ) ; CD34+ cells and MSCs were also isolated from bone marrow ( BM ) during surgical intervention ( Permit Numbers: EK300/13 and EK128/09; RWTH Aachen University ) ; and serum from MDS patients or healthy controls was collected in Düsseldorf and Aachen , respectively ( Permit numbers: 2972 and EK206/09 ) ., The mathematical model developed in this study considers interaction of normal hematopoiesis and myelodysplastic cells in the bone marrow ., It is based on a previously proposed model of the hematopoietic system 18 that was extended to describe dynamics of aberrant clones , as in MDS development 26 ., The model is based on a system of ordinary differential equations describing the flux of cells through different maturation stages for both normal and malignant cells ., The structure of the model is depicted in Figure 1 and a detailed description of the model is given in Text S1 ., CD34+ cells were isolated from fresh umbilical cord blood using the human CD34 Micro Bead Kit ( Miltenyi Biotec GmbH , Bergisch-Gladbach , Germany ) as described before 31 ., Alternatively , CD34+ cells were isolated from human bone marrow aspirate from the femur obtained during orthopaedic surgery ., MSCs were isolated from the caput femoris and cultured as described before 32 , 33 ., For co-culture experiments , we have used MSCs of passage 3 to 6 ( 10–15 population doublings ) ., Serum samples from 57 MDS patients and 5 healthy controls were obtained from the Department of Hematology of Heinrich Heine University in Düsseldorf ., Additionally , serum of 12 healthy controls was obtained from the Department of Gynaecology at RWTH Aachen University ., Generation of serum was performed as described in detail before 31 ., Relevant patient data are summarized in Table 1 in Text S1 ., Hematopoietic progenitor cells were expanded for up to seven days as described previously 34 in StemSpan culture medium supplemented with 10 ng/mL stem cell factor ( SCF; PeproTech GmbH , Hamburg , Germany ) , 20 ng/mL thrombopoietin ( TPO; PeproTech ) , 10 ng/mL fibroblast growth factor 1 ( FGF-1; PeproTech ) and 10 µg/mL heparin ( Roche GmbH , Mannheim , Germany ) 32 ., For co-culture experiments , addition of cytokines was not performed as MSCs alone activate proliferation ., Culture medium was always supplemented with 10% serum of individual MDS patients or control samples as described in our previous work 31 ., Freshly isolated CD34+ cells ( either from CB or BM ) were labelled with carboxyfluorescein diacetate N-succinimidyl ester ( CFSE; Sigma-Aldrich , Hamburg , Germany ) to monitor cell divisions as previously described 34 ., After five days , CFSE intensity was measured by flow cytometry ., For immunophenotypic analysis , cells were stained with CD34-allophycocyanin , CD133-phycoerythrin and CD45-V500 and analyzed using a FACS Canto II ( BD ) 32 ., Further details on immunophenotypic analysis are provided in Text S1 ., Colony forming unit ( CFU ) frequency was determined to estimate culture expansion on HPCs ., In brief , 12 , 500 CD34+ cells were grown for seven days in StemSpan medium supplemented with SCF , TPO , FGF , heparin and 10% patient serum ., The progeny was harvested and analyzed in the CFU-assay as described before 31 ., Concentrations of SCF , TPO and FGF in patient serum were determined with RayBio Human ELISA Kits ( RayBiotec , Norcross , GA , USA ) according to the manufacturers instructions ., Concentration of erythropoietin ( EPO ) was measured by the laboratory diagnostic center of RWTH Aachen University with a chemoluminescent-immunometric assay ( IMMULITE 1000 EPO ) ., All results are expressed as mean ± standard deviation ( SD ) or ± standard error of the mean ( SEM ) ., To estimate the probability of differences , we have adopted the two-sided Students T-test ., Probability value of p<0 . 05 denoted statistical significance ., We propose a mathematical model to address the relevance of self-renewal and proliferation rates for MDS development ., The model describes interaction of, 1 ) normal hematopoietic cells , which progress along long-term repopulating stem cells ( LT-HSCs ) , short-term repopulating stem cells ( ST-HSCs ) , multipotent progenitor cells ( MPPs ) , committed progenitor cells ( CPCs ) , precursors and mature cells ( Figure 1A ) , with, 2 ) cells of the MDS clone which progress through analogous steps of differentiation except for mature cells ( MDS-LT-HSCs , MDS-ST-HSCs , MDS-MPPs , MDS-CPCs and dysplastic precursors; Figure 1B ) ., We assume that proliferation is regulated in normal and malignant cells by feedback signals acting on all developmental stages - it is inversely correlated with the number of mature cells in peripheral blood ( PB ) ., On the other hand , we assume that self-renewal is regulated by cellular density in a virtual stem cell niche occupied exclusively by the more primitive cells in the marrow – it is inversely correlated with the number of cells in the three more primitive compartments ( LT-HSCs , ST-HSCs , MPPs , MDS-LT-HSCs , MDS-ST-HSCs , and MDS-MPPs; Figure 1C ) ., A wide range of values of each parameter has been examined ., The simulations consistently demonstrate that high self-renewal of MDS-initiating cells is crucial for MDS development ., Only if MDS-LT-HSCs have a higher self-renewal potential than normal LT-HSCs , they eventually outcompete healthy hematopoiesis ., In contrary , increased proliferation of MDS-cells alone is not sufficient ., Notably , we have assumed that the proliferation rate of MDS-HSCs is lower than in normal LT-HSCs ( maximal cell division rate every 100 versus every 50 days , respectively ) - even then the MDS clone gains predominance if the self-renewal rate is higher than in normal LT-HSCs ( maximal self-renewal rate 90% versus 70% , respectively ) ., Nevertheless , high proliferation rates in MDS cells - although not required for establishment of the disease – would accelerate expansion of a cell population if self-renewal is also increased ., Our results indicate that increased self-renewal is most essential for MDS , whereas an additional increase of proliferation accelerates the impairment of hematopoiesis ., MDS is usually a slow progressive disease which occurs particularly in elderly people ., Simulated examples with input parameters derived from our previous work 18 , 35 demonstrated that clonally derived MDS cells may increase over approximately 15 to 17 years without clinically relevant changes in bone marrow or blood counts ( Figure 2A ) ., After 17 years , the BM will contain about 1 . 66×10−6% MDS-LT-HSCs , 0 . 39% MDS-ST-HSCs , 1 . 12% MDS-MPPs , 5 . 38% MDS-CPCs and 8 . 15% dysplastic progenitors ., Then , within few years , the number of mature cells in PB drops significantly ( Figure 2B ) ., Correspondingly , the percentage of normal hematopoietic cells in the bone marrow declines ( Figure 2C ) ., The simulated dynamics of disease development are as follows:, 1 ) Initially , a single MDS cell expands very slowly due to higher self-renewal compared to normal LT-HSCs ., 2 ) Consequently , the number of cells in the bone marrow niche increases which leads , via feedback signaling , to reduced self-renewal of cells in the niche ., 3 ) This indirectly results in suppression of normal hematopoiesis and cytopenia ., 4 ) The low number of mature cells triggers proliferation of normal and malignant cells and thereby enhances disease progression ( Figure 2D ) ., In this model , we consider apoptosis rates of mature cells and of dysplastic progenitors only ., However , due to the increasing number of dysplastic precursors which die within 10 days , the percentage of apoptotic cells in the bone marrow increases to 5 . 6% ( under the assumption that apoptosis takes 24 h; Figure 2E ) ., Alternatively , we modeled MDS including further maturation of MDS cells and high apoptosis on the level of committed progenitors ., We assume that MDS derived mature cells have higher apoptosis rates than normal mature cells ( half-life time only 16 h ) , which is in line with higher apoptosis rates in bone marrow and peripheral blood observed in MDS patients 3 , 15 , 36 ., These simulations lead to qualitatively similar results: in all cases , enhanced self-renewal of disease initiating cells is crucial for establishment of the disease ., This indicates that increased apoptosis is compatible - but not required - for MDS development in our approach ( Figure 1 in Text S1 ) ., The percentage of CD34+ cells in healthy bone marrow , low-risk MDS , and high-risk MDS was 1 . 4±0 . 2% , 3 . 4±0 . 7% and 7 . 8±1 . 9% , respectively ( Figure 2 in Text S1 ) ., In our model , we assume that LT-HSCs , ST-HSCs , and MPPs , as well as MDS-LT-HSCs , MDS-ST-HSCs , and MDS-MPPs correspond to CD34+ cells – they are not pure stem cell fractions but they are all influenced by the self-renewal signal ., The percentage of primitive cells is compatible with dynamics of the mathematical model , but it rapidly increases over time ., It has been previously suggested that the percentage of blasts , defined as CD117+ or CD34+ cells , has prognostic value for survival 2 , 37 ., In this regard , it might be speculated that high-risk MDS is characterized by higher cell-intrinsic self-renewal ., Based on our mathematical model , we assumed that serum of MDS might comprise signaling molecules related to the systemic feedback which stimulate proliferation of CD34+ cells ., These cells can be expanded in vitro – particularly if co-cultured with MSCs – but this is associated with further loss of stemness ( Figure 3 in Text S1 ) ., We isolated serum of 57 MDS patients and 12 healthy controls ., CB-derived CD34+ cells were then stained with CFSE and cultured in parallel with culture media supplemented with 10% of individual serum samples ., After five days , the cells were analyzed by flow cytometry ( Figure 3A ) ., Overall , proliferation rate of CD34+ cells , and hence dilution of CFSE , was significantly higher in MDS serum ( p\u200a=\u200a0 . 007 ) ., When we subdivided MDS patients into high risk ( sAML , RAEBI and RAEBII ) , low risk ( RCMD and RCMD-RS ) , CMML I , and 5q chromosomal deletion increased proliferation was particularly observed using serum of low-risk MDS ( p\u200a=\u200a0 . 041; Figure 3B ) ., These results were reproduced with all patient sera using HPCs of three different cord blood samples ., Especially serum derived from leukopenic and anemic patients enhanced proliferation of HPCs ( p\u200a=\u200a0 . 05 and p\u200a=\u200a0 . 004 , respectively ) , whereas this trend was less pronounced with serum from thrombopenic patients ( Figure 4 ) ., However , under co-culture conditions with MSCs , the growth-supporting effect of MDS serum was obscured by the overall growth-stimulation of stromal cells , even though we did not use cytokines in these experiments ( Figure 4 in Text S1 ) ., MDS is rather observed in elderly patients and it is conceivable that age-matched HPCs respond differently to feedback signals ., Therefore , we performed two additional experiments with HPCs from adult bone marrow using all patient sera ., In analogy to our results with CB-derived HPCs , BM-derived CD34+ cells revealed significantly higher proliferation if stimulated with serum from MDS-patients ( del ( 5q ) : p\u200a=\u200a0 . 0005; high-risk MDS: p\u200a=\u200a0 . 0007; low-risk MDS: p\u200a=\u200a0 . 0019; Figure 5 in Text S1 ) ., Overall , the results support the notion that the number of mature cells is inversely correlated with the proliferative effect of patient serum - which is in agreement with our model ., Computer simulations demonstrated that our mathematical model recapitulates clinical observations under the assumption that the feedback signal for self-renewal decays if malignant cells accumulate in the stem cell niche ., Therefore , we reasoned that MDS patient serum might also impair maintenance of the primitive immunophenotype in vitro ., To this end , we have only measured expression of CD34 and CD133 in CB-HPCs which underwent five cell divisions to exclude bias by proliferation ., In fact , CD34 and CD133 expression was moderately decreased with MDS serum ( Figure 3B ) ., In contrast , expression of CD45 was not influenced by MDS serum ., A similar effect was also observed using BM-HPCs ( Figure 5 in Text S1 ) ., Although effects of MDS serum on immunophenotype were rather moderate , they are in agreement with the proposed decrease of the self-renewal signal ., The impact of MDS serum was further analyzed with regard to maintenance of colony forming units ( CFUs ) : CD34+ CB-HPCs were cultured in vitro for 7 days , and this was performed in parallel with medium supplemented with 10% of individual serum samples ., The cells were then reseeded in methylcellulose medium and 12 to 14 days later , colony types and numbers were detected ., Comparing MDS serum and control serum , no significant differences were found in colony-initiating cells ( CFU-G , CFU-M , CFU-GM , and CFU-GEMM ) ., Only the number of erythroid colonies ( BFU-E and CFU-E ) was significantly increased when exposed to serum of MDS patients ( p\u200a=\u200a0 . 004 and p\u200a=\u200a0 . 02 respectively; Figure 5A ) ., Thus , colony assays provide no support for the presence of circulating factors in MDS patient serum that increase colony formation initiated by the most primitive hematopoietic progenitors ., The proposed feedback signals may involve growth factors ., Therefore , we have analyzed serum levels of stem cell factor ( SCF ) , thrombopoietin ( TPO ) and fibroblast growth factor ( FGF ) which support expansion of CD34+ cells in vitro 32 , and of erythropoietin ( EPO ) which stimulates hematopoietic differentiation ., Concentrations of SCF , TPO and FGF were higher in MDS serum than in control serum , but this trend did not reach statistical significance ( Figure 6 in Text S1 ) ., However , the EPO-concentration was significantly higher in MDS patient serum and this is in line with previous reports ( Figure 5B ) 12 , 38 ., Our study indicates that increased self-renewal of MDS-initiating cells is the most critical parameter to initiate MDS development ., This may also explain why the disease seems to be stem cell derived as stem cells already reveal relatively high self-renewal rates ., The central question in this process is the nature of feedback signals regulating hematopoiesis ., Our models suggest that cure of MDS would only be achieved if the self-renewal rate can be specifically down-regulated in the malignant cells - particularly in the tumor-initiating MDS-LT-HSCs ., Therefore , better understanding of the MDS-niche interaction is crucial to identify new therapeutic targets .
Myelodysplastic syndromes ( MDS ) are triggered by an aberrant hematopoietic stem cell ( HSC ) ., It is , however , unclear how this clone interferes with physiologic blood formation ., In this study , we followed the hypothesis that the MDS clone impinges on feedback signals for self-renewal and differentiation and thereby suppresses normal hematopoiesis ., Based on the theory that the MDS clone affects feedback signals for self-renewal and differentiation and hence suppresses normal hematopoiesis , we have developed a mathematical model to simulate different modifications in MDS-initiating cells and systemic feedback signals during disease development ., These simulations revealed that the disease initiating cells must have higher self-renewal rates than normal HSCs to outcompete normal hematopoiesis ., We assumed that self-renewal is the default pathway of stem and progenitor cells which is down-regulated by an increasing number of primitive cells in the bone marrow niche – including the premature MDS cells ., Furthermore , the proliferative signal is up-regulated by cytopenia ., Overall , our model is compatible with clinically observed MDS development , even though a single mutation scenario is unlikely for real disease progression which is usually associated with complex clonal hierarchy ., For experimental validation of systemic feedback signals , we analyzed the impact of MDS patient derived serum on hematopoietic progenitor cells in vitro: in fact , MDS serum slightly increased proliferation , whereas maintenance of primitive phenotype was reduced ., However , MDS serum did not significantly affect colony forming unit ( CFU ) frequencies indicating that regulation of self-renewal may involve local signals from the niche ., Taken together , we suggest that initial mutations in MDS particularly favor aberrant high self-renewal rates ., Accumulation of primitive MDS cells in the bone marrow then interferes with feedback signals for normal hematopoiesis – which then results in cytopenia .
Myelodysplastic syndromes are diseases which are characterized by ineffective blood formation ., There is accumulating evidence that they are caused by an aberrant hematopoietic stem cell ., However , it is yet unclear how this malignant clone suppresses normal hematopoiesis ., To this end , we generated mathematical models under the assumption that feedback signals regulate self-renewal and proliferation of normal and diseased stem cells ., The simulations demonstrate that the malignant cells must have particularly higher self-renewal rates than normal stem cells – rather than higher proliferation rates ., On the other hand , down-regulation of self-renewal by the increasing number of malignant cells in the bone marrow niche can explain impairment of normal blood formation ., In fact , we show that serum of patients with myelodysplastic syndrome , as compared to serum of healthy donors , stimulates proliferation and moderately impacts on maintenance of hematopoietic stem and progenitor cells in vitro ., Thus , aberrant high self-renewal rates of the malignant clone seem to initiate disease development; suppression of normal blood formation is then caused by a rebound effect of feedback signals which down-regulate self-renewal of normal stem and progenitor cells as well .
blood cells, medicine and health sciences, cell cycle and cell division, cell processes, hematologic cancers and related disorders, mathematical computing, mathematics, stem cells, cell growth, mesenchymal stem cells, adult stem cells, computer and information sciences, cell potency, animal cells, stem cell niche, computing methods, myelodysplastic syndromes, hematopoietic progenitor cells, hematology, hematopoietic stem cells, cell biology, biology and life sciences, cellular types, physical sciences, molecular cell biology, hematopoiesis