Figure 1.
Schematic presentation of parameters of the mathematical model.
(A) Normal hematopoiesis is assumed in six maturation steps: 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. Grey arrows exemplarily demonstrate the balance of self-renewal and differentiation at each step. The maximal proliferation rates and the maximal self-renewal rates were adopted from our previous work [18]. (B) MDS development is simulated in five compartments (MDS-LT-HSCs, MDS-ST-HSCs, MDS-MPPs, MDS-CPCs and dysplastic precursors). For MDS-LT-HSCs, we assume that the proliferation rate is even lower than for normal LT-HSCs (maximal once per 100 d versus maximal once per 50 d), whereas the self-renewal rate is higher (up to 90% versus up to 70%). (C) Signal intensity for proliferation is assumed to be inversely correlated to mature cells in peripheral blood, whereas the self-renewal signal is inversely correlated to the number of stem and progenitor cells in the BM-niche compartment. Both signals affect normal hematopoiesis as well as the malignant clone.
Figure 2.
Simulation of MDS development.
(A) Simulated MDS development under the assumptions depicted in Figure 1. Initially, a single MDS-LT-HSC is present. Evolution of cell numbers in the different compartments is simulated over 55 years. (B) A sharp decline of mature cells is achieved after about 17 years – this would correspond to clinical manifestation of MDS. (C) Simulated cellular composition in the BM at the relevant time frame. (D) Corresponding signal intensities for self-renewal and proliferation are presented. Self-renewal decays due to the accumulation of malignant cells in the BM-niche; proliferation is activated due to ineffective hematopoiesis. (E) The percentage of apoptotic cells in the bone marrow is presented.
Figure 3.
Serum of MDS patients stimulates proliferation of CD34+ cells.
(A) CD34+ HPCs from umbilical cord blood were stained with CFSE and subsequently cultivated for five days in vitro in culture medium supplemented with 10% serum of individual patients or of healthy controls. Cell division history was monitored by residual CFSE-staining and dotted lines indicate five cell divisions. (B) Mean fluorescence intensities after five cell divisions were normalized to control samples. In each experiment, 57 MDS serum supplements and 17 control serum supplements were tested in parallel. Mean and standard deviation were calculated over measurements with three different cord blood samples (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
MDS serum of cytopenic patients enhances proliferation.
The growth-stimulatory effect of MDS serum on CD34+ cord blood cells was correlated to (A) leucocyte counts, (B) thrombocyte counts, and (C) hemoglobin level (Hb). For statistical analysis of individual experiments (with different CB samples) CFSE intensity was normalized to healthy controls (blue circles resemble MDS serum, red circles individual control samples). Increased proliferation (i.e. less residual CFSE intensity) was observed with serum from leukopenic patients, leukocytotic patients (which rather resemble secondary AML with lower number of mature cells in PB), and anemic patients. Reference ranges used for classification: leucocytes: 3,800–10,500/µL; thrombocytes: 1.5×105–4.5×105/µL; Hb: male: 13–18 g/dL, female: 11–16 g/dL. Error bars represent SD (*p<0.05, **p<0.01, ***p<0.001).
Figure 5.
CFU-Frequency upon stimulation with MDS serum.
(A) CD34+ HPCs from cord blood were cultured with 10% serum supplements of individual MDS patients or controls. After seven days, cells were re-seeded in methylcellulose medium and after two weeks, the numbers of erythrocyte (BFU-E and CFU-E), granulocyte (CFU-G), macrophage (CFU-M) and combined (CFU-GM and CFU-GEMM) colonies were counted. (B) Erythropoietin (EPO) concentration was significantly higher in MDS-derived serum samples. (C) The growth promoting effect on CD34+ cells was then plotted against the EPO concentration. CFSE intensity was normalized to healthy controls of the corresponding experiment. Error bars represent SD (*p<0.05, **p<0.01, ***p<0.001).