Additional file 4 of Correction: Quantification of hematopoietic stem and progenitor cells by targeted DNA methylation analysis

Additional file 4. Table S6. Application for NNLS model with 6 CpGs

Development of a cyclin A1-transgenic mouse model.

<p>A. Schematic overview about the constructs used to develop transgenic mouse lines. The driver mouse line SCL-tTA expresses the tetracycline-dependent transactivator protein (tTA) in hematopoietic stem cells under the control of the stem cell leukemia-factor enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref024" target="_blank">24</a>]. In the novel cyclin A1-bi-luciferase responder mouse line, the cDNA of cyclin A1 and luciferase as reporter gene were expressed in absence of tetracycline in parallel and inducibly under control of the bidirectional tTA-responsive promoter element Pbi-1. B. Bars indicate expression levels of human cyclin A1 expression as detected by qRT-PCR in bone marrow cells that were transduced with a retroviral tTA-containing construct and cultured with or without tetracycline in methylcellulose for 10 days (n = 2 for each sample). C. Three months old mice carrying either cyclin A1 alone (control) or together with the driver construct SCL-tTA (SCL-tTAxcyclinA1-tg; n = 3 for each genotype) were induced for seven weeks and investigated for luciferase activity and cyclin A1 mRNA in the bone marrow and spleen. High luciferase activity was only detectable in induced SCL-tTAxcyclinA1-tg bone marrow and spleen cells. Numbers indicate mean luciferase levels.</p

Cyclin A1 expression does not significantly influence PML-RARα-driven leukemogenesis.

<p>A. Kaplan-Meier survival curves of heterozygous PML-RARα-knockin mice with (SCL-cyclinA1-tg; n = 12) or without (control; n = 14) ectopic human cyclin A1 expression in the bone marrow. Although there was a trend towards an accelerated disease in the presence of ectopic cyclin A1 expression, latency and penetrance did not differ significantly between the two genotypes (p = 0.282, log-rank test). Also, the phenotype of acute myeloid leukemia was not changed by the presence of cyclin A1 (B). Shown here are examples of FACS analysis of bone marrow (upper panels) and spleen cells (lower panels) of diseased mice. Murine PML-RARα-driven leukemic blasts are characterized by the surface expression of CD34 and GR-1 which does not occur in non-leukemic mice. The number of CD34⁺/GR-1⁺ cells in diseased mice did not alter significantly upon cyclin A1 expression. SCL, Stem Cell Leukemia enhancer driving tTA expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>]; FI, fluorescent intensity; P/Rα-KI, PML-RARα-knockin mice. C. Kaplan-Meier survival curves of PML-RARα-knockin mice with wild type (PML-RARα-KI/cyclin A1^+/+; n = 30) or cyclin A1-knockout (PML-RARα-KI/cyclin A1^-/-; n = 35). Cyclin A1^-/- mice without PML-RARα-expression did not develop a lethal phenotype (control/cyclinA1^-/-; n = 3). Absence of murine cyclin A1 did not affect PML-RARα-driven leukemia. D. The PML-RARα-leukemic phenotype was not altered by the absence or presence of murine cyclin A1. May-Grünwald staining of blood smears showed the same distribution of leukemic blasts and high numbers of differentiated myeloid cells (upper panels). FACS analysis revealed comparable numbers of myeloid cells in the blood (CD11b⁺/GR-1⁺, lower panels).</p

Cyclin A1 expression in human and murine leukemic blasts.

<p>A. and B. Cyclin A1 was analyzed in mRNA microarray expression data from the purified fraction of human mononuclear bone marrow cells after Ficoll density centrifugation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref019" target="_blank">19</a>]. A. The expression of cyclin A1 was significantly increased in AML blasts compared to normal bone marrow (NBM) (p<0.001, t-test). Shown here are log arbitrary units. B. The expression of cyclin A1 was significantly decreased in AML blasts with complex karyotype and increased in AML M3 blasts compared with normal karyotype and (*p<0.001, t-test). C. Cyclin A1 expression was significantly induced in bone marrow cells from human AML patients with FAB subtype M3 (p = 0.015, t-test) and with FAB subtype M5/5a/5b (p = 0.05, two-tailed t-test) compared to normal bone marrow cells (NBM). Cyclin A1 expression was determined by qRT-PCR and normalized to GAPDH expression level. D. In bone marrow cells of PML-RARα-knockin mice, cyclin A1 expression was significantly upregulated upon a full-blown leukemic phenotype compared to non-leukemic PML-RARα-knockin mice (p = 0.04, t-test).</p

Additional file 1: Figure S1. of Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

Supplementary material & methods

Serum of myeloproliferative neoplasms stimulates hematopoietic stem and progenitor cells

<div><p>Background</p><p>Myeloproliferative neoplasms (MPN)—such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF)—are typically diseases of the elderly caused by acquired somatic mutations. However, it is largely unknown how the malignant clone interferes with normal hematopoiesis. In this study, we analyzed if serum of MPN patients comprises soluble factors that impact on hematopoietic stem and progenitor cells (HPCs).</p><p>Methods</p><p>CD34⁺ HPCs were cultured in medium supplemented with serum samples of PV, ET, or MF patients, or healthy controls. The impact on proliferation, maintenance of immature hematopoietic surface markers, and colony forming unit (CFU) potential was systematically analyzed. In addition, we compared serum of healthy young (<25 years) and elderly donors (>50 years) to determine how normal aging impacts on the hematopoiesis-supportive function of serum.</p><p>Results</p><p>Serum from MF, PV and ET patients significantly increased proliferation as compared to controls. In addition, serum from MF and ET patients attenuated the loss of a primitive immunophenotype during <i>in vitro</i> culture. The CFU counts were significantly higher if HPCs were cultured with serum of MPN patients as compared to controls. Furthermore, serum of healthy young <i>versus</i> old donors did not evoke significant differences in proliferation or immunophenotype of HPCs, whereas the CFU frequency was significantly increased by serum from elderly patients.</p><p>Conclusion</p><p>Our results indicate that serum derived from patients with MPN comprises activating feedback signals that stimulate the HPCs–and this stimulatory signal may result in a viscous circle that further accelerates development of the disease.</p></div

Effects of serum from myelofibrosis patients on hematopoietic stem and progenitor cells.

<p>(A) Exemplary histogram to demonstrate higher proliferation of CD34⁺ HPCs in culture medium supplemented with serum of MF patients as compared to serum from healthy controls. After five days, residual staining of carboxyfluorescein succinimidyl ester (CFSE) is lower with MF-serum. Each peak corresponds to one cell division. (B) Mean fluorescence intensity (MFI) of flow cytometric measurements of HPCs that were cultured for 5 days in parallel with 12 MF and 15 control samples. Values were normalized by the mean MFI of healthy controls. (C) Immunophenotypic analysis in relation to the number of cell divisions (according the residual CFSE staining). The numbers provide estimates for cell divisions. (D) CD34⁺ cells were cultured for seven days in parallel with serum-supplements of MF patients (n = 9) or controls (n = 12) and the number of colony forming units (CFUs) was then analyzed. * p<0.05, ** p<0.01, ***p<0.001.</p

Effects of serum from polycythemia vera patients on hematopoietic stem and progenitor cells.

<p>(A) Residual CFSE staining after five days indicated that serum of PV patients had only a moderate effect on proliferation of CD34⁺ cells as compared to serum of healthy donors. (B) Mean fluorescence intensity (MFI) of flow cytometric measurements of HPCs that were cultured for 5 days in parallel with 8 PV and 15 control samples (normalized to controls). (C) Immunophenotypic analysis in relation to the number of cell divisions (according the residual CFSE staining). The numbers provide estimates for the number of cell divisions. (D) Cell culture with serum of PV patients (n = 8) resulted in higher colony forming unit (CFU) frequency than serum of healthy controls (n = 15). * p<0.05, ** p<0.01, ***p<0.001.</p

Moderate association of stimulatory effects of serum with corresponding blood counts.

<p>Mean fluorescence intensities (MFI) were normalized to healthy controls and fold changes were correlated with clinical parameters. (A) Serum taken from patients with low red blood cell (RBC) counts had in tendency higher stimulatory effect on proliferation of HPCs. MFI of residual CFSE staining was normalized by the mean of the healthy control. (B) Proliferation of HPCs was higher in serum with lower platelet counts (PLC). (C) Serum of patients with low RBC counts supported maintenance of CD34 expression better than serum of patients with high RBC counts. (D) White blood counts (WBC) revealed moderate anti-correlation with maintenance of CD133 expression in HPCs upon culture with corresponding serum samples. (E-G) Cytoreductive therapy did not impact on the stimulatory effect of patient serum with regard to (E) proliferation, (F) CD34 expression, and (G) CD133 expression (mean ± standard deviation; n.s. = not significant).</p

Serum of elderly donors stimulates maintenance of CFU-frequency.

<p>(A) Serum of young and old healthy donors had similar effect on proliferation of hematopoietic stem and progenitor cells (exemplary histogram, residual CFSE was analyzed after five days). (B) Mean fluorescence intensity (MFI) of flow cytometric measurements of HPCs that were cultured for 5 days in parallel with serum of 14 young (< 25 years) and 15 elderly (>50 years) donors (normalized to young donors). (C) Immunophenotypic analysis in relation to the number of cell divisions (according the residual CFSE staining). The numbers provide estimates for the number of cell divisions. (D) Cell culture with serum of elderly donors (n = 15) resulted in higher colony forming unit (CFU) frequency than serum of younger donors (n = 14). * p<0.05, ** p<0.01, ***p<0.001.</p