Fig 1.
Effects of serum from myelofibrosis patients on hematopoietic stem and progenitor cells.
(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.
Fig 2.
Effects of serum from essential thrombocythemia patients on hematopoietic stem and progenitor cells.
(A) Hematopoietic stem and progenitor cells proliferate faster if cultured with serum of ET-patients as compared to healthy controls. Residual CFSE staining 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 15 ET 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 cell divisions. (D) Cell culture with serum of ET patients (n = 15) resulted in higher colony forming unit (CFU) frequency than serum of healthy controls (n = 15). * p<0.05, ** p<0.01, ***p<0.001.
Fig 3.
Effects of serum from polycythemia vera patients on hematopoietic stem and progenitor cells.
(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.
Fig 4.
Moderate association of stimulatory effects of serum with corresponding blood counts.
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).
Fig 5.
Serum of elderly donors stimulates maintenance of CFU-frequency.
(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.